HK1178205A - Dac hyp compositions and methods - Google Patents

Description

DAC HYP compositions and methods

1. Background of the invention

Daclizumab (DAC) is a humanized IgG₁Monoclonal antibodies, which bind to the human high affinity interleukin-2 (IL-2) receptor alpha subunit (CD25 or Tac), are expressed on the surface of activated, but not resting, t-and B-lymphocytes. When bound to CD25 on activated cells, DAC blocks the formation of high affinity IL-2 receptor complexes, thereby blocking IL-2 induced proliferation of activated cells.

DAC with an approximate binding affinity (K) of 0.3nM as determined by a direct binding assay for PHA blasts_D) Binds to CD25 and inhibits PHA blast proliferation in a dose-dependent manner (Hakimi et al, 1993, j. immunol.151 (2): 1075-85). At suboptimal IL-2 doses (2.5ng/mL), proliferation of the IL-2 dependent cell line Kit225/K6 was inhibited by 50% by 15nM DAC (Pilson et al, 1997, J.Immunol.159 (3): 1543-56). In the IL-2 dependent, antigen-induced T cell proliferation assay, 50% inhibition of proliferation was observed using DACs in the range of 0.5-1. mu.g/mL (3-7nM) (Junghans et al, 1990, Cancer Res.50 (5): 1495-502).

One form of DAC was previously manufactured by Hoffman-La as ZENAPAX^TMIs commercially available under the trade name of seq id No. 1, which is used as an adjuvant to immunosuppressive regimens including cyclosporine and corticosteroids to treat acute allograft rejection in kidney transplant patients. ZENAPAX is provided as a concentrate for further dilution and intravenous administration. Concentrate per tubeContains 5mL of a solution containing 5mg/mL DAC, 3.6mg/mL sodium phosphate monobasic monohydrate, 11mg/mL disodium phosphate dodecahydrate, 4.6mg/mL sodium chloride, 0.2mg/mL polysorbate 80, and HCl and/or NaOH sufficient to adjust the pH to pH 6.9. The recommended dose for adult and pediatric patients is 1.0mg/kg by diluting a calculated volume of 25mg/5mL ZENAPAX concentrate with 50mL of sterile 0.9% sodium chloride solution and administering intravenously over a period of 15 minutes through the peripheral or central vein.

DAC also shows efficacy in the treatment of uveitis (Nussenblatt et al, 2004, FOCIS 2004 meeting; Jul 18-23, Montreal, QC.Abstract 4688; Nussenblatt et al, 2003, J.Autoimmun.21: 283-93) and multiple sclerosis (see, e.g., Bielekova et al, 2004, Proc.Nat' I.Acad.Sci.USA 101 (23): 8705-. Although DAC has proven safe and effective, it is desirable to obtain high concentration liquid formulations that have long shelf life and can be conveniently administered without further formulation or manipulation, as well as new molecules of daclizumab with improved properties (such as enhanced safety) compared to ZENAPAX DAC.

2. Brief description of the invention

As mentioned in the background section, daclizumab is a humanized IgG that specifically binds to the human interleukin-2 receptor (IL-2R) alpha subunit (also known as CD25 or Tac)₁Antibodies, the human interleukin-2 receptor is an important mediator of lymphocyte activation. One form of daclizumab was previously manufactured by Hoffman-La as ZENAPAX^TMIs commercially available under a trade name of (a), which when used as an adjuvant to immunosuppressive regimens including cyclosporine and corticosteroids, has been shown to be safe and effective in treating renal allograft rejection (see, e.g., the european medicines agency ("EMEA") market authority for ZENAPAX), and also in treating multiple sclerosis (see, e.g., Bielekova et al, 2004, proc.nat' l.acad.sci.usa 101 (23): 8705-8708, a; rose et al, 2007, Neurology 69: 785-789; U.S. patent No.7,258,859). ZENAPAX DAC is expressed in GS-NS0 (murine myeloma) cells according to EMEA and purified using procedures including Q-Sepharose chromatography, S-Sepharose chromatography, diafiltration, Q-Sepharose II chromatography, ultrafiltration, S-300 gel filtration chromatography and ultrafiltration. It has now been found that daclizumab expressed in NS0 cell line adapted to grow in serum-free, cholesterol-free and other animal product-free medium and isolated by a different process has properties and properties different from, and in some cases superior to, ZENAPAX daclizumab ("ZENAPAX DAC"). This novel daclizumab, referred to herein as DAC HYP, has: an isotype spectrum different from ZENAPAX DAC (as determined by cation exchange chromatography); a different N-linked glycosylation profile than ZENAPAX DAC (even though both forms of daclizumab are expressed in NS0 cells); and ADCC cytotoxicity in bioassay lower than ZENAPAX DAC.

For example, the isoforms of daclizumab may arise from heterogeneity at the N-and C-termini of the heavy chains. Mature V of daclizumab_HThe amino acid sequence of the chain begins at position 20 of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 4). Mature V_HThe N-terminal glutamine (Q) of the chain (bold, underlined text in FIG. 2) can be cyclized to form pyroglutamic acid (pE). In some cases, the signal peptide sequence may be truncated, leaving a residue corresponding to mature V_HA valine-histidine-serine (VHS) sequence linked to the N-terminal glutamine residue of the chain. Since each daclizumab molecule comprises two V s_HThe chain, the various N-terminal isoforms of daclizumab may include forms comprising: (1) two glutamine residues (Q/Q); (2) a glutamine residue and a VHS sequence (Q/VHS or VHS/Q); (3) two VHS sequences (VHS/VHS); (4) a glutamine residue and a pyroglutamic acid residue (Q/pE or pE/Q); (5) a pyroglutamic acid residue and a VHS sequence (pE/VHS or VHS/pE); and (6) two pyroglutamic acid residues (pE/pE). Different C-terminal isoforms are also possible, containing 0, 1 or 2C-terminal lysine (K) residues (0K, 1K or 2K), resulting in a complex isoform spectrum.

Quite surprisingly, when ZENAPAX DAC V_HWhen the N-terminal glutamine of the chain is fully cyclized to pyroglutamic acid, DAC HYP does not reach full cyclization. DAC HYP cation exchange chromatography is therefore characterized by pE/Q and Q/VHS isoform peaks. While not wishing to be bound by any theory, it is believed that these unique pE/Q and Q/VHS isoforms may be affected by the leader sequence used to express DAC HYP. Thus, in one aspect, the disclosure provides a composition of daclizumab, as determined by cation exchange chromatography, wherein the pE/Q isoform comprises from 3% to 17%, from 3% to 15%, from 5% to 15%, more preferably from 5% to 12% or from 7% to 12% of the N-terminal isoform, and/or wherein the Q/VHS isoform comprises from 1% to 15%, more preferably from 3% to 12% of the N-terminal isoform.

In certain embodiments, the daclizumab composition is characterized by a cation exchange chromatography profile substantially similar to the dachpy profile of fig. 18 or 23.

Daclizumab has an N-linked oligosaccharide attached to heavy chain residue Asn 296. When these N-linked oligosaccharides were released using the amidase PNGaseF and analyzed by HPLC, DAC HYP exhibited a different glycosylation profile than ZENAPAX DAC, despite the fact that both were recombinantly produced in the NS0 cell line. Indeed, the glycosylation profile of DAC HYP is exceptionally uniform. Referring to the upper panel of fig. 21, the glycosylation profile of ZENAPAX DAC is characterized by peaks representing the oligosaccharides G0-GlcNAc, G0, G1, Man5, G2, Man6, Man7 and sialylated oligosaccharides. The lower panel of fig. 21 shows that the glycosylation profile of DAC HYP is characterized by two major peaks corresponding to the G0-GlcNAc glycoform and the G0 glycoform, and a minor peak corresponding to the G1 glycoform. The G0-GlcNAc glycoform can comprise about 5% to about 20% of the AUC, more typically about 7.2% to 14.6% of the AUC. The G0 glycoform may constitute 70% to 99.2% of the AUC, more typically 80.9% to 99.2% of the AUC. The G1 glycoform may account for 1% to 9% of the AUC, more typically 1.4% to 3.8% of the AUC. Sialylated oligosaccharides accounted for 1.0% or less of the total AUC.

High levels of immunogenicity and effector function are a problem for drugs that are administered over a long period of time. In addition, rapid clearance rates can reduce the availability of the drug. As is well known to the skilled person, differences in the glycosylation pattern of therapeutic antibodies can lead to differences in immunogenicity. Antibodies with highly homogeneous glycosylation patterns (such as DAC HYP) can provide beneficial immunogenicity profiles, ADCC levels and clearance rates. In addition, biologics with a more uniform glycosylation pattern reduce batch-to-batch variation and may improve consistency and stability.

Thus, in another aspect, the disclosure provides a composition of daclizumab characterized by a uniform N-linked glycosylation profile. In one embodiment, the daclizumab composition is characterized by an N-linked glycosylation profile, as determined by HPLC, that includes a G0-GlcNAc glycoform for a total AUC of about 5-20%, in certain embodiments a G0-GlcNAc glycoform for a total AUC of about 5% -18% or about 7-15% (e.g., 7.2% -14.6% or 6.9% to 14.7%), in certain embodiments a G0-GlcNAc glycoform for a total AUC of about 7.3%, and a G0 glycoform for a total AUC of about 70% -99.2%, in certain embodiments a G0 glycoform for a total AUC of about 75% -90%, about 75-92%, or about 81-88% (in certain embodiments a G0 glycoform for a total AUC 86%). Alternatively, the G1 peak is less than about 10% of the total AUC, less than about 5% of the total AUC, less than about 4% of the total AUC, or less than about 3% of the total AUC, and in certain embodiments, from about 1% to about 4% (e.g., 1.4% to 3.8%) or from about 1% to about 3%. The Man5 glycoform preferably comprises about 3% or less of the total AUC. In other embodiments, the daclizumab composition is characterized by an HPLC N-linked glycoform profile substantially similar to the profile shown in fig. 19.

In certain aspects, the disclosed daclizumab compositions are characterized by a total of two or more glycoform peaks. In certain embodiments, the disclosed daclizumab compositions are characterized by (a) two major peaks corresponding to the G0-GlcNAc glycoform and the G0 glycoform that collectively comprise about 75% to about 100%, about 80% to about 100%, or about 85% to about 100% of the total AUC and/or (b) peaks corresponding to the Man5, Man6, and Man7 glycoforms that collectively comprise about 6% or less of the total AUC and/or (c) peaks corresponding to the Man6 and Man7 glycoforms that collectively comprise about 2% or less of the total AUC. In such embodiments, the percentage of G0-GlcNAc, G0, G1, and/or Man5 may be present in the amounts described in the preceding paragraph.

The binding and inhibitory properties of DAC HYP, as well as the potency of DAC HYP function as evaluated in assays measuring inhibition of IL-2 induced T cell proliferation, were similar to those of ZENAPAX DAC. However, quite surprisingly, DAC HYP showed significantly lower ADCC cytotoxicity than ZENAPAX DAC, possibly due, at least in part, to the difference in their non-fucosylated mannose glycosylation levels (see figure 21). As shown in figures 22A and 22B, DAC HYP showed at least 25% lower ADCC cytotoxicity than ZENAPAX DAC as determined in the cellular assay. As the skilled person will appreciate, the reduced ADCC cytotoxicity of DAC HYP is beneficial for indications involving long-term administration of unwanted cell death, such as the treatment of multiple sclerosis and uveitis. In these cases of long-term application of therapy, e.g., as in the treatment of multiple sclerosis and other non-neoplastic indications, DAC HYP therapy may be compared to the use of zennapax^TMThe treatment is safer.

Thus, in another aspect, the disclosure provides a daclizumab composition characterized by exhibiting less than about 30%, 25%, 20%, 15%, 10%, 5% or even less ADCC cytotoxicity at a concentration of 1 μ g/mL, wherein ADCC cytotoxicity is determined in an in vitro assay using a 25: 1, 40: 1, 50: 1 or 60: 1 effector cell to target cell ratio, such as when using Kit225/K6 as a target cell and/or when using PBMC effector cells from 3 or more, 6 or more, 10 or more or 50 or more healthy donors. In particular embodiments, the disclosure provides a daclizumab composition characterized by exhibiting ADCC cytotoxicity in the range of 5-30%, 10-30%, 15-30%, 5-25%, 10-25%, 20-30%, 15-25%, 15-35%, or 20-35% at a concentration of 1 μ g/mL, wherein ADCC cytotoxicity is in a ratio of effector cells to target cells using a 25: 1, 40: 1, 50: 1, or 60: 1 ratio, such as when using Kit225/K6 as a target cell and/or when using a fraction from 3 or more, 6, or 3 or moreAbove, 10 or above or 50 or above healthy donors in vitro. Considering that DAC HYP is IgG₁The low level of ADCC cytotoxicity observed with DAC HYP compared to ZENAPAX DAC was surprising in immunoglobulins and without the framework mutations known to reduce ADCC cytotoxicity.

The safety of DAC HYP compared to ZENAPAX DAC can be further improved by using a high yield serum-free process that allows the production of high purity products without Bovine Serum Albumin (BSA). Accordingly, the disclosure herein provides a composition of daclizumab that is BSA-free and/or is a product of a cell culture process in the absence of BSA.

A composition of daclizumab (DAC HYP composition) characterized by 1 or more of the properties discussed above may be conveniently obtained by recombinant expression in mammalian cells. While not wishing to be bound by any particular theory of operation, it is believed that 1 or more of the unique features and/or characteristics discussed above may be due, at least in part, to the use of a high-yielding recombinant expression system. This can be achieved in any way, such as by gene amplification using DHFR, or using a selectable marker gene under the control of a weak promoter, preferably in combination with a strong promoter that drives expression of the protein of interest, preferably a secreted protein. Without being bound by theory, it is believed that selection of a marker under the control of a weak promoter facilitates identification of stable transfectants in which the expression vector is integrated into a chromosomal region where transcription is active, thereby achieving high expression levels of the protein of interest. In one embodiment, the weak promoter driving expression of the selectable marker is the SV40 promoter (Reddy et al, 1978, Science 200: 494-502), in which the activity of one or both enhancer regions has been reduced or eliminated, e.g., by partial or complete deletion, optionally in combination with a strong promoter driving expression of the protein of interest, e.g., the CMV IE promoter (Boshart et al, 1985, Cell 41 (2): 52130).

Thus, in another aspect, the disclosure provides vectors for the production of recombinant cell lines stably expressing high levels of daclizumab, such as DAC HYP, wherein expression of a selectable marker is under the control of the SV40 promoter in which enhancer function has been reduced, such as by partial deletion of one or both enhancer sequences (designated dE-SV 40). A specific dE-SV40 promoter sequence which can be used for the generation of stable expression cell lines is located at position 6536-6735 of the vector pHAT. IgG1.rg. dE (SEQ ID NO: 5), which is shown in FIGS. 3A-3D and 3E (SEQ ID NO: 12). Various embodiments of specific vectors that can be used to generate stably expressing cell lines are described in U.S. application No. 61/565,419 filed on 30/11/2011 and in international application No. PCT/US11/62720 filed on 30/11/2011, which are incorporated herein by reference.

Generally, a vector for expressing a daclizumab, such as DAC HYP, will comprise one or more features exemplified by phat.igg1.rg.de (described in section 5.1 below), such as a promoter. The two strands of daclizumab may be placed under independent transcriptional control, but are preferably on the same vector, and the coding region may be genomic DNA or cDNA containing introns and exons. As an alternative to independent transcriptional regulation, the two chains may be expressed as a single transcript or a single open reading frame, the coding regions of which are separated by an internal ribosomal entry site or a self-cleaving internal protein sequence, in which case the heavy and light chain coding sequences are under the control of a single promoter. Exemplary promoters are the CMV IE promoter and enhancer (at positions 0001. igg1.rg. de (SEQ ID NO: 5) 0632 and 3982. 4604). Other features include a transcription initiation site (if no promoter is selected), a transcription termination site, and an origin of replication. Examples of these features are shown in table 1, where the elements of pHAT. IgG1.rg. dE are summarized.

Particular embodiments for expressing the heavy and light chains of a daclizumab, such as DAC HYP, from a single exogenous nucleic acid in NS0 cells use a selectable marker operable in mammalian cells, such as neomycin phosphotransferase (neo)^r) Hygromycin B phosphotransferase (hyg)^r) Hygromycin B phosphotransferase (Hph), puromycin-N-acetyltransferase (pu)ro^r) Blasticidin S deaminase (bsr)^r) Xanthine/guanine phosphoribosyltransferase (gpt), Glutamine Synthetase (GS) or herpes simplex virus thymidine kinase (HSV-tk). In a preferred embodiment, the selectable marker in the disclosed vectors is the E.coli guanine phosphoribosyltransferase selectable marker under the control of the SV40 promoter with enhancer attenuation, the coding sequence of which can be found at position 6935-7793 of pHAT. IgG1.rg. dE (SEQ ID NO: 5) shown in FIGS. 3A-3D.

In another aspect, the disclosure provides a host cell transfected with a vector for recombinant production of daclizumab, such as DAC HYP. The host cell may be any mammalian cell, including, for example, Chinese Hamster Ovary (CHO) cells, NS0 murine myeloma cells, Sp2/0 cells, per.c6 cells, Vero cells, BHK cells, HT1080 cells, COS7 cells, WI38 cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, MDCK cells, and 293 cells. Once transfected, the vector can integrate into the genome to produce a stably producing cell line. The skilled person will appreciate that it is not desirable to include animal products in compositions intended for administration to humans. Thus, host cells that do not require serum or other animal products (such as cholesterol) for growth are preferred. Host cells in need of such animal products can be adapted to utilize serum-free and other animal product-free media. Methods for adapting murine myeloma NS0 cells to growth in serum-and cholesterol-free medium are described in Hartman et al, 2007, Biotech. & bioeng.96 (2): 294, 306 and Burky et al, 2007, Biotech. & Bioeng.96 (2): 281 and 293. The specific NS0 cell line adapted to growth in serum-free and cholesterol-free medium has been stably transfected with the vector described above that can be used to produce DAC HYP (clone 7a11-5H 7-14-43).

As the skilled artisan will recognize, the basal and feed media used to culture the recombinant protein-producing cells, as well as other variables such as feed schedule, growth rate, temperature, and oxygen level, may affect the yield and quality of the expressed protein. Methods of optimizing these conditions are within the purview of the skilled artisan; exemplary conditions are set forth in exemplary embodiments of the disclosure. Preferably, the cells are adapted to grow in a medium free of cholesterol, serum and other animal-derived components; in this case the base and feed media preferably comprise limiting chemicals which can replace these components. It has also been found that a medium containing high levels of glucose, such as 10-35g/L glucose, advantageously increases the productivity of the cell culture. In specific embodiments, the basal medium has about 10-20g/L, more preferably about 15g/L of glucose and/or the feed medium has 22-35g/L, more preferably about 28g/L of glucose. As known in the art, the feed medium may be added to the cells over a period of 8-15 days, preferably 9-13 days, or most preferably 10-13 days, according to a gradual ascending feed schedule.

For DAC HYP expressed in NS0 producer strain 7A11-5H7-14-43, the composition of the growth and feed media and other variables affecting expression and production have been optimized. Accordingly, the disclosure also provides optimized basal media, feed schedules, and other culture methods and conditions for producing daclizumab in high yield and purity. These media and culture parameters and methods are described in more detail in section 5.3.

It has also been found that purifying daclizumab from a cell culture using a combination of certain chromatography steps results in a purified daclizumab and DAC HYP drug substance composition, as well as a liquid daclizumab and DAC HYP drug formulation that is stable to storage in liquid form at high concentrations, wherein typically the nominal concentration of daclizumab or DAC HYP is at least about 100mg/mL ± 10-15%, in some embodiments 150mg/mL ± 10-15% (as determined by UV spectroscopy or refractive index).

Stable high concentration pharmaceutical formulations of daclizumab are generally prepared by exchanging a concentrated daclizumab formulation with an exchange buffer having an osmolality in the range of about 267-327mOsm/kg (e.g., 270-310mOsm/kg) and a pH at 25 ℃ in the range of about pH 5.8-6.2 (e.g., 5.9-6.1 at 25 ℃) to produce an intermediate formulation, followed by dilution of the intermediate formulation with a polysorbate dilution buffer to obtain a stable high concentration liquid formulation, wherein the high concentration liquid formulation comprises about 100mg/mL ± 10% daclizumab (e.g., DAC HYP), in certain embodiments at least about 150mg/mL daclizumab (e.g., DAC HYP), as determined by UV spectroscopy or refractive index. The dilution buffer is the same as the exchange buffer, but contains about 0-10% (w/v) polysorbate 80, which is used in an amount such that the final stable high concentration daclizumab formulation has a calculated polysorbate 80 concentration (nominal concentration) that is in the range of 0.02-0.04%, and in certain embodiments about 0.03% (w/v). A variety of different buffers and excipients may be included in the exchange and dilution buffers to achieve osmolarity and pH within defined ranges. A specific non-limiting example of an exchange buffer suitable for formulating a stable, high concentration liquid pharmaceutical formulation of daclizumab and DAC HYP contains about 40mM succinate and about 10mM NaCl and has a pH of about 6.0 at 25 ℃. A specific non-limiting example of a dilution buffer suitable for use with this exchange buffer contains about 40mM succinate, about 100mM NaCl, and about 1% (w/v) polysorbate 80, and has a pH of about 6.0 at 25 ℃. The pH of the final formulation can be adjusted using acid or base to achieve an actual pH of about 6.0 at 25 ℃.

Stable, high concentration liquid formulations of daclizumab are characterized by low levels of aggregation, as determined by size exclusion chromatography, which typically contain at least 95% monomer and less than 3% aggregate, sometimes less than 1.5% aggregate, more typically greater than 99% monomer and less than 0.8% aggregate. Other purity characteristics of high concentration liquid daclizumab pharmaceutical formulations are described in more detail in section 5.6.

The high concentration pharmaceutical formulations of daclizumab are further characterized by a long shelf life, being stable for up to 54 months or more, such as at least 5 years, at 2-8 ℃, storage under accelerated conditions (23-27 ℃/60 ± 5% relative humidity), and storage under pressurized conditions (38-42 ℃/75 ± 5% relative humidity), without producing greater than 5% degradation and formation of greater than 3% aggregates (as determined by SDS-PAGE and size exclusion chromatography, respectively), for up to 9 months or more.

As described above, a stable high concentration liquid formulation of daclizumab may be prepared by diluting the intermediate formulation with a polysorbate dilution buffer to obtain a finished pharmaceutical formulation of daclizumab. Thus, in another aspect, the disclosure provides a purified daclizumab (preferably DAC HYP) intermediate formulation without a polysorbate, containing at least 150mg/mL of daclizumab, and in certain embodiments about 170-190mg/mL of daclizumab, that may be diluted with a polysorbate dilution buffer to obtain a stable high concentration liquid pharmaceutical formulation of daclizumab as described herein. In particular embodiments, the concentrated polysorbate-free intermediate formulation nominally contains about 155mg/mL or about 180mg/mL of daclizumab (preferably DAC HYP), about 40mM sodium citrate, and about 100mM NaCl, at a pH of 6.0 at 25 ℃. In particular embodiments, the concentrated polysorbate-free intermediate formulation nominally contains about 155mg/mL or about 180mg/mL of daclizumab (preferably DAC HYP), about 40mM sodium succinate, and about 100mM naci, at a pH of 6.0 at 25 ℃. The daclizumab compositions are characterized by low aggregate levels, which are further described below.

It has been found that concentration of daclizumab by ultrafiltration induces the formation of aggregates, which can result in high concentration pharmaceutical formulations of daclizumab containing unacceptable levels (e.g., > 3%) of aggregates. Therefore, it is preferred to utilize a "refining" step to remove aggregates prior to concentrating the daclizumab drug substance. The acceptable aggregate level prior to concentration will depend on the concentration of the daclizumab drug substance to be concentrated, the desired concentration in the final daclizumab drug formulation, and the acceptable aggregate level in the final daclizumab drug formulation. For example, if it is desired to obtain a 150mg/mL daclizumab formulation containing less than 3% aggregates, and the daclizumab drug substance must be concentrated 10 to 30 fold (e.g., 20 fold) to achieve this completed daclizumab formulation, the daclizumab composition to be concentrated should contain < 0.3% aggregates, preferably < 0.2% aggregates, preferably even lower levels, such as about 0.1% aggregates.

The starting daclizumab pharmaceutical substance composition containing acceptable aggregate levels may be obtained for concentration into the concentrated daclizumab intermediate and final pharmaceutical formulations described herein using a variety of known techniques, including, for example, strong cation exchange chromatography and hydrophobic interaction chromatography. However, it has been surprisingly found that weak cation exchange chromatography reduces the aggregate level of a daclizumab composition containing daclizumab in the range of 4-12mg/mL and up to 2.5% aggregates to very low levels, typically to about 0.1% aggregates. The use of weak cation exchange to remove aggregates is environmentally milder compared to hydrophobic interaction chromatography with nitrogen-containing solutions (e.g., ammonium sulfate solutions).

Thus, in another aspect, the disclosure provides methods of refining a daclizumab composition to remove aggregates, the refined composition so obtained typically comprising about 4 to 15mg/mL of daclizumab, as determined by size exclusion chromatography, wherein 0.3% or less (e.g., 0.2% or less or 0.1% or less) is in the form of aggregates. The method generally comprises flowing a composition of daclizumab comprising about 4-10mg/mL, typically about 8-9mg/mL, preferably about 8.5mg/mL, daclizumab and > 0.5% aggregates through a weak cation exchange resin in a suitable buffer to adsorb the daclizumab, and eluting the adsorbed daclizumab with an elution buffer. Useful weak cation exchange resins include, but are not limited to, CM-650M (Tosohbiosciences), CM-Sepharose, CM-HyperD. The composition of the equilibration, wash and elution buffers depends on the weak cation exchange resin used, as will be apparent to those skilled in the art. For CM-650M resin (Tosoh Biosciences, product No. 101392), equilibration and wash buffers at pH 4.5 containing about 20mM sodium citrate and elution buffers at pH 4.5 containing 20mM sodium citrate and 75mM sodium sulfate work well. The flow rate used depends on the choice of resin and the column size. For a CM-650M resin cylindrical column having a bed height in the range of about 10-30CM (e.g., 17-19CM), a flow rate in the range of about 50-200CM/hr (e.g., 90-110CM/hr, preferably about 100CM/hr) works well. Chromatography may be carried out at room temperature or lower, such as temperatures in the range of 4 °, 10 °, 15 °, 20 ° or 25 ℃. The temperature range generally used is 18-25 deg.C (e.g., 18-22 deg.C).

The purification process of ZENAPAX DAC according to ZENAPAX EMEA comprises the following 12 steps:

(i) concentrating the culture medium;

(ii) Q-Sepharose chromatography;

(iii) S-Sepharose chromatography;

(iv) low pH treatment to inactivate virus;

(v) concentration/diafiltration;

(vi) DV50 filtration to remove virus;

(vii) Q-Sepharose II chromatography;

(viii) viresolve chromatography to remove virus;

(ix) performing ultrafiltration concentration;

(xi) S-300 gel filtration chromatography;

(xii) Concentrating by ultrafiltration

(xiii) Filling into bottles in a sterile manner.

The process is inefficient and the purification yield is low. It has been found that a process with fewer steps can achieve higher yields while achieving higher purity, which enables the resulting daclizumab drug substance to be formulated into the high concentration pharmaceutical formulations described above. Accordingly, the disclosure herein also provides improved methods of isolating and/or purifying a daclizumab drug substance and a high concentration pharmaceutical formulation. This process utilizes a combination of protein A affinity chromatography, strong anion exchange chromatography (Q-Sepharose), and weak cation exchange chromatography (CM-650M), which allows for continuous flow processing without dilution of the process intermediates. An improved method for obtaining a purified daclizumab drug substance comprises the steps of:

(i) protein a affinity chromatography to isolate daclizumab from other cell culture components;

(ii) low pH viral inactivation;

(iii) strong anion exchange (Q-Sepharose) chromatography to remove DNA

(iv) Weak cation exchange (CM-650M) chromatography to reduce aggregates; and

(v) filtering to remove virus.

The exact volume, column size and operating parameters will depend in part on the scale of purification, as is well known in the art. Specific volumes, column sizes and operating parameters for large scale purification are described in section 5.4.

Crude daclizumab to be purified and optionally formulated by the methods described above can be harvested from the cell culture using a variety of conventional methods, such as microfiltration, centrifugation, and depth filtration directly from the bioreactor. However, it has been found that crude daclizumab can be conveniently harvested by flocculating the cells by lowering the pH of the cell culture to about pH5 at a temperature below 15 ℃, which flocculated cells can be removed by centrifugation. In one embodiment, crude daclizumab is harvested by lowering the pH of the cell culture to about pH5, cooling the culture to less than 15 ℃ (e.g., 4 ℃) for 30-90 minutes, and centrifuging the resulting suspension to remove cells. This process is generally applicable to any cell culture that secretes a recombinant protein into the culture medium, and is not particularly applicable to cultures that produce daclizumab or therapeutic antibodies. A variety of different acids, including weak or strong organic acids, or weak or strong inorganic acids, can be used to adjust the pH of the culture. For the daclizumab cultures, citric acid has been found to work well. The pH of the culture may be adjusted prior to harvest using a concentrated citric acid solution, such as a 0.5M-2M solution.

Purification of dacyphp was accomplished by using three chromatography steps, virus inactivation, virus filtration and final ultrafiltration. Protein a affinity chromatography is the first step in the purification process, which removes most of the process-related impurities. In order for the protein a affinity column to be reusable, it must be regenerated and sterilized. It has been found that aqueous NaOH is effective in accomplishing regeneration and disinfection of the column. However, the use of NaOH solution may degrade the protein a resin, increasing the overall production cost. It has also been found that the use of solutions containing NaOH and benzyl alcohol to disinfect protein a affinity chromatography resins gives good results and leads to a significant increase in the number of purification cycles. Accordingly, the disclosure also provides a sanitizing solution and method for regenerating and sanitizing protein a affinity columns and resins. The buffer typically contains about 100 to 500mM sodium citrate, about 10 to 30mM NaOH, and about 0.5 to 3% (v/v) benzyl alcohol, and has a pH in the range of about pH 10 to 13. The buffer may also optionally include other components, such as salts and/or detergents. Sodium citrate and benzyl alcohol are important for both protecting the protein a resin from NaOH and for enhancing microbicidal activity. In a specific embodiment, the protein a disinfection buffer comprises about 200mM sodium citrate, about 20mM NaOH, and about 1% (v/v) benzyl alcohol. As described in section 5.4.2, the disinfectant solution containing benzyl alcohol and sodium hydroxide has beneficial antimicrobial effects and can be used to disinfect a protein a column during any antibody purification process.

The sterilization buffer may be used to sterilize protein a chromatography resin in a batch process, wherein the resin is washed with an excess (e.g., 1.5-2X volume) of the sterilization buffer, followed by incubation in an excess (e.g., 1.5-2 volumes) of the sterilization buffer for about 30-45 minutes, followed by equilibration with an equilibration buffer or a storage buffer. The sterilization buffer may also be used to sterilize a prepared protein A chromatography column, wherein the column is washed with an excess (e.g., 1.5-2 column volumes) of the sterilization buffer at a suitable flow rate (e.g., about 110-. Suitable equilibration and storage buffers are described in section 0.

The sterilization of protein a columns with the sterilization buffer described herein significantly increases the number of purifications that a single batch of resin can be used. A single batch of protein a resin can typically only sustain about 30 purification cycles, as sterilized with conventional NaOH buffers (e.g., 50mM NaOH, 0.5M NaCl), whereas a protein a column sterilized with the sterilization buffer described herein can use more than 100 purification cycles. While not wishing to be bound by any theory of operation, it is believed that the sterilization buffer described herein partially protects the immobilized protein a from NaOH-induced degradation, thereby increasing the useful life of the resin. Thus, while improvements are expected for all protein a resins, including those using mutant protein a strains designed to resist NaOH degradation (e.g., MabSuRe resin), the sterilization buffers described herein are particularly beneficial when used to sterilize protein a resins and columns using unmodified immobilized protein a or protein a that has not been engineered to be NaOH stable. The disclosure further provides methods comprising performing more than 30, more than 35, or more than 40 antibody purification runs, and in some cases up to 50 or up to 100 protein purification cycles using a protein a affinity resin, comprising performing the purification runs as disclosed herein and washing the resin with a disinfecting solution.

As described above, daclizumab specifically binds to CD25 expressed on activated but not resting T and B lymphocytes and blocks the binding of IL-2 to CD25, thereby inhibiting the formation of high affinity IL-2 receptor complexes and inhibiting the proliferation of activated T-and B-cells. The DAC compositions and formulations described herein, particularly DAC HYP compositions and formulations, also bind specifically to CD25 and exhibit similar biological properties. The DAC compositions and formulations described herein, particularly DAC HYP, may thus be used in any of the tests and treatment methods described generally for daclizumab, particularly ZENAPAX. Accordingly, the disclosure also provides methods of inhibiting activated T-and B-cell proliferation using the DAC compositions and formulations described herein, particularly DAC HYP compositions and high concentration stable liquid formulations, in vitro and in vivo as therapeutic methods for treating diseases in which activated T-and B-cell proliferation plays a role, such as treating and preventing allograft rejection, treating uveitis, and treating multiple sclerosis.

The methods generally comprise contacting activated T-and/or B-cells with a composition or formulation of daclizumab described herein in an amount sufficient to inhibit proliferation thereof.

For methods of treatment, the method generally comprises administering to the subject an amount of a daclizumab composition, a DAC HYP composition as described herein or a high concentration DAC formulation, to provide a therapeutic effect. In a particular embodiment, the daclizumab compositions and formulations may be used alone or in combination with other agents, such as interferon-beta, for the treatment of multiple sclerosis. The DAC compositions described herein can be administered subcutaneously to a patient at a dose ranging from 75mg to 300mg (e.g., 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, or 300mg) or from 1mg/kg to 4mg/kg from weekly to monthly (e.g., weekly, biweekly, twice monthly, four weeks, or monthly). The composition may be provided in a pre-filled syringe for subcutaneous use, preferably at a nominal concentration of daclizumab of 100mg/mL + -10-15% or 150mg/mL + -10-15%. The concentrated DAC compositions may also be diluted for intravenous administration.

3. Brief description of the drawings

FIG. 1 provides the DAC-HYP light chain cDNA (SEQ ID NO: 1) and the translated amino acid sequence (SEQ ID NO: 2). The bold, underlined aspartic acid (D) residue is the first amino acid of the properly processed mature protein, and the amino acid sequence upstream of this residue corresponds to the signal sequence.

FIG. 2 provides the DAC-HYP light chain cDNA (SEQ ID NO: 3) and the translated amino acid sequence (SEQ ID NO: 4). The bolded, underlined glutamine (Q) residue is the first amino acid of the properly processed mature protein, and the amino acid sequence upstream of this residue corresponds to the signal sequence.

FIG. 3A-FIG. 3D together provide the full nucleotide sequence (SEQ ID NO: 5) of the vector pHAT. IgG1.rg. dE.

FIG. 3E provides a specific embodiment of the dESV40 promoter (SEQ ID NO: 12) that can be used to select high producing strains.

Figures 4A-4B provide a schematic representation of the vector pHAT. IgG1.rg. dE (figure 4A) derived from the vector pABX. gpt (figure 4B) modulated to express any heavy and light chain genes or even non-antibody polypeptides.

Fig. 5 provides an exemplary production process for DAC HYP.

Figure 6 shows UV (280nm), pH and conductivity monitoring of the product fractions during protein a affinity chromatography.

FIG. 7 shows UV (280nm), pH and conductivity monitoring of the product fractions during Q-Sepharose chromatography.

FIG. 8 shows UV (280nm), pH and conductivity monitoring of the product fractions during CM cation exchange chromatography.

Fig. 9 provides a schematic illustration of a DAC HYP ultrafiltration system.

Figure 10 provides a 0-60 minute DAC HYP peptide chromatogram. The reference spectra are 100mg/mL of the DACHYP preparation, and Lot 1 and Lot 2 correspond to 150mg/mL of the DAC HYP preparation.

FIG. 11 provides a chromatogram of DAC HYP peptide from 55 to 115 minutes. The reference spectrum is 100mg/mL of DAC HYP preparation, with batch 1 and batch 2 corresponding to 150mg/mL of DAC HYP preparation.

FIG. 12 provides a 110-170 minute DAC HYP peptide chromatogram. The reference spectrum is 100mg/mL of DAC HYP preparation, with batch 1 and batch 2 corresponding to 150mg/mL of DAC HYP preparation.

FIG. 13 provides overlaid circular dichroism spectra for DAC HYP150mg/mL batch 1 and batch 2. Reference is a 100mg/mL preparation of DAC HYP.

Fig. 14A-14B provide the overlaid zero-order uv spectrum and overlaid second-derivative uv spectrum, respectively. The reference spectrum is 100mg/mL of DAC HYP preparation, with batch 1 and batch 2 corresponding to 150mg/mL of DAC HYP preparation. All three spectra are shown in each of fig. 14A and 14B, but appear as a single spectrum once overlaid on each other.

FIGS. 15A-15B provide size exclusion chromatograms at full scale and expanded scale, respectively. The reference spectrum is 100mg/mL of DAC HYP preparation, with batches 1 and 2 corresponding to 150mg/mL of DACHYP preparation.

FIG. 16 is a plot of DAC HYP aggregation as a function of time.

FIG. 17 shows reduced and non-reduced SDS-PAGE (left and right panels, respectively). The reference spectrum is 100mg/mL of DAC HYP preparation, with batches 1 and 2 corresponding to 150mg/mL of DACHYP preparation.

Figure 18 shows a cation exchange chromatogram of DAC HYP. The reference spectra are 100mg/mL of the DACHYP preparation, and Lot 1 and Lot 2 correspond to 150mg/mL of the DAC HYP preparation. The peak markers correspond to different N-and C-terminal isoforms.

FIG. 19 shows an HPLC chromatogram of N-linked oligosaccharides cleaved from DAC HYP. The reference spectrum is 100mg/mL of DAC HYP preparation, with batches 1 and 2 corresponding to 150mg/mL of DACHYP preparation.

Figure 20 shows ADCC response curves for DAC HYP. The reference spectra are 100mg/mL of the DACHYP preparation, and Lot 1 and Lot 2 correspond to 150mg/mL of the DAC HYP preparation.

Figure 21 shows HPLC chromatograms of N-linked oligosaccharides released from DAC HYP (lower panel) and ZENAPAX DAC (upper panel) displaying their different glycosylation profiles.

Figures 22A-22B provide a comparison between ADCC activity of two DAC HYP preparations (referred to as DAC HYP batch 3 and DACHYP batch 4), DAC Penzuburg and zennapax DAC, using an ADCC assay format with variable effector to target cell ratio (figure 22A) and variable antibody concentration (figure 22B).

Figure 23 provides a comparison of DAC HYP, DAC Penzuburg, and ZENAPAX DAC charge equivalents.

4. Detailed description of the invention

The disclosure provides, among other things, DAC compositions having particular properties, high concentration DAC formulations that are stable for storage at different temperatures that are particularly useful for certain modes of administration, vectors and host cells useful for producing DAC compositions, optimized culture broths and culture conditions useful for producing DAC compositions, methods of purifying DAC compositions and high concentration DAC formulations, and methods of using DAC compositions and high concentration formulations, for example, to inhibit proliferation of activated T-and/or B-cells and to treat and/or prevent diseases mediated by activated T-and/or B-cells, such as multiple sclerosis.

Daclizumab (DAC), as used herein, refers to a humanized IgG1 monoclonal antibody having the light (VL) chain sequence shown in FIG. 1 (positions 21-233 of SEQ ID NO: 2) and the heavy (VH) chain sequence shown in FIG. 2 (positions 20-465 of SEQ ID NO: 4). The CDR sequences of DACs are as follows:

V_LCDR#I：S A S S S I S Y M H (SEQ ID NO：6)

V_LCDR#2：T T S N L A S (SEQ ID NO：7)

V_LCDR#3：H Q R S T Y P L T (SEQ ID NO：8)

V_HCDR#1：S Y R M H (SEQ ID NO：9)

V_HCDR#2：Y I N P S T G Y T E Y N Q K F K D (SEQ ID NO：10)

V_HCDR#3：G G G V F D Y (SEQ ID NO：11)

certain daclizumab molecules, one specific form of DAC that was previously marketed under the trade name ZENAPAX by Hoffman La Roche, inc, have been reported in the literature as adjuvants to immunotherapy (including cyclosporine and corticosteroids) for the prevention of allograft rejection in kidney transplant patients. The DAC form sold under the tradename ZENAPAX is referred to herein as "ZENAPAXDAC.

Another form of DAC, although never commercially available, produced by an agency in Penzberg, germany has been used in certain clinical trials. This form of DAC is referred to herein as "DAC Penzberg".

As described herein, the disclosure herein relates in part to a new form of DAC having characteristics and properties that are different from, and in some cases superior to, those of ZENAPAX DAC and DAC Penzberg. Accordingly, the disclosure herein relates in part to novel DAC compositions. The novel DAC compositions are characterized by one or more of the following features as described more fully in the brief description section:

(1) characteristic pE/Q and/or Q/VHS N-terminal isoforms;

(2) a homogeneous N-linked oligosaccharide profile characterized by two major peaks and one minor peak;

(3) reduced ADCC cytotoxicity compared to zenpax DAC and DAC Penzberg; and

(4) have low levels of aggregate form (< 3%) when formulated up to a nominal concentration of 150 + -10-15%.

DAC compositions having one or more of these characteristics and/or properties are referred to herein as "DAC HYP" compositions. To enumerate the various aspects and features of the invention described herein, specific DAC HYP having all four of the above properties is described, as well as specific compositions and methods of producing and purifying the same. It will be understood, however, that the DAC HYP compositions need not have all four of the above-described characteristics to fall within the scope of the disclosure herein. In particular embodiments, DAC HYP has at least two of the above-described properties (1) to (4) (e.g., at least (1) and (2), (1) and (3), (1) and (4), (2) and (3), (2) and (4), or a combination of (3) and (4)) or at least three of the above-described properties (1) to (4) (e.g., at least (1), (2) and (3), (1), (2) and (4), (1), (3) and (4), (2), (3), and (4)). Such DAC HYP compositions may also have < 3% aggregates, < 2% aggregates and even lower levels, such as < 1% aggregates, when formulated at concentrations of 100mg ± 10-15% or even 150 ± 10-15%.

Furthermore, while certain aspects and embodiments of the invention described herein are shown and enumerated using DAC HYP, the skilled artisan will appreciate that it is not limited to DAC HYP and may be used in daclizumab compositions generally, nor to IgG2, IgG3, and IgG4 anti-CD 25 antibodies having CD 25-specific binding properties similar to DAC, and non-humanized anti-CD 25 antibodies suitable for administration to humans. These various anti-CD 25 antibodies are referred to herein as "DAC analogs". Such DAC analogs often include the 6 DAC CDRs mentioned above, but may include other CDR sequences.

The characteristics and properties of DAC HYP compositions can be confirmed using standard tests and methods. For example, N-terminal and C-terminal isotype profiles can be assessed using cation exchange chromatography and detection at 220 nm. In one specific method, 100 μ L of a test sample (1 mg/mL antibody in buffer A) was resolved at room temperature on a ProPac WCX-10 column (Dionex corporation) equipped with a ProPac WCX-10G guard column (Dionex corporation) using the following separation gradient (column equilibrated with buffer A):

buffer a 15mM sodium phosphate pH 5.9

Buffer B250 mM NaCl, 15mM sodium phosphate pH5

The N-linked glycosylation profile can be assessed by cleaving the N-linked oligosaccharide with the amidase PNGase F, derivatizing the oligosaccharide with a fluorescent label and analyzing the resulting mixture by normal phase HPLC for fluorescence detection. In one specific method, anthranilic acid-derivatized cleaved N-linked glycans were resolved on a 250X 4.6mm poly-amine bonded Asahipak NH2P-504E column (5 μm particle size, Phenomenex, Cat. No. CHO-2628) at 50 ℃ using the following elution gradient (using 100 μ L sample injection volume; the column was equilibrated with 85% buffer A/15% buffer B):

buffer a ═ 1% v/v tetrahydrofuran, 2% v/v acetic acid, in acetonitrile

Buffer B ═ 1% v/v tetrahydrofuran, 5% v/v acetic acid, 3% v/v triethylamine in water

Purity can be confirmed by reduced SDS-PAGE (preformed 14% Tris-glycine gradient minigel, Invitrogen product No. 601632) and colloidal blue staining and/or size exclusion chromatography and detection at 280 nm. Specifically, 15. mu.L of the test sample (20 mg/mL antibody in elution buffer) can be resolved at room temperature on a 7.8mM X30 cm TSK G3000SWXL column (Tosoh Biosciences, product number 601342) equipped with a 0.5 μm pre-column filter (Upchurch product number A-102X) using an isocratic elution buffer gradient (200mM KPO4, 150mM KCl, pH6.9) at a flow rate of 1 mL/min.

The DAC HYP compositions and other DAC formulations described herein, such as the stable, high concentration liquid DAC formulations described herein, are useful for treating a variety of diseases and conditions believed to be mediated at least in part by activated T-and/or B-cells, including, for example, allograft rejection and multiple sclerosis. Specific patient populations, formulations, methods and dosages and schedules for treating and/or preventing allograft rejection are described in U.S. patent No. 6,013,256, which is incorporated herein by reference. Specific patient populations, formulations, methods of administration, dosages, and schedules useful for treating multiple sclerosis patients are described in U.S. patent No.7,258,859, which is incorporated herein by reference. All of these formulations, methods of administration, dosages and schedules, as well as the specific patient populations and combination therapies disclosed, are equally applicable to the DAC HYP compositions and the high concentration DAC formulations described herein when available.

The DAC HYP compositions and formulations described herein are administered in an amount capable of providing a therapeutic effect. Therapeutic effects include, but are not limited to, treatment of the underlying disease. Therapeutic efficacy may also include amelioration or reduction of symptoms or side effects of a particular disease, as assessed using standard diagnostic and other tests. For multiple sclerosis, various methods of assessing treatment efficacy are described in U.S. patent No.7,258,859, which is incorporated herein by reference, including, for example, assessment of brain lesions and/or assessment of disability progression using magnetic resonance imaging. All of these different tests can be used to assess the efficacy of a treatment in the context of a patient suffering from multiple sclerosis.

Stable, high concentration DAC formulations, whether prepared from DAC HYP, DAC in general, or a DAC analog, are particularly useful for subcutaneous administration to treat chronic diseases, such as multiple sclerosis. The formulations may be conveniently administered as a rapid subcutaneous injection or diluted for intravenous administration. The formulation may be administered subcutaneously to a patient weekly to monthly (e.g., weekly, biweekly, twice monthly, four weeks or monthly) at a dose ranging from 75mg to 300mg (e.g., 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg) or 1mg/kg to 4 mg/kg. The composition may be provided in a pre-filled syringe for subcutaneous use. The diluted formulation may be administered intravenously at an appropriate dose at the same frequency as subcutaneous administration.

5. Exemplary embodiments

Various aspects and features of the inventions described herein are further described below in the form of exemplary embodiments. It will be appreciated that although the exemplary embodiments utilize specific cell culture media, cell culture conditions, column chromatography resins, and equilibration, washing, and elution buffers, routine modifications may be made. Furthermore, while a variety of cell culture methods are exemplified with a particular producer strain (clone 7A11-5H7-14-43), it is contemplated that other DAC or DAC analog producers, with or without routine optimization, can be successfully used. Moreover, the described features may be combined with other described features in specific embodiments (whether in the foregoing summary or in the following exemplary embodiments) without substantially affecting the desirable characteristics of the disclosed methods and compositions, and further, unless expressly excluded from the context, the various embodiments may be combined and used in various ways. Accordingly, it is to be understood that the exemplary embodiments provided below are intended to be illustrative, not limiting, and that they should not be considered limiting on the claims.

The manufacturing process listed below was used to produce 150mg/mL of DAC HYP drug substance. For the manufacture of 100mg/mL of DAC HYP drug substance, small process variations can be introduced:

the cell culture used to produce 100mg/mL DAC HYP (see section 5.3) did not contain a defoaming emulsion, while the 150mg/mL DAC HYP cell culture in a 10,000L bioreactor used a low concentration of Dow Corning Antifoam C to minimize foaming. When producing DAC HYP formulations with a final antibody concentration of 100mg/mL, CM-650M columns were sterilized with 0.5M NaOH, 0.5M sodium sulfate buffer (see section 5.4.5); when DAC HYP formulations were produced with a final concentration of 150mg/mL antibody, sodium sulfate was removed from the disinfection buffer. For the manufacture of 100mg/mL DAC HYP, at the end of the downstream process, one step ultrafiltration/diafiltration (UF/DF) was used just before adding polysorbate 80 and diluting the drug substance to the final volume (see section 5.4.7), while for the manufacture of 150mg/mL concentration of drug substance, two steps UF/DF were used.

The following example shows a comparative analysis between batches of DAC HYP at 100mg/mL and 150 mg/mL. In several studies, a 150mg/mL batch of DAC HYP was compared to a 100mg/mL batch of DAC HYP manufactured at 10,000L scale, referred to below as reference standard batch RS 0801.

DAC HYP expression constructs

Hybridomas producing anti-Tac, mouse IgG2a monoclonal antibodies were prepared by fusing the murine myeloma cell line NS-1 with splenocytes from mice immunized with a human T-cell line established from a T-cell leukemia patient (Uchiyama et al, 1981, j. immunol.126 (4): 1393-7). anti-Tac was chosen because of its reactivity with activated T-cells, but not resting T-cells or B-cells. anti-Tac was then shown to react with the alpha subunit of the human IL-2 receptor (Leonard et al, 1982, Nature 300 (5889): 267-9).

The mouse anti-Tac light and heavy chain variable region amino acid sequences were determined from the respective cDNAs (Queen et al, 1989, Proc. nat' l Acad. Sci. USA 86 (24): 10029-33). The binding affinity of mouse anti-Tac is retained in the humanized form as described in Queen et al. The Complementarity Determining Regions (CDRs) of the murine anti-Tac are first transferred to the acceptor framework of the human antibody Eu. With the aid of a three-dimensional model, mouse framework residues that determine the conformation of the CDRs and thus the binding affinity are identified and replaced in the acceptor framework with human counterparts. In addition, atypical amino acids in the acceptor framework were substituted with consensus residues at the corresponding positions in humans to eliminate potential immunogenicity.

The DAC HYPVL and VH genes were constructed as small exons by annealing and extension of overlapping oligonucleotides as described by Queen et al (1989). For expression of DAC HYP in the form of IgG1, the resulting VL and VH genes were cloned into a single expression vector to construct pHAT.IgG1.rg. dE (see FIG. 3 and FIG. 4A), as outlined by Cole et al (1997, J.Immunol.159 (7): 3613-21) and Kostelny et al (2001, int.J.cancer93 (4): 556-65). Plasmid pHAT.IgG1.rg.dE contains the genes for the heavy and kappa light chains of daclizumab IgG1, each under the control of the human Cytomegalovirus (CMV) promoter. This plasmid contains the E.coli guanine phosphoribosyltransferase (gpt) gene as a selectable marker. Genetic elements in pHAT. IgG1.rg. dE are described in Table 1 below.

The dESV40 promoter spans positions 6535 to 6735 of pHAT. IgG1.rg. dE (6536-6562 is 27 residues of the 72bp enhancer A; 6566-6629 is a three 21bp repeat; 6536-6735 is the reverse complement of 5127-1 and 1-133 in Genbank: J02400.1 (simian virus 40 whole genome)). The nucleotide sequences of the DAC HYP light and heavy chain genes in the expression vector were verified by DNA sequencing.

DAC HY0 stable cell line

The mouse myeloma NS0 cell line was obtained from the European Collection of cell cultures (ECAC)C directory #85110503, Salisbury, Wiltshire, UK). Vials of these NS0 cells were thawed into DMEM supplemented with 10% FBS. Cells were 7.5% CO at 37 ℃₂The lower was kept in a humidified incubator. Subsequently, the cells were cultured in SFM-3 basal medium supplemented with 1mg/mL BSA. SFM-3 is a 1: 1 mixture of DEME and Ham's F-12 supplemented with 10mg/mL insulin and 10. mu.g/mL transferrin. NS0 cells were adapted to SFM-3 without supplements by gradually reducing the amount of FBS present in the culture medium until it was eliminated, and then finally removing BSA in a single step over a period of about 3 months. The resulting host cell lines were passaged 15 to 20 times in SFM-3 and frozen stocks were prepared.

SFM-3-adapted cells were transfected by electroporation with pHat. igg1.rg. de (linearized with FspI enzyme (New England Biolabs, catalogue No. R0135L, batch 43)). Briefly, 30-40. mu.g pHAT. IgG1.rg. dE was added to 1X 10⁷Exponentially growing adapted NS0 cells and pulsed twice at 1.5kV, 25. mu.F using a GenePulser instrument (BioRad, Richmond, Calif.). Following electroporation, cells were plated at 20,000 cells/well in DMEM ± 10% FBS on five 96-well plates at a density that favors the formation of single colonies per well after mycophenolic acid ("MPA") selection. Such as Hartman et al 2007, Biotech.&

Bioeng96 (2): 294-306, transfectants that have stably integrated the vector are selected in the presence of mycophenolic acid. Starting from NS0 stable transfectants that produce high levels of DAC HYP, three consecutive rounds of subcloning were performed in PFBM-1 containing 2.5% or 5% fetal bovine serum (FBS; HyClone, Logan, UT) by limiting dilution cloning or Fluorescence Activated Cell Sorting (FACS). In each round of subcloning, one best producer was used for the next round of subcloning. After the third subcloning round, the final producer cell line was selected (7A11-5H 7-14-43). Then 1X 10 per tube by dissolving in 1mL 90% FBS/10% DMSO (Sigma, St. Louis, Mo.)⁷Individual cells were frozen to prepare seed stocks of the final producer cell line.

Recombinant production of DAC HYP

5.3.1. Cell culture and recovery

Cells were thawed from single cell stocks and expanded in progressively larger volumes within T-bottles, roller bottles, and bioreactors until production scale. After completion of the production culture, the cell culture fluid is clarified by centrifugation and depth filtration and transferred to a harvest preservation vessel. The duration of the production culture is about 10 days.

Cell culture and recovery can be performed in a variety of different cell culture facilities using standard equipment, as is known in the art. In another embodiment, cells are thawed from a single cell reservoir and expanded in progressively larger volumes within shake flasks and bioreactors until production scale is reached. After completion of the production culture, the cell culture fluid is clarified by centrifugation and depth filtration and transferred to a harvest preservation vessel. The duration of the production culture is about 10 days.

5.3.1.1. Preparation of inoculum

The production batch is initiated by thawing the individual cell stock tubes. The cells were transferred to a T-flask containing the chemically defined Medium Protein Free basic Medium-2 (PFBM-2). A custom powder of PFBM-2 can be prepared by ordering from Invitrogen via a Hybridoma-SFM medium powder prepared requiring NaCl, phenol red, transferrin and insulin free, containing the sodium salt of iron (III) EDTA in an amount that yields a concentration of 5mg/L when reconstituted, while the amounts of the remaining components are adjusted to the same concentration as the Hybridoma-SFM after reconstitution when reconstituted. The prepared PFBM-2 medium comprises the following components: 8g/L custom powder, 2.45g/L sodium bicarbonate, 3.1Sg/L NaCl and 16.5g/L D-glucose monohydrate (15g/L glucose).

Cells were expanded by serial passage into roller bottles or spinner bottles every two days thereafter. The T-bottle, roller bottle and roller bottle were placed in an incubator at a temperature set point of 37 ℃ at 7.5% CO for the T-bottle and roller bottle₂And 5% CO for roller bottles₂Operates in the environment of (1).

Flasks were supplemented with 5% CO, depending on the volume of the cell culture, either by addition to the headspace or by injection into the culture₂The impeller speed is controlled to a constant number of Revolutions Per Minute (RPM). The target inoculum density for all inoculum expansion passages was about 2.5X 10⁵Viable cells/mL.

In addition, the inoculum can be prepared according to methods known in the art using a variety of standard culture vessels, volumes, and conditions. For example, a production batch is initiated by thawing individual cell stock tubes. The cells can be transferred to shake flasks containing the chemically defined Medium Protein Free basic Medium-2 (PFBM-2). A custom powder of PFBM-2 can be prepared by ordering from Invitrogen via a Hybridoma-SFM medium powder prepared requiring NaCl, phenol red, transferrin and insulin free, containing the sodium salt of iron (III) EDTA in an amount that yields a concentration of 5mg/L when reconstituted, while the amounts of the remaining components are adjusted to the same concentration as the Hybridoma-SFM after reconstitution when reconstituted. The prepared PFBM-2 medium comprises the following components: 8g/L custom powder, 2.45g/L sodium bicarbonate, 3.15g/L NaCl and 16.5g/L D-glucose monohydrate (15g/L glucose). Alternatively, copper sulfate heptahydrate may be added at a concentration of, for example, 0.04mg/L at the bioreactor stage.

Cells were expanded by serial passage into shake flasks every two days thereafter. The shake flask was placed in an incubator at a temperature set point of 37 ℃ at 7.5% CO₂Operates in the environment of (1).

Shake flasks on a shaker in an incubator at a constant number of Rotations Per Minute (RPM). The target inoculum density for all inoculum expansion passages was about 2.2-2.5X 10⁵Viable cells/mL.

Approximately 14 days after thawing of the cell stock, when a sufficient number of viable cells have been produced, the first of several, typically 3 or 4, stainless steel stirred vessel seed bioreactors is inoculated. Before use, the seed bioreactor was cleaned in situ, steamed in situ, and an appropriate volume of PFBM-2 medium was added. In situ steam of a bioreactorBefore treatment, the pH and dissolved oxygen probes were calibrated. Seeding the first bioreactor with a sufficient number of cells to achieve 2.0-2.5X 10⁵Target of initial cell density of viable cells/mL. Growth in each reactor was about 2 days and reached 2.0-2.5X 10⁵After an initial cell density target of viable cells/mL, continuous transfer to larger volumes is performed (typically 100L to 300L followed by 1,000L bioreactors, or 60L to 235L, 950L and 3750L bioreactors). By automatically controlling the addition of CO₂Gas or 1M sodium carbonate (Na)₂CO₃) Thereby maintaining the culture pH. The target operating conditions for the seed and production bioreactors included a temperature set point of 37 ℃, pH 7.0 and 30% dissolved oxygen (as a percentage of air saturation). 100L, 300L and 1000L bioreactors were agitated at 100rp, 80rpm and 70rpm, respectively. In some cases, the target operating conditions for the seed and production bioreactors include a temperature set point of 37 ℃ to inject CO₂And pH of 7.0 with added base control and dissolved oxygen at 30% (as a percentage of air saturation). The larger volume bioreactor may be agitated at a speed of 100rpm, 80rpm, 70rpm or 40 rpm.

5.3.2. Bioreactor for producing cell cultures

After approximately 2 days of culture in a1,000L seed bioreactor, the inoculum was transferred to a stainless steel stirred vessel production bioreactor. The production bioreactor has a working volume of about 10,000L. Before use, the bioreactor was cleaned in situ, steamed in situ, and approximately 4,000L of PFBM-2 medium was added. The pH and dissolved oxygen probes were calibrated before the bioreactor was subjected to in situ steam treatment.

In another embodiment, the inoculum is grown in a 3750L bioreactor prior to transfer to a stainless steel stirred vessel production bioreactor having a working volume of about 15,000L, the production bioreactor is cleaned in situ, steamed in situ, and added with about 4,000 and 7,000L of PFBM-2 medium prior to use.

The production bioreactor has a target inoculation density of 2.0-2.5 × 10⁵Living cellIn the range of/mL. Chemically defined Protein Free Feed Medium concentrate (PFFM-3) (a chemically defined concentrated Feed Medium prepared by reconstituting subfractions 1 and 2 of PFFM3, L-glutamine, D-glucose, disodium hydrogen phosphate heptahydrate, L-tyrosine, folic acid, hydrochloric acid and sodium hydroxide) was added during the cultivation.

PFFM3 contained the components shown in table 4:

PFFM3 subcomponent 1 contained the components shown in table 5 below:

pFFM3 subcomponent 2 contained the components shown in table 6 below:

the timing and amount of PFFM-3 addition to the culture occurred as shown in Table 7 below:

by automatically controlling the addition of CO₂Gas and 1M sodium carbonate (Na)₂CO₃) Thereby maintaining the culture pH at about pH 7.0, preferably between pH 7.0 and pH 7.1. The dissolved oxygen is allowed to drop to about 30% of air saturation. The oxygen/air mixture is injected into the culture to achieve a constant total gas flow rate and dissolved oxygen is controlled by adjusting the air to oxygen ratio as needed and by increasing the agitation speed after the maximum oxygen to air ratio is reached. In another embodiment, the agitation is adjusted to maintain a constant power/volume ratio. Simethicone-based antifoam emulsion was added to the bioreactor in the required amount based on the foam level. Periodic sampling to test cell density, cell viability, product concentration, glucose and lactate concentration, dissolved O₂Dissolved CO₂pH and osmolarity. Approximately 10 days after inoculation, the bioreactor culture was harvested. Before harvesting, the contents of the bioreactor were sampled as untreated bulk material.

5.3.3. Harvesting and cell removal

Just prior to harvest, the production bioreactor is first cooled to < 15 ℃, then adjusted to pH 5.0 ± 0.1 with 0.5M or 1M or 2M citric acid and held for a period of about 30-90 or 45-60 minutes to flocculate the cells and cell debris prior to transfer to the harvest vessel. The pH adjusted harvest was then clarified by continuous centrifugation, which was run at preset parameters of bowl speed (bowl speed) and flow rate as defined in the batch record file.

The centrate was filtered through a depth filter followed by a 0.22 μm membrane filter and collected in a pre-sterilized container. The cell-free harvest was adjusted to about pH6.4 using 1-2M Tris solution and stored at 2-8 ℃ for further processing. In some cases, this pH adjustment occurs within 12 hours of adjusting the initial bioreactor pH to pH 5.0.

Purification of DAC HYP

5.4.1. Overview

The DAC HYP purification and formulation process was designed to increase efficiency relative to the ZENAPAX production process and ensure continuous removal of product and process related impurities. The following section describes the purification process. Purification is based on three chromatographic techniques (protein a affinity chromatography, Q Sepharose anion exchange chromatography, and CM-650(M) cation exchange chromatography) and low pH virus inactivation, virus filtration, ultrafiltration/diafiltration, and formulation steps. All steps take place in a closed apparatus. An overview of the DAC HYP purification process is presented in fig. 5 and discussed below.

5.4.2. Protein A chromatography

The protein a affinity chromatography step is the first purification step in the downstream sequence of operations. This step occurs in one or more cycles, depending on the size of the column, typically two or three cycles for the columns described in table 8A (i.e., the cell-free harvest is split into two aliquots, each aliquot is then separately loaded onto the protein a column and eluted). Recombinant protein a affinity chromatography resin binds specifically to IgG and separates the antibody from other components of the cell culture harvest.

After equilibration of the protein a column with equilibration buffer, the neutralized cell-free harvest is passed through the column to bind the antibodies to the column resin. The equilibration buffer was 20mM sodium citrate, 150mM sodium chloride, pH 7.0. The column is loaded to a capacity of no more than 35 grams of antibody (protein) per liter of packing resin. After loading, the column was washed with equilibration buffer to remove unbound and loosely bound impurities from the resin and pre-eluted with citrate buffer to adjust the concentration of citric acid and sodium chloride on the column. The citrate buffer was 10mM sodium citrate, pH 7.0. The bound antibody was then eluted from the column with a pH step change using 10mM sodium citrate elution buffer pH 3.5. The summary of protein a chromatography conditions is listed in table 8A:

the absorbance of the effluent was monitored at 280nm as the product eluted from the column and used to direct the collection of product fractions (see figure 6).

The use of a disinfecting buffer containing sodium hydroxide and benzyl alcohol advantageously kills a wide range of microorganisms while minimizing the impact on the quality of the protein a resin. To illustrate this, a variety of antiseptic solutions are mixed with a variety of microorganisms and incubated for a period of time. At different incubation intervals, a portion of the mixed disinfection buffer was neutralized and the microbial titer was determined and compared to a control. Microbial activity is expressed as a reduction in the log of the microorganism over time. Table 8B shows the reduction in microbial titer as a function of contact time with disinfectant buffer (20mM sodium hydride, 200mM sodium citrate, and 1% benzyl alcohol).

Table 8C shows microbial reduction with different disinfection buffers:

A＝50mM NaOH，0.5M NaCl(60min)

b20 mM NaOH, 200mM sodium citrate, 1% benzyl alcohol (60min)

Ca 200mM sodium citrate, 0.5% benzyl alcohol (48hr)

D2% benzyl alcohol (24hr)

The previous data show that the disinfectant solution containing benzyl alcohol and sodium hydroxide is very effective in killing a large number of microorganisms, including gram negative and gram positive bacteria, spore forming bacteria, yeast and fungi. Greater than 5 log counts (log) were observed on E.coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans after 30 minutes of routine sterilization₁₀) Is reduced. Although killing of fungi (Aspergillus niger) takes a long time, there are also few fungal infections in cell culture fluids. The most common microorganisms isolated from biotechnological facilities are bacillus, pseudomonas and staphylococcus. After 30 minutes of contact time, these are effectively killed by the disinfecting solution. In contrast, sodium hydroxide or benzyl alcohol alone is not effective in killing all microorganisms. Moreover, the sodium hydroxide disinfectant solution does not kill spore-forming bacillus.

5.4.3. Maintaining low pH to inactivate viruses

This step is designed to inactivate endogenous virus-like particles and viruses that are sensitive to low pH. The protein a eluate from each protein a cycle is eluted into a collection vessel, to which 0.5M HCl is added until ph3.5 ± 0.1 is reached. The product was transferred to a holding vessel where the pH was verified by another pH meter. The low pH holding step is closely controlled to pH3.5 + -0.1 or + -0.2 (e.g., pH 3.35-3.64) for 30-120 minutes or 30-240 minutes. After 30-120 minutes of holding, the virus-inactivated eluate is neutralized to pH 7.8. + -. 0.1 or. + -. 0.3 (e.g.pH 8.05-8.34) using 1M Tris base and subsequently transferred through a 0.22 μ M filter to a product pool vessel. A summary of the low pH virus inactivation conditions is listed in table 9:

alternatively, the pH target after neutralization may be 25 ℃, 8.2.

Q sepharose anion exchange flow through chromatography

The Q Sepharose anion exchange chromatography step serves to reduce product and process related impurities (e.g., nucleic acids, host cell proteins, product aggregates, leached protein a ligands, etc.) and provides additional virus clearance for the purification process. The loading conductivity and pH are selected in such a way that the antibody flows through the column and negatively charged impurities, such as host cell proteins and cellular DNA, bind to the positively charged resin.

The anion exchange column was equilibrated with 20mM tris, 20mM sodium chloride, pH 7.8 equilibration buffer. The pH-adjusted product from the low pH holding step is loaded onto the column to a capacity of no more than 60 grams of antibody (protein), or no more than 30-60 grams of antibody (protein), per liter of packed resin. After loading was complete, unbound antibody and impurities were removed from the column with equilibration buffer.

The collection of the product was guided by monitoring the absorbance of the effluent at a wavelength of 280nm (see FIG. 7).

The flow rate of sterilization is 100cm/hr, and the retention time is 60 min.

The summary of O sepharose chromatography conditions is listed in Table 10:

for some applications, the storage buffer volume setpoint is 3, while the column storage temperature is set to 5-25 ℃.

CM-650(M) cation exchange chromatography

This chromatography step is the final step utilized in the process to reduce trace levels of process and product related impurities. In addition to reducing aggregates and cleaved fragments of the antibody, this step also reduces process-related impurities, such as host cell nucleic acids and proteins and leached protein a.

The column was equilibrated with 20mM sodium citrate, pH 4.5 equilibration buffer. The anion exchange eluent pool is adjusted to pH 4.5 + -0.1 or + -0.2 (e.g., 4.35-4.64) with 0.5M citric acid and loaded onto the column to a target loading capacity of no more than 25 or 30 grams of antibody (protein) per liter of packing resin. After the binding step, the column is washed with equilibration buffer to remove any unbound or loosely bound impurities from the resin. The bound antibody was then eluted from the column in a one-step elution mode using 20mM sodium citrate, 75mM sodium sulfate, pH 4.5 elution buffer. Collection of peaks was guided by monitoring the absorbance of the effluent at a wavelength of 280nm (see FIG. 8)

A summary of CM-650(M) chromatography conditions is listed in Table 11:

optionally, no dilution buffer is used.

5.4.6. Nanofiltration

The purpose of the nanofiltration step is to provide additional virus clearance capacity for the purification process. In this step viruses and virus-like particles are removed by size exclusion mechanisms. The pores of the filter are designed to allow the antibodies to pass through the filter while retaining the virus-like particles and virus on the upper side of the filter.

The cation exchange eluate, which had been filtered through a 0.22 μm or 0.1 μm filter, was passed through a nanofilter retaining the parvovirus, followed by rinsing the filter with DAC HYP formulation buffer (40mM succinate, 100mM sodium chloride, pH 6.0) without polysorbate 80. A buffer flush step was applied to recover residual antibody in the line and filter mesh.

A summary of the nanofiltration parameters is listed in table 12:

or > 50L/m²

Or > 50L/m²

Or about 50L/m²。

5.4.7. Ultrafiltration/diafiltration (UF/DF)

This step was designed to concentrate the product and exchange the buffer in the product with the dachpy formulation buffer without polysorbate 80. This step was run in tangential flow mode using a 30kDa nominal molecular weight cut-off membrane. Two ultrafiltration/diafiltration stages were used to produce a 150mg/mL formulation at the expected product volume based on final concentration and relative hold-up volume for each UF system.

The first stage treatment was performed using a large UF system (see fig. 9). The nanofiltration filtrate was first concentrated to about 30mg/mL and then diafiltered into exchange buffer (formulation buffer without polysorbate 80). The diafiltered antibody solution was further concentrated to about 100mg/mL and subsequently recovered from the UF/DF system at a concentration of about 55mg/mL and transferred through a 0.22 μm filter. The diafiltered antibody solution may also be recovered at a concentration of about 20-60 mg/mL. A summary of the first stage parameters is listed in table 13:

0.1M NaOH

Or 2 times for 40min

Or 20-70.

The second stage treatment was performed with a smaller UF system, but using the same 30kDa cut-off membrane. The DACHYP solution (typically 55mg/mL) was concentrated to about 180mg/mL, recovered from the UF system, and subsequently transferred through a 0.22 μm filter. The UF system was flushed with formulation buffer without polysorbate 80 and transferred through a 0.22 μm filter to obtain about 170mg/mL or about 150-170mg/mL of purified drug substance.

A summary of the second stage parameters is listed in table 14:

or 2 times 40 min.

5.5. Formulating DAC HYP

The final process step is to dilute the purified drug substance to a final target concentration of 150 or 100mg/L ± 10%, i.e. 150 ± 15mg/mL (in the case of a 150mg/mL formulation) or 100 ± 10mg/mL (in the case of a 100mg/mL formulation) in a buffer containing polysorbate 80 at an appropriate concentration. The formulation is carried out in stages.

For example, polysorbate 80-free formulation buffer (40mM sodium succinate, 100mM sodium chloride, pH 6.0) is first added to the purified drug substance to reach the target volume of 90% of the formulated drug substance. A calculated amount of polysorbate 80 dilution buffer (40mM succinate, 100mM sodium chloride, 1% polysorbate 80, pH 6.0) was then added to reach the target polysorbate 80 concentration of 0.03% (w/v) in the final formulation. Finally, the product volume was adjusted with formulation buffer without polysorbate 80 (made from succinate and succinic acid for the 150mg/mL formulation and succinate and HCl for the 100mg/mL formulation) to reach a final antibody concentration of 150 ± 15mg/mL (preferably 150 ± 8 mg/mL). 100mg/mL drug product is similarly formulated to a final concentration of 100 + -10mg/mL (preferably 100 + -5 mg/mL).

The formulated drug substance was filtered through a 0.22 μm filter into a BioProcess Container (TM) placed in a semi-rigid cylindrical supportA bag (or equivalent). The support closes the BPC with a lid and provides a protective barrier between the flexible bag and the external environment. The formulated drug substance is stored at 2-8 ℃ in an access-controlled cooler for drug product fill/finish operations.

A summary of formulation conditions is listed in table 15:

1mL of the drug product was filled into a bottle or syringe. A summary of the components in the finished 150mg/mL and 100mg/mL products had the components shown in Table 16 (all amounts are nominal):

characterization of DAC HYP drug substance

DAC HYP is glycosylated at amino acid 296 of both heavy chain subunits, with the primary oligosaccharide form existing as a core fucosylated dual-antenna structure lacking a terminal galactose.

The N-terminus of the DAC HYP heavy chain exists in three major forms: 1) n-terminal glutamine (predicted from the DNA sequence), 2) N-terminal pyroglutamic acid (resulting from cyclization of the N-terminal glutamine) and 3) N-terminal valine, histidine and serine residues as well as the predicted N-terminal glutamine residue (resulting from incomplete cleavage of the signal peptide).

The C-terminus of the DAC HYP heavy chain is present with and without a C-terminal lysine residue. Its major form lacks the C-terminal lysine residue, producing C-terminal glycine.

DAC HYP has a molecular weight of 144kDa, calculated based on the primary amino acid composition defined by the nucleotide sequence. The corresponding molecular weight of the reduced heavy chain was 48.9kDa and the reduced light chain was 23.2 kDa. These molecular weights do not take into account carbohydrate content or post-translational modifications.

DAC HYP binds with high specificity to CD25, with CD25 being expressed on activated, but not resting, T and B lymphocytes. Binding of DAC HYP to CD25 on these activated cells blocked the binding of IL-2 to CD25 and the subsequent formation of a high affinity IL-2 receptor complex. Thus, IL-2-induced proliferation of activated cells is blocked. It is believed that the observed and potential therapeutic efficacy of DAC HYP depends in large part on its inhibitory effect on the proliferation of activated autoreactive T cells. However, DAC HYP may also exert therapeutic effects by its blocking effect on other CD 25-bearing cell types (e.g., eosinophils).

To confirm that high concentration 150mg/mL DAC HYP formulations are suitable for clinical studies, a comprehensive physicochemical and biological evaluation was performed to characterize two batches of DAC HYP150mg/mL drug substance referred to herein as batch 1 and batch 2 (or batch 150-1 or batch 150-2, respectively) and compare with a reference standard batch RS0801 from a DAC HYP 100mg/mL batch manufactured at 10,000L scale.

The results indicate that the 150mg/mL batch of DAC HYP drug product is highly pure, similar to the 100mg/mL batch, and suitable for use in clinical studies. A summary of these properties is shown in table 17:

5.6.1. color, appearance and clarity

The appearance of DAC HYP drug substances was evaluated by visually examining the color and clarity of the solution under direct light against a black and white background without magnification. The presence of visible particles in the solution was also evaluated. Typical appearances of batches of DAC HYP drug products are described in table 17.

pH determination

The pH of DAC HYP was determined according to United states Pharmacopeia protocol <791 >. The pH ranges for the various batches of DAC HYP drug product are summarized in table 17.

Determination of product concentration by UV Spectroscopy

The concentration of DAC HYP was determined by UV spectroscopy. DAC HYP samples were diluted by weight with buffer. The UV absorbance of each diluted sample solution was measured for buffer blank at 278 nm. And calculating the protein concentration of the sample by using the DAC HYP absorption coefficient. Protein concentrations of the DAC HYP drug products of the various batches are summarized in table 17.

N-terminal sequencing

DAC HYP150mg/mL batches were evaluated by N-terminal sequencing. Samples were analyzed using an automated Edman degradation sequencer.

The expected amino acid sequence DIQMTQSPSTLSASV (SEQ ID NO: 13) for the first 15 residues of the light chain was confirmed in all samples.

Most of the heavy chains in DAC HYP are blocked by pyroglutamic acid residues (pE) and will not generate the N-terminal heavy chain sequence. The next most prevalent N-terminal heavy chain sequence in DAC HYP starts with a valine, histidine, serine (VHS) sequence, which results from the heavy chain signal peptide without processing of the three terminal residues. Fourteen of the first fifteen N-terminal residues of the VHS heavy chain sequence (VHSQVQLVQSGAEVK (SEQ ID NO: 14)) were confirmed in all samples. The fourth residue glutamine could not be confirmed due to the large amount of glutamine detected from LC in previous sequencing cycles. Evidence that the heavy chain has an N-terminal glutamine is also present in all samples. This sequence is the result of the native N-terminal heavy chain glutamine residue not being cyclized to the pyroglutamic acid form. The results of N-terminal sequencing of the 150mg/mL batches were consistent with the sequences predicted from the heavy and light chain coding sequences. Similar results were obtained for the 100mg/mL batch.

5.6.5. Mass analysis of heavy and light chains

The molecular weights of the DAC HYP150mg/mL batches and the reference standard RS0801 heavy and light chains were evaluated by liquid chromatography mass spectrometry (LC-MS) analysis. All batches were deglycosylated with amidase PNGasesF, reduced with dithiothreitol, alkylated with iodoacetic acid, and isolated by reverse phase chromatography. The theoretical masses of the heavy and light chains were calculated from the protein sequences. As shown in Table 18 below, the molecular weights of the observed samples were within 1Da of the calculated molecular weight:

as described in the preceding subsection, the two most common heavy chain forms of DAC HYP are known to contain an N-terminal pyroglutamic acid (pE) residue or valine, histidine, serine (VHS) sequence and no C-terminal lysine. The mass obtained for the two major heavy chain variants and light chains in the 150mg/mL batch was similar to the reference standard RS0801 and was consistent with the mass predicted from the protein sequence.

The mass results for the heavy and light chains, together with the peptide mapping results presented in the following section, confirm the presence of the expected primary structure of the light and heavy chains in the DAC HYP150mg/mL batch.

5.6.6. Peptide mapping

The reverse phase HPLC peptide profile was used to evaluate DAC HYP150mg/mL batches and reference standard RS0801 (DAC HYP produced from 100mg/mL drug substance manufactured at 10,000L scale). All batches were reduced with dithiothreitol, alkylated with iodoacetic acid, and enzymatically digested with trypsin. The resulting peptides were separated by reverse phase chromatography and detected by ultraviolet absorbance at 215nm to generate a peptide map.

To determine the primary amino acid sequence, the peptide profile of the 150mg/mL batch was compared to a reference standard RS 0801. In the peptide profile of reference standard RS0801, peptides corresponding to ninety-eight percent of the expected heavy and light chain residues have been previously identified by mass spectrometry. Residues not taken into account in the peptide map are individual amino acids or residues in the very polar dipeptide, which are not expected to be retained by the reverse phase column. Masses consistent with pyroglutamic acid, glutamine and VHS sequences at the N-terminus of the heavy chain N-terminal peptide have been identified in reference standards. DAC HYP contains a consistent N-linked glycosylation site at Asn296 in the heavy chain Fc portion, and for peptides containing Asn296 residues, masses consistent with the core structure of linked complex dual-antenna oligosaccharides have been identified.

Peptide profiles comparing DAC HYP150mg/mL batches with reference standard RS0801 are shown in fig. 10(0 to 60min), fig. 11(55 to 115 min) and fig. 12(110 to 170 min). The peptide profile of the DAC HYP150mg/mL batch was similar to the reference standard and confirmed the presence of the expected primary structure in the 150mg/mL batch.

5.6.7. Circular dichroism spectrum

DAC HYP150mg/mL batches and reference standard RS0801 were analyzed by far ultraviolet circular dichroism (far-UV CD) to assess secondary structure. Before analysis, the samples were diluted with water to a final protein concentration of 0.2 mg/mL. Spectra were acquired at 195 to 260nm using a 0.1cm cell and the signals obtained were converted to molar ellipticity after subtraction of buffer.

The coverage far-UV CD spectra of DAC HYP150mg/mL batches 1 and 2 and reference standard RS0801 are shown in figure 13. The spectra of all batches showed a positive band at about 202nm and a negative band at about 217 nm. The negative band at 217nm is characteristic of the beta-sheet conformation, the main conformation of the IgG molecule. The far-UV CD spectra of the batches were similar, supporting the same secondary structure between the DAC HYP150mg/mL batch and the reference standard RS 0801.

5.6.8. Ultraviolet spectrum

The DAC HYP150mg/mL batches and reference standard RS0801 were analyzed by Ultraviolet (UV) spectroscopy to assess tertiary structure. Before analysis, samples were diluted to a final protein concentration of 0.5mg/mL with formulation buffer (40mM succinate, 100mM sodium chloride, 0.03% polysorbate 80, pH 6.0). Spectra at 250nm to 350nm were taken using a 1cm pathlength quartz cuvette and normalized to absorbance at 280nm of 1.0.

The overlaid zero-order and second-order derivative UV spectra (calculated from the smoothed zero-order data) are shown in fig. 14A and 14B, respectively. The zero and second derivative spectra of all batches evaluated were superposable, which supports the same tertiary structure between DAC HYP150mg/mL batches 1 and 2 and the reference standard RS 0801.

5.6.9. Size exclusion chromatography

By using with aqueous flowThe porous silica columns of the phases were subjected to Size Exclusion Chromatography (SEC) and the UV absorbance was measured at 280 nm. Specifically, elution buffer (200mM KPO) was used₄150mM KCl, pH6.9) at a flow rate of 1mL/min at a TSK G3000SW of 7.8mm × 30cm equipped with a 0.5 μm pre-column filter (Upchurch product No. A-102X)_xL15 μ L of test sample (20 mg/mL antibody in elution buffer) was analyzed on a column (Tosoh Biosciences, product No. 601342) at room temperature.

As shown in fig. 15A (full scale) and fig. 15B (expanded scale), the chromatogram of the DAC HYP150mg/mL batch was similar to the reference standard RS 0801. All batches showed the same major and minor aggregate peaks corresponding to the daclizumab monomers. As shown in table 19 below, the aggregate results for the 150mg/mL batch of DAC HYP were similar to the 100mg/mL batch of DAC HYP:

the 150mg/mL batch and reference standard RS0801 were analyzed by SEC (SEC-MALS) with multi-angle light scattering detection to determine aggregate peak molecular weight. The aggregate peak molecular weight obtained was about 300kDa for all batches, consistent with antibody dimer.

Aggregate formation in DAC HYP was monitored over a period of 18 months. The level of aggregates in the formulation increased when stored at 5 ℃ for 18 months, but the percentage of aggregates was stable and did not exceed about 1.5% (see fig. 16). The new data for the new batch showed that about 1.8-1.9% of aggregates could appear at 6 weeks when stored at 5 ℃, but for all samples tested, only less than 3% of aggregates were formed within 5 years when stored at 5 ℃.

5.6.10 sedimentation rate analysis type ultracentrifugation

Monomer and aggregates were characterized in DAC HYP150mg/mL batches and 100mg/mL batches using sedimentation rate analytical ultracentrifugation (SV-AUC). The sedimentation coefficient values and relative abundances of the monomers and each aggregate are shown in tables 20 and 21 below.

Monomer was the major component observed in each batch. The sedimentation coefficients of the monomer peaks were highly consistent from batch to batch, indicating that the conformation of the monomers was similar between the 150mg/mL and 100mg/mL batches. The monomer content of the 150mg/mL batch was similar to the 100mg/mL batch.

The major aggregate species in each batch had a sedimentation coefficient consistent with that of the antibody dimers. This is consistent with the SEC-MALS results, indicating that the SEC aggregate peak is composed primarily of antibody dimers (see previous section). Low levels of two larger aggregate species were also observed in each batch by AUC, with sedimentation coefficients consistent with trimers and tetramers. The dimer, trimer and tetramer content in the 150mg/mL batch was similar to the 100mg/mL batch.

5.6.11 quantitative reduction of SDS-PAGE

Purity was determined by SDS-PAGE using 4-20% (usually 14%) tris-glycine gel and colloidal blue staining. The samples were analyzed under reducing conditions with a sample loading of 10. mu.g. Purity was calculated by dividing the sum of the heavy and light chain band areas as determined by densitometry by the total band area.

As shown in Table 22 below, the 150mg/mL batch was of high purity and similar to the 100mg/mL batch:

5.6.12 qualitative SDS-PAGE

The purity of DAC HYP was assessed by reducing and non-reducing gel electrophoresis. Analysis was performed on preformed 14% or 8-16% Tris-glycine gels. Two aliquots of 150mg/mL DAC HYP formulations were compared to the reference batch as previously described. Reduced and non-reduced gels for analysis of DAC HYP purity are shown in fig. 17. The band pattern of the 150mg/mL ratio was similar to the reference standard RS0801, and no new band was detected in the 150mg/mL ratio.

5.6.13 cation exchange chromatography

The charge equivalence profiles of DAC HYP150mg/mL batches and 100mg/mL batches were analyzed using cation exchange Chromatography (CEX). CEX was performed using a non-porous, carboxylate-functionalized weak cation exchange column and detected at 220 nm. mu.L of the test sample (1 mg/mL of antibody dissolved in buffer A) was resolved at room temperature on a ProPacWCX-10 column (Dionex) equipped with a ProPac WCX-10G guard column (DioneX corporation) using the following separation gradient (column equilibrated with buffer A):

buffer a 15mM sodium phosphate pH 5.9

Buffer B250 mM NaCl, 15mM sodium phosphate pH5

As shown in FIG. 18, the CEX chromatogram of the 150mg/mL batch was consistent with the reference standard RS0801, and no new charge isoforms were detected in the 150mg/mL batch. The five major isoforms present in the CEX chromatogram are due to heterogeneity at the N-terminus of the heavy chain, including: 1) two pyroglutamic acid residues (pE/pE); 2) one pyroglutamic acid residue and one glutamine residue (pE/Q); 3) one pyroglutamic acid residue and one VHS sequence (truncated VHS signal peptide located before the N-terminal glutamine residue of the mature heavy chain) (pE/VHS); 4) a glutamine residue and a VHS sequence (Q/VHS); 5) two VHS sequences (VHS/VHS). The C-terminal lysine (K) isoform is also resolved and identified in FIG. 18. Each of the N-terminal isoforms described above may exist as different C-terminal isoforms (0, 1, or 2K). Due to the complexity of the mixture, only the C-terminal isoforms of the major pE/pE and pE/VHS isoforms are clearly resolved and determinable using the described methods.

Quantitative results for the N-and C-terminal isoforms for the 150mg/mL batch and the 100mg/mL batch, respectively, are provided in tables 23 and 24, where the percentages reported are based on the area under the curve (AUC) for a particular peak as compared to the total AUC for all peaks:

5.6.14. oligosaccharide map

The oligosaccharide distribution of DAC HYP150mg/mL batches and 100mg/mL batches was analyzed by oligosaccharide profiling. The N-linked oligosaccharide was cleaved off from heavy chain Asn296 using the amidase PNGaseF. The oligosaccharides are then derivatized with a fluorescent label (in this case anthranilic acid) and separated from the antibody by a nylon membrane. Derivatized, cleaved N-linked glycans 250X 4.6mm poly-amine bonded Asahipak Amino NH with fluorescence detection at 50 ℃₂P-504E column (5 μm particle size, Phenomenex, Cat number CHO-2628) utilizationThe following elution gradient was used for resolution (100. mu.L sample injection volume; column equilibrated with 85% buffer A/15% buffer B):

buffer a ═ 1% v/v tetrahydrofuran, 2% v/v acetic acid in acetonitrile

Buffer B ═ 1% v/v tetrahydrofuran, 5% v/v acetic acid, 3% v/v triethylamine in water

A chromatogram comparing the 150mg/mL batch with the reference standard RS0801 is shown in FIG. 19. Oligosaccharide peaks at least 1.0% of the total peak area are labeled and reported in table 25:

all batches consisted primarily of G0 and G0-GlcNAc (G0 lacking GlcNAc on one arm of the dual antenna structure), which represents the DAC HYP process. Sialylated oligosaccharides eluted at about 68 minutes, which was less than 1.0% in all tested batches. The uncharacterized oligosaccharides, designated peak 3, were present in similar abundance in all samples tested.

5.6.15. Oxidation by oxygen

The potential methionine oxidation of the dachpy batches was evaluated by monitoring the presence of oxidized and non-oxidized tryptic peptides in the peptide profile. The non-oxidized and oxidized peak areas of each methionine-containing peptide were determined by mass spectrometric ion chromatography. For each methionine residue, the percentage of oxidized methionine was calculated by dividing the mass spectrum peak area of the oxidized peptide by the sum of the peak areas of the oxidized and non-oxidized peptides.

As shown in Table 26 below, the methionine oxidation results were similar for the 150mg/mL batch and the five 100mg/mL batches:

the heavy chains Met251 and Met427 were least stable and showed the greatest degree of oxidation. In batches tested simultaneously to evaluate comparability, the oxidation levels of Met251 and Met427 did not exceed 4.8% and 1.8%, respectively.

5.6.16. Binding potency (binding of CD25)

The binding of DAC HYP150mg/mL and 100mg/mL batches to IL-2 receptor alpha subunit (CD25) was evaluated by ELISA and potency was determined as part of the release assay. Soluble CD25 was immobilized onto microtiter plates and incubated with varying amounts of DAC HYP. And detecting the bound DAC HYP by using a goat anti-human IgG antibody coupled with horseradish peroxidase and a 3, 3 ', 5, 5' -tetramethylbenzidine substrate in sequence. Using 4-parameter fitting, log of the obtained absorbance value to DAC HYP concentration₁₀Plotting, using parallel line analysis yields the percentage of relative potency values.

The drug substance results are summarized in the following table:

the binding potency results for the 150mg/mL batch were similar to the 100mg/mL batch.

5.6.17. Surface plasmon resonance (combination of CD25)

Surface plasmon resonance analysis was performed to determine the affinity constant (K) for the binding interaction of DAC HYP with the IL-2 receptor alpha subunit (CD25)_D)。

Goat anti-human IgG Fc antibodies were immobilized on the chip surface to capture DAC HYP samples, after which different concentrations of soluble CD25 were injected in duplicate onto the captured DAC HYP using an automated method. Binding data were collected and corrected using a reference flow chamber and buffer blank and fitted with BIAEvaluation software using a 1: 1 Langmuir model to obtain equilibrium constants.

The results for DAC HYP150mg/mL batches and reference standard RS0801 are summarized in Table 28:

association constant (K) for 150mg/mL batches_a) Dissociation constant (K)_d) And the affinity constant (K)_D) The values are similar to the reference standard RS 0801.

5.6.18. Functional efficacy

As part of the release test, the functional efficacy of DAC HYP150mg/mL batches and 100mg/mL batches were evaluated. The functional potency assay measures the inhibition of IL-2-induced T-cell proliferation by DAC HYP binding to the IL-2 receptor alpha subunit (CD 25). Varying amounts of DACHYP were incubated with IL-2 receptor-expressing KIT-225K6 cells in the presence of IL-2 (Hori et al, 1987, Blood 70: 1069-1072). Inhibition of T-cell proliferation by DAC HYP was subsequently detected using alamar blue. Using 4-parameter fitting to obtain the log of the fluorescence value to the DAC HYP concentration₁₀Plotting, using parallel line analysis results in relative efficacy value percentage.

The results of functional efficacy are summarized in table 29:

the functional efficacy results for the 150mg/mL batch were similar to the 100mg/mL batch.

5.6.19. Antibody-dependent cellular cytotoxicity

Two batches of DAC HYP150mg/mL formulations were evaluated relative to the reference standard RS0801100mg/mL DAC HYP.

By using⁵¹Cr-labeled IL-2 receptor-expressing KIT-225K6 cells, followed by incubation with DAC HYP. Different amounts of human effector cells (PBMC) were added to obtain different ratios of effector cells to target cells (KIT-225K 6). Monocytes bearing Fc receptors interact with the DAC HYP Fc domain and subsequently cause lysis of the target cells. The degree of cytotoxicity is determined by⁵¹Cr release from target cells was determined and expressed as a percentage of maximal cell lysis.

PBMCs from multiple donors were used for each sample. For each donor, the percentage ADCC activity of the sample was calculated relative to the reference standard RS0801 based on the percentage cytotoxicity. ADCC results are summarized in table 30 below:

response curves for the 150mg/mL batch, reference standard RS0801, positive and negative control antibodies, and no antibody control (antibody independent cytotoxicity or AICC) are shown in figure 20.

The ADCC activity of the 150mg/mL batch was similar to that of the reference standard RS 0801.

5.6.20. Residual protein A

Residual protein a was determined by ELISA method, in which standards, sample controls, blanks, and test samples were diluted with denaturing buffer and placed in a boiling water bath to dissociate protein a and to denature and precipitate daclizumab. After boiling, standards, controls and samples were cooled, centrifuged, and added to microtiter plates coated with polyclonal anti-protein a capture antibody. The residual protein A in the sample was then detected using biotinylated anti-protein A antibody followed by streptavidin alkaline phosphatase and p-nitrophenyl phosphate (PNPP) substrate. The concentration of protein A was determined by analyzing the plate in a spectrophotometric microplate reader and generating a log-1og standard curve. Test sample results are reported in parts per million (ppm) units. Parts per million results were calculated by dividing the results for ng/mL protein a by the concentration of test sample antibody in mg/mL.

DNA content 5.6.21

Detection of mouse DNA was determined in a contract laboratory using a quantitative polymerase chain reaction (Q-PCR) test method. In this method, a sample is subjected to DNA extraction. The specific fragments of the mouse DNA repeat elements were then amplified using mouse specific primers and probes, and the sample extracts were analyzed by Q-PCR. DNA amplification results in the generation of a fluorescent signal that is detected. DNA in the sample is quantitated by comparison to a standard curve generated using known amounts of mouse DNA. Results are expressed as picograms of DNA per milligram of antibody. The average DNA content in different batches of DAC HYP drug product is summarized in table 17.

5.6.22. Host Cell Protein (HCP)

The residual host cell protein in the product was quantified using a commercially available kit. NS0HCP was captured and detected using affinity purified goat polyclonal antibody against NS0 cell lysate. HCP standards were generated by harvesting cell-free harvests from the mock production process. A standard curve was prepared using HCp working standards and samples containing HCP were serially diluted to reach the range of the standard curve. Standards, sample controls and test samples were added to plates coated with anti-NS 0HCP polyclonal antibody. Host cell proteins were then detected using an anti-NS 0HCP polyclonal antibody conjugated to horseradish peroxidase (HRP) followed by a 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) substrate. The plate was then analyzed on a spectrophotometric microplate reader and a four parameter curve fit was generated to quantify the amount of HCP in the sample.

The results of the NS0HCP ELISA test are reported in parts per million (ppm) units. The results were calculated in parts per million by dividing the results for ng/mL HCP by the mg/mL antibody concentration. The average HCPs for each batch of DAC HYP drug product are summarized in table 17.

5.6.23. Concentration of polysorbate 80

Polysorbate 80 was quantified spectrophotometrically, based on the formation of a colored cobalt thiocyanate complex with polysorbate 80. A standard curve was constructed using a series of polysorbate 80 standards. The concentration of polysorbate 80 in the sample was determined from the standard curve. The concentration ranges for polysorbate in the various batches of DAC HYP drug products are summarized in table 17.

5.6.24. Osmolarity

Osmolarity was determined using a vapor pressure let down osmometer. Prior to analyzing the sample, the osmometer is calibrated using an osmolarity standard having the desired osmolarity of the sample. The osmolarity ranges for the various batches of DAC HYP drug product are summarized in table 17.

5.6.25. Conclusion

The physicochemical and biological analyses performed provided a comprehensive evaluation of the 150mg/mL and 100mg/mL formulations of DAC HYP and DACHYP. So far, the physicochemical and biological properties of all test batches were similar.

The first 15 amino acids of the heavy and light chains, peptide mapping and molecular weight analysis, determined by N-terminal sequencing, were consistent with the daclizumab gene sequence for all DAC HYP batches.

The aggregate levels and size distributions of the aggregate species were similar in all the 150mg/mL and 100mg/mL batches tested, as determined by SEC-MALS and SV-AUC, as were their purities as tested by gel electrophoresis.

The charge isotype profile for the 150mg/mL batches was similar to the 100mg/mL batches with only slight differences in the relative amounts of the N-terminal isotypes pE/VHS (150mg/mL batch 31% pE/VHS; 100mg/mL batch 34 to 42% pE/VHS) and Q/VHS (150mg/mL batch 11 to 12% Q/VHS; 100mg/mL batch 4 to 9% Q/VHS). The characteristics of DAC HYP are as follows:

n-terminal isoform of CEX:

pE/pE：31-46％

pE/Q：7-12％

pE/VHS：31-42％

Q/VHS；3-12％

VHS/VHS：1-17％

c-terminal isoform of CEX:

0K：53-80％

1K：14-28％

2K：5-19％

the N-linked glycan profile of DAC HYP is as follows:

G0-GlcNAc：7.2％-14.6％

G0：80.9-88.2％

peak 3: 1.3-1.7%

G1：1.4-3.8

The measured oxidation level of DAC HYP was low.

DAC HYP is biologically active and is capable of inhibiting IL-2 induced T-cell proliferation as demonstrated in ELISA and surface plasmon resonance CD25 binding experiments. DAC HYP is also characterized by low aggregate levels in storage.

Stability of DAC HYP

High concentration DAC HYP formulations are storage stable. The following table provides stability data for a 150mg/mL batch of dachy drug substance.

Table 31 below provides stability data after storage in 50mL bags under recommended conditions (2-8 ℃). Table 32 below provides accelerated stability data stored in 50mL bags at 23-27 ℃. Table 33 below provides the pressurization stability data.

5.8. Comparison between different forms of daclizumab

One manufactured by Hoffman-La Roche Inc. ("Roche") as ZENAPAX^TMMarketed as daclizumab for intravenous use in the treatment of allograft rejection (terminated). DAC Penzberg is a 100mg/mL subcutaneous formulation of daclizumab for use in clinical trials by PDL BioPharma (see CHOICE study described in section 5.9.1, below).

A comparison between DAC HYP, ZENAPAX DAC and DAC Penzberg formulations is shown in table 34. In the table, the formulation buffer is the buffer into which DAC was diafiltered to give the final formulation. Thus, the recorded concentrations are nominal concentrations:

several features of DAC HYP were compared to those of ZENAPAX DAC and DAC Penzberg.

A comparison between glycosylation of DAC HYP and ZENAPAX DAC is shown in figure 21. Analysis of exemplary batches of three forms of daclizumab is shown in table 35:

DAC HYP also has significantly lower mannose sugar levels (e.g., Man5, Man6, Man7) and sialylation sugar levels (see e.g., fig. 21) than ZENAPAX DAC.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an in vitro test that can be used to assess the potential cytotoxicity of Fc-dependent activity and antibody-target binding. ADCC activity of various daclizumab preparations was determined in a variable effector-to-target cell ratio or variable antibody concentration format using Peripheral Blood Mononuclear Cells (PBMCs) from six healthy donors as effector cells and a KIT225/K6 cell line expressing CD25 as target cells.

For forms in which the ratio of effector cells to target cells is variable, the⁵¹Cr-labeled KIT225/K6 cells (12.500 cells/well) were preincubated with 1. mu.g/mL antibody (final concentration) in a v-bottom 96-well plate for 30 minutes at 4 ℃ in 100. mu.L volume of ADCC test medium (containing 435mL RPMI-1640, 5.0mL L-glutamine, 50mL heat-inactivated fetal bovine serum, 500. mu.L 1000X 2-mercaptoethanol, 5.0M penicillin-streptomycin (100X) and 5.0mL HEPES (1M stock solution) per 500 mL). Control blood cells were incubated in ADCC test medium without antibody to subsequently determine antibody dependence⁵¹And releasing Cr.

PBMC (Effector cells) were serially diluted in ADCC assay medium in additional 96-well polypropylene plates to obtain 6.25X 10 per 100. mu.L⁵Single cell, 3.13X 10⁵1.56X 10 cells⁵Individual cell, 7.81X 10⁴Single cell or 3.91X 10⁴Cell concentration of individual cells. Adding a 100. mu.L volume of PBMC suspension to each well⁵¹Plates of Cr-labeled KIT225/K6 and antibody gave effector to target (E: T) ratios of 50: 1, 25: 1, 12.5: 1, 6.25: 1 and 3.13: 1. In addition, a volume of 100. mu.L of ADCC test medium per well was added separately (without mAb) to the medium containing⁵¹Preparation of Cr-labeled KIT225/K6 and monoclonal antibodyIn the board to determine⁵¹Spontaneous release of Cr. The test plate was spun at 50RCF for 2 minutes and at 7.5% CO₂Is incubated at 37 ℃ for 4 hours.

30 minutes before the end of the 4-hour incubation, a volume of 25. mu.L of 8% TritonX-100 was added to the appropriate control wells to confirm⁵¹Maximum release of Cr from target cells. Once the 4 hour incubation was complete, the plates were spun at 350RCF for 5 minutes and a volume of 100 μ Ι _ of supernatant per well was transferred to a vial. Each vial was inserted into a scintillation vial and counted in a Beckman Gamma 5500B counter or equivalent for 1 minute.

For forms in which the antibody concentration is variable, the⁵¹Cr-labeled KIT225/K6 cells (12.500 cells/well, target cells) were preincubated with different doses of antibody (5, 1, 0.2, 0.04, 0.008, and 0.0016 μ g/mL) to mab (final concentration) in V-bottomed 96-well plates in a volume of 100 μ L ADCC assay medium for 30 minutes at 4 ℃. Control wells were incubated with ADCC test medium alone (no mAb) to subsequently determine antibody independence⁵¹And releasing Cr.

PBMC (Effector cells) were serially diluted to 3.13X 10 in ADCC assay medium in an additional 96-well polypropylene plate⁵Individual cells/100. mu.L concentration. Adding a 100. mu.L volume of PBMC suspension to each well⁵¹Cr-labeled KIT225/K6 and mAb plates, a 25: 1 ratio of effector cells to target cells (E: T) was obtained. In addition, a volume of 100. mu.L of ADCC test medium per well was added separately (non-responder cells) to the medium containing⁵¹Cr-labeled KIT225/K6 and monoclonal antibody to determine⁵¹Spontaneous release of Cr. The test plate was spun at 50RCF for 2 minutes and at 7.5% CO₂Is incubated at 37 ℃ for 4 hours.

30 minutes before the end of the 4-hour incubation, a volume of 25. mu.L of 8% TritonX-100 was added to the appropriate control wells to confirm⁵¹Maximum release of Cr from target cells. Once the 4 hour incubation was complete, the plates were spun at 350RCF for 5 minutes and a volume of 100 μ Ι _ of supernatant per well was transferred to a vial. Inserting each small tubeInto a scintillation vial and counted in a Beckman Gamma 5500B counter or equivalent for 1 minute.

ADCC results are shown in figure 22A (variable effector to target cell ratio) and figure 22B (variable antibody concentration). These data show that the maximum ADCC activity achieved with DAC HYP tested at graded concentrations is about 30-40% lower than that stimulated by the same concentrations of ZENAPAX DAC and DAC Penzberg.

A comparison of charge-equivalent spectra (corresponding to N-terminal variants) of DAC HYP versus ZENAPAX DAC versus DAC Penzberg is shown in fig. 23.

Clinical trials of DAC HYP

Study of CHOICE

The choicee trial was a randomized, double-blind, placebo-controlled phase 2 trial in which daclizumab was added to interferon beta therapy in 230 patients with relapsed MS. This trial tested two dosage regimens of DACPenzberg (see description of the product in section 5.8 above) administered as subcutaneous injections: 1mg/kg of daclizumab is administered every four weeks, 2mg/kg of daclizumab is administered every two weeks. The results of the study show that the addition of daclizumab at 2mg/kg every two weeks in interferon beta therapy results in a significant reduction of new or enlarged, gadolinium-enhanced lesions at 24 weeks compared to interferon beta therapy alone.

The results of the choicee study are described in Wynn et al, 2010, Lancet neurol.9 (4): 381-90. Daclizumab therapy is generally well tolerated. Common adverse events were similar in all treatment arms. Grade 3 adverse events were observed in 24% of DAC/interferon β treated patients and 14% of placebo/interferon β treated patients. The most common grade 3 adverse event was infection, which occurred in 7% of DAC/interferon beta treated patients and 3% of placebo/interferon beta treated patients. There were no occasional infections or deaths, and all infections were resolved with standard therapy.

The CHOICE test shows that the interferon beta-a 1 is in the background of the treatmentIn MS patients, the tolerability of daclizumab was good, resulting in a dose-dependent reduction of 72% of new/expanded, gadolinium-enhanced lesions compared to interferon beta-a 1 alone. Potency and immunomodulatory CD56 assay^brightSignificant expansion of Natural Killer (NK) cells is involved.

SELECT study

A randomized, double-blind, placebo-controlled dose range Study (SELECT) was performed to determine the safety and efficacy of two different dose levels of DAC HYP.

For an overview. The study was conducted in 76 centers in the czech republic, germany, hungary, india, poland, russia, ukrains and english. Each patient's care included a neurologist for treatment, a nurse for treatment (or research assistant), a neurologist for examination, an MRI technician, and a pharmacist (or authorized prescriber). Randomization is performed in all sites with a centralized Interactive Voice response system. 150 patients were subjected to protocol-defined interim benefit-free (protocol-defined internal health) analysis after 24 weeks of follow-up.

A patient. Patients meeting eligibility criteria are 18-55 years of age, have clinically confirmed relapsing remitting multiple sclerosis (according to 2005McDonald criteria # 1-4; see Polman et al, 2005Ann Neurol 58: 840-846), have a baseline Expanded Disability Status Scale (EDSS) of 0-0.50(Kurtzke, 1983, Neurology 33 (11): 1444-52) and have at least one MS relapse within 12 months prior to randomization, or have a new Gd + lesion found by brain MRI within 6 weeks prior to randomization. These patients were randomized to receive DAC HYP (150mg or 300mg) or placebo as subcutaneous injections every 4 weeks for 52 weeks. Patients with pregnancy potential need to practice effective contraceptive measures.

If a patient has primary-progressive, secondary-progressive or progressive-relapsing MS, there is a history of malignancy, severe allergy or anaphylaxis or known drug hypersensitivity or has a condition that will not appear to the investigator to beOther important medical situations where DAC HYP can be administered are excluded. Additional exclusion criteria included previous use of DAC HYP or ZENAPAX^TMTotal lymphatic radiation, cladribine (cladribine), mitoxantrone (mitoxantrone), T cell or T cell receptor immunization, or any therapeutic mab other than natalizumab or rituximab (rituximab). At the time of randomization, patients could not have been treated with cyclophosphamide or rituximab within the previous year; (ii) failure to receive treatment with natalizumab, cyclosporine, azathioprine, methotrexate, intravenous immunoglobulin, plasmapheresis, or cell apheresis within the first 6 months; or live virus vaccine can not be inoculated in 3 months before randomization, and glatiramer acetate (IFN beta) and interferon alpha can not be treated; or the previous 30 days without treatment with corticosteroids, 4-aminopyridine or related products.

The group is characterized as follows:

DAC: (ii) daclizumab; HYP, high-yield treatment; SD, standard deviation; MS: multiple sclerosis; EDSS: expanding the disability status scale; gd +, gadolinium reinforcement.

Patients who had not previously received treatment for MS other than steroids.

Placebo n 203, DAC HYP150mg n 206; DAC HYP300mg n 206 (all p values compared between groups > 0.05).

Terminal point. The primary objective of this study was to determine whether DAC HYP monotherapy could reduce recurrence of MS, as defined by Annual Recurrence Rate (ARR) at 52 weeks. Relapse is defined as a new or recurrent neurological symptom (not associated with fever or infection) that persists for > 24 hours and is examined by neurologistsThe assessment was accompanied by new neurological findings. All suspected relapses were evaluated by the Independent board for neurological evaluation (INEC) consisting of three blinded MS neurologists to determine whether the protocol for MS relapse was satisfactory. Primary analysis (primary analysis) included only inci-approved recurrences.

A secondary objective was to determine whether DAC HYP could effectively reduce the number of accumulated new Gd + lesions when performing brain MRI scans on a small group of patients at weeks 8, 12, 16, 20 and 24; reducing the number of new or newly enlarged T2 ultrastrong (hyperintense) lesions at week 52; reducing the proportion of relapsing patients between baseline and week 52; and improving quality of life (QoL) (as determined by change in physiological impact score from baseline at week 52 in 29 multiple sclerosis impact Scale (MSIS-29) (Hobart et al, 2001, Brain 124(pt 5): 962-73)). Confirmed progression of disability was assessed by changes in EDSS scores between baseline and week 52 (1.0 point increase in EDSS at baseline EDSS ≧ 1.0, or 1.5 point increase at baseline EDSS ≧ 0 and lasting 12 weeks). EDSS evaluations were performed every 12 weeks, and at weeks 20, 52, 60, and 72.

Additional QoL endpoints are changes at week 52 in the overall assessment of subject health as determined by the EQ-visual analogue scale (EQ-VAS) (EuroQol-a new institution determining health-related quality of life, 2011, 17.11.11 logs http:// www.euroqol.org /), as well as the EQ-5D health survey (EuroQol-a new institution determining health-related quality of life, 2011, 17.11.11 logs http:// www.euroqol.org /), 12 plain forms of health survey SF-12(Ware et al, 1996, Medical Care (34), (3): 220-33), and the MSIS-29 mental scale (Hobart et al, 2001, Brain 124(pt 5): 962-73).

Additional MRI endpoints are the number of Gd + lesions at week 52, the volume of total and new or newly enlarged T2 ultrastrong lesions at weeks 24 and 52, the volume of total and new T1 ultrastrong lesions "black holes" at weeks 24 and 52, defined as lesions of equal intensity/inferior to gray matter and that do not enhance after administration of gadolinium, and the percentage of total brain volume change as assessed by the SIENA method (Smith et al, 2001, J Comput Assist tomog 25 (3): 466-75).

Subsets of lymphocytes were assayed at various time points using validated FACS tests. Will CD56^brightNK cells defined as CD3^-/CD16⁺/CD56^brightA lymphocyte. Immunogenicity to DAC HYP was assessed using a standard ELISA to screen for anti-drug antibodies, followed by a cell test to test for neutralizing antibodies on all positive samples.

Statistical analysis. A sample size of approximately 600 patients was selected with a 90% ability to detect a 50% reduction in ARR between the DAC HYP treated group and the placebo group as estimated by simulations and two-sided testing assuming a negative two-term distribution with 10% off-rate, 5% type I error rate. The ARR of the placebo group appeared to be 0.476 based on the most recently completed trial on the RRMS subjects. All reported p values are two-tailed.

The primary analysis evaluated the difference in ARR between each DAC HYP group versus placebo. Relapse occurred after rescue treatment with alternative MS drugs was examined. Differences were assessed using a negative binomial regression model that adapted for the number of relapses one year prior to study entry, baseline EDSS (EDSS. ltoreq.2.5 vs EDSS > 2.5), and baseline age (. ltoreq.35 vs > 35 years). The secondary analysis tested treatment differences using negative second term regression (number of new Gd + lesions between weeks 8 and 24; number of new or newly expanded T2 super-strong lesions), Cox proportional hazards model (time before first recurrence, time before disease development) and anova model (change in EDSS, volume of new or newly expanded T2 lesions, volume of new T1 strong lesions, QoL), and proportional dominance model (number of new Gd + lesions at week 52). The proportion of patients without relapse was estimated from the Kaplan-Meier survival curve profile.

For the cumulative number of new Gd + lesions between weeks 8 and 24, if the patient missed 1 or 2 consecutive scans or all scans, the last non-baseline observation was preceded or the average number of lesions within each treatment group was used, respectively. For other MRI endpoints, missing data was entered as an average within each treatment group. For MSIS-29, if the patient misses < 10 entries, the score is entered with the average of the missed entries. For patients who missed ≧ 10 entries and for other QoL determinations, the missed data was estimated using a stochastic slope and intercept model.

Statistical testing of efficacy endpoints utilized separate comparisons of DAC HYP300mg vs placebo and DAC HYP150mg vs placebo. The overall type I error rate due to the various comparisons is controlled using a closed-ended sequential (sequential closed) test procedure.

Efficacy analysis was evaluated in the intent-to-treat (intent-to-treat) population including all patients undergoing randomization. However, prior to completion of the study, 21 patients from a single study center were previously excluded from the ITT population because evidence that they received the wrong dose at the center was identified prior to completion of the study (all patients at the center are receiving effective treatment). In one sensitivity analysis, all primary and secondary efficacy analyses were repeated using all randomized patients. All safety analyses were based on a safe population defined as all patients receiving at least one dose of study drug and having at least one post-randomization evaluation.

A preplanned futile analysis was performed after 150 subjects completed the week 24 follow-up to provide an opportunity to terminate in cases where the postulated effect of DAC HYP was not apparent. Since efficacy may vary over the duration of the study, there is no plan to terminate the study early for evidence of superiority at the time of the futile analysis. The benefit was assessed by assessing the cumulative number of new Gd + lesions and the conditional efficacy of the ARR endpoint between 8 and 24 weeks for each dose group, respectively. The safety monitoring committee reviewed the data at the time of analysis and recommended continued research based on overall consistency of the data and risk benefit assessment.

Summary of results。

From 2/15/2008 to 5/14/2010, eligible participants are randomized. The baseline characteristics of the three treatment groups were similar, although patients in the DAC HYP150mg group tended to have more T2 and Gd + T1 lesions than those in the DAC HYP300mg group. Of all randomized patients, a total of 577 (93%) completed a treatment session in which the proportion of DAC HYP patients completing the study and placebo-treated patients were similar.

Detailed results。Clinical efficacy. At week 52 (primary endpoint), patients randomized to receive either DAC HYP150mg (0.21) or 300mg (0.23) were lower for ARR compared to placebo group (0.46) (table 36), showing a 54% reduction in DAC HYP150mg to placebo (95% CI, 31% to 69%, p < 0.0001) and a 50% reduction in DAC HYP300mg to placebo (95% CI, 26% to 66%, p ═ 0.0002) (table 36). Within 52 weeks, the proportion of relapsing patients was reduced by 36% in the DAC HYP150mg (19%) and 300mg (20%) groups compared to the placebo group (p ≦ 0.01 for both comparisons) (Table 36). Compared to placebo, 3 months of sustained disability progression at week 52 was reduced by 57% (risk ratio 0.43, 95% CI, 0.21 to 0.88, p 0.021) in DAC HYP150mg and 43% (risk ratio 0.57, 95% CI, 0.30 to 1.09, p 0.091) in DAC HYP300 mg.

A relative increase in MSIS-29 physiological score of 4.0 for DAC HYP150mg versus placebo was observed at week 52, with less significant changes in DAC HYP300mg patients (p < 0.0008 and p ═ 0.1284 versus placebo, respectively, table 36). Similar improvements were observed in other measures of health-related quality of life, including measures of physiological and psychological function and overall health (table 36).

MRI. DAC HYP decreased the viability of new MS lesions as determined by MRI in the entire study population and in the groups that had MRI performed monthly between weeks 8 and 24 (table 36). In contrast to the clinical endpoints, the point efficacy estimates were slightly stronger in the 300mg dose group compared to the 150mg dose group, even after adjustment for potential baseline imbalances. Longitudinal analysis showed that Gd + lesion viability was higher in the 150mg dose group compared to the 300mg dose group in the first few months of treatment, but similar at week 52 (table 36). Sensitivity analyses involving 21 patients from excluded study sites gave similar results in all efficacy analyses.

Safety feature. Adverse Events (AE) occurred in patients with similar proportions of the DAC HYP150mg group (73%), DAC HYP300mg group (76%), and placebo (79%) groups (table 37). Severe AE occurred in 26% of patients in placebo, 15% in DAC HYP150mg and 17% in DAC HYP300 mg. Excluding MS recurrence, SAE occurred in 6%, 7% and 9% of patients per group (table 37). AEs occurring in > 0.5% of DAC HYP patients are shown in table 37. The incidence of severe infections was 2% in patients treated with DAC HYP and 0% in placebo. Of 7 patients with severe infection while continuing to take medication, 1 stopped treatment due to severe infection and 6 re-entered treatment after the infection subsided. The incidence of skin events was 18% in the DAC HYP150mg group, 22% in the DAC HYP300mg group, and 13% in the placebo group (table 37). Severe skin events occurred in 1% of patients treated with DAC HYPIn (1). 1 patient recovering from severe rash treated with DAC HYP died due to a complication of lumbar muscle abscess. At necropsy, previously undiagnosed lumbar muscle abscesses were found to spread to the mesenteric artery and lead to local thrombosis and acute ischemic colitis. 5 malignancies occurred during the trial: 2 cases of cervical cancer (1 in each of placebo and DAC HYP150mg groups); 1 thyroid tumor in DAC HYP150mg group was a non-severe thyroid nodule; and 2 melanoma tumors in the DAC HYP300mg group. Melanoma cases were treated by local excision and no recurrence was reported.

Laboratory findings. At week 52, total NK cell counts (cells/mm) in patients treated with DAC HYP compared to placebo³) Increase (median: 42.0(150mg DAC HYP), 46.5(300mg DAC HYP), p < 0.001 over 4.5 placebo. Increase in total NK cell number and CD56^brightThe increase in NK cell selectivity was correlated with a median of 7.77 at baseline to 44.84 at the end of treatment. In contrast, CD^dimNK cells were only slightly altered (median was changed from 122.68 to 123.70). CD56 in the two DAC HYP arm-to-placebo group^brightNK cells were clearly amplified (p < 0.0001) at the first time point (week 4) after baseline. CD56^brightThe median NK cells expanded from 0.6% of lymphocytes at baseline to 2.8% at week 52. In contrast, B-cell and total lymphocyte counts were moderately reduced in patients treated with DAC HYP (table 38). CD4 in DAC HYP-treated patients at week 52⁺And CD8⁺Reduction in T cell count by about 7-10%, CD4⁺/CD8⁺The ratio remains constant during the treatment.

Liver Function Test (LFT) abnormalities are above 5X ULN, which occurs in 4% of patients treated with DAC and < 1% with placebo. These abnormalities usually occurred late in the treatment period (median +308 days from start) and resolved within the median of 62 days. Of the 17 patients treated with DAC HYP who elevated > 5X ULN, 6 continued or re-treated with DAC HYP for at least 6 months after resolution, during which time all patients did not relapse. In 2 patients, elevated LFTs were associated with infection (1 hepatitis b and 1 cytomegalovirus infection).

Immunogenicity. At week 24, DAC HYP neutralizing antibodies were detected in 6 (2%) patients treated with DAC HYP (5 patients in the 150mg dose group, 1 subject in the 300mg dose group). In some patients, these antibodies were transient, with DAC HYP neutralizing antibodies appearing only in 1 subject per DAC HYP dose group at week 52.

Conclusion. Antagonism of CD25 with monotherapy with monthly subcutaneous administration of DAC HYP showed potent and clinically meaningful effects on MS disease activity over 1 year as measured by reduction in recurrence rate in treatment, MRI-determined new lesions, and disability progression, primarily in the untreated MS patient population.

6. Detailed description of the preferred embodiments, incorporated by reference

Aspects disclosed herein are described in embodiments set forth in the following numbered paragraphs.

1. A modified NS0 cell adapted to be grown in serum and cholesterol free medium and engineered to express a recombinant protein, said cell being capable of achieving a volumetric productivity of more than 100 mg/L/day of recombinant protein in 100L culture during a 10 day batch feed when grown in serum and cholesterol free medium.

2. The modified NS0 cell of embodiment 1, which when grown in a medium free of cholesterol and animal-derived components is capable of achieving a volumetric productivity of more than 100 mg/L/day of recombinant protein in 1,000L culture during a 10 day fed batch.

3. The modified NS0 cell of embodiment 1, which when grown in a medium free of cholesterol and animal-derived components is capable of achieving a volumetric productivity of more than 100 mg/L/day of recombinant protein in 16,000L culture during a 10 day fed batch.

4. The modified NS0 cell of any one of embodiments 1-3, wherein the feed medium is added according to the following schedule, wherein the volume added represents a percentage of the initial cell culture volume:

sky	Volume of addition
		0	0

1	0
		2	4
3	7.8
		4	7.8
5	7.8
		6	11
7	13
		8	15
9	15
		10	0

5. The modified NS0 cell of embodiment 1, which is capable of achieving a volumetric productivity of more than 200 mg/L/day of recombinant protein in 100L culture during 13 days of fed batch.

6. The modified NS0 cell of embodiment 5, which when grown in cholesterol-free medium is capable of achieving a volumetric productivity of more than 200 mg/L/day of recombinant protein in 1,000L culture during 13 days of fed batch.

7. The modified NS0 cell of embodiment 5, which when grown in serum and cholesterol free medium is capable of achieving a volumetric productivity of more than 100 mg/L/day of recombinant protein in 16,000L culture during a 10 day batch feed.

8. The modified NS0 cell of embodiment 1, stably transfected with a nucleic acid useful for expressing an anti-CD 25 monoclonal antibody.

9. The modified NS0 cell of embodiment 8, wherein the anti-CD 25 monoclonal antibody comprises a heavy chain variable region corresponding in sequence to SEQ ID NO: 2 VL chain at positions 21-233 and sequences corresponding to SEQ ID NO: 4 VH chain of positions 20-465.

10. The modified NS0 cell of embodiment 1, transformed with the vector pabx.

11. A modified NS0 cell of embodiment 1, transformed with the vector phat.

12. The modified NS0 cell of embodiment 1, designated clone 7a11-5H 7-14-43.

13. A method of producing a recombinant protein comprising culturing the modified NS0 cell of any one of embodiments 1-12.

14. The method of embodiment 13, wherein the modified NS0 cells are cultured under conditions capable of producing at least 100 mg/L/day recombinant protein in 100L, 1,000L, or 16,000L culture during a 10 day batch feed or at least 200 mg/L/day recombinant protein in 100L, 1,000L, or 16,000L culture during a 13 day batch feed.

15. The method of embodiment 13 or embodiment 14, wherein the modified NS0 cells are cultured in the absence of serum and cholesterol.

16. The method of embodiment 15, wherein the modified NS0 cells are cultured in the absence of tropolone and hydrocortisone.

17. The method of embodiment 13 or embodiment 14, wherein the modified NS0 cells are cultured in basal and/or feed medium containing 10-35g/L glucose.

18. The method of embodiment 17, wherein the modified NS0 cells are cultured in a basal medium comprising 15g/L glucose and/or a feed medium comprising 28g/L glucose.

19. The method of embodiment 18, wherein the basal medium consists of PFBM2 ± 10% components.

20. The method of embodiment 18, wherein the feed medium consists of components of PFFM3 ± 10%.

21. The method of embodiment 19 or embodiment 20, wherein the cells are cultured in a basal medium for 1-3 days, followed by culture in a feed medium for 10-13 days.

22. The method of embodiment 18, wherein the feed medium is added according to the schedule set forth in table 7 ± 10%.

23. A vector useful for recombinant expression of a protein of interest comprising a weak promoter driving expression of a selectable marker operable in mammalian cells and a strong promoter driving expression of the protein of interest.

24. The vector of embodiment 23, wherein the protein of interest is a therapeutic antibody.

25. The vector of embodiment 24, wherein the protein of interest is an anti-CD 25 antibody.

26. The vector of embodiment 25, wherein the anti-CD 25 antibody comprises a daclizumab CDR.

27. The vector of embodiment 26, wherein the anti-CD 25 antibody is daclizumab.

28. A method of obtaining a mammalian host cell with high volumetric productivity of a protein of interest comprising transfecting the cell with the vector of any one of embodiments 23-27 and selecting a cell that is capable of producing at least 100 mg/L/day of the protein of interest in 100L, 1,000L, or 16,000L culture during a 10 day batch feed or at least 200 mg/L/day of recombinant protein in 100L, 1,000L, or 16,000L culture during a 13 day batch feed.

29. Composition comprising daclizumab, wherein daclizumab is characterized by the presence of a pE/Q heavy chain N-linked isoform and/or a Q/VHS heavy chain N-terminal isoform

30. The composition of embodiment 29, wherein the pE/Q heavy chain N-terminal isoform comprises about 6-15% of daclizumab.

31. The composition of embodiment 29, wherein the pE/Q heavy chain N-terminal isoform comprises about 7-12% of daclizumab.

32. The composition of any one of embodiments 29 to 31, wherein said Q/VHS heavy chain N-terminal isoform comprises about 1-15% of daclizumab.

33. The composition of embodiment 32, wherein the Q/VHS heavy chain N-terminal isoform comprises about 3-12% of daclizumab.

34. The composition of embodiment 29, wherein said daclizumab heavy chain is present in the following N-terminal isoform:

same type	Incidence rate
		pE/pE	25％-50％
pE/Q	6％-15％
		pE/VHS	25％-48％
Q/VHS	1％-15％
		VHS/VHS	0.5％-25％

35. The composition of embodiment 29, wherein said daclizumab heavy chain is present in the following N-terminal isoform:

same type	Incidence rate
		pE/pE	31-46％
pE/Q	7％-12％
		pE/VHS	31％-42％
Q/VHS	3％-12％
		VHS/VHS	2％-17％

36. The composition of embodiment 29, wherein said daclizumab is characterized by a cation exchange chromatography isotype profile substantially similar to figure 18.

37. The composition of embodiment 29, wherein said daclizumab is DAC HYP.

38. A composition comprising daclizumab, wherein said daclizumab is characterized by an N-linked glycosylation HPLC profile comprising two major peaks, one corresponding to oligosaccharide G0 GlcNAc and one corresponding to oligosaccharide G0, wherein the AUC of the combination of the two peaks comprises about 88-99.5% of the total AUC of all the peaks.

39. The composition of embodiment 38, wherein the AUC of the G0 GlcNAc peak is about 5-18% of the total AUC of all peaks and the AUC of the G0 peak is about 75-92% of the total AUC of all peaks.

40. The composition of embodiment 39, wherein the AUC of said G0 GlcNAc peak is about 6-16% of the total AUC of all peaks and the AUC of said G0 peak is about 78-90% of the total AUC of all peaks.

41. The composition of any one of embodiments 38-40, wherein said N-linked glycosylation profile has a Man5 of less than about 3%.

42. The composition of embodiment 41, wherein said N-linked glycosylation profile has less than about 0.5% of G2, Man6, and/or Man 7.

43. The composition of embodiment 38, wherein said N-linked glycosylation profile comprises a third peak corresponding to sialylated oligosaccharides whose AUC comprises 1% or less of the total AUC of all peaks.

44. The composition of embodiment 38, wherein said N-linked glycosylation profile comprises a third peak corresponding to oligosaccharide G1, said AUC of the G1 peak comprising about 1-5% of the total AUC of all peaks.

45. The composition of embodiment 44, wherein the AUC of the G1 peak is about 1-2% of the total AUC of all peaks.

46. The composition of embodiment 38, wherein said daclizumab has an N-linked glycosylation HPLC profile substantially similar to that of figure 19 or figure 21 below.

47. The composition of embodiment 38, wherein said daclizumab is DAC HYP.

48. Compositions comprising daclizumab, which exhibit an ADCC mean cytotoxicity of less than 35% as determined in an in vitro cell assay using effector cells from at least 3 healthy donors and Kit225K6 cells as target cells, with a concentration of 1 μ g/mL of daclizumab and a ratio of effector cells to target cells of about 25: 1. .

49. The composition of embodiment 48, wherein said daclizumab exhibits an ADCC mean cytotoxicity of 10% to 30% in said assay.

50. The composition of embodiment 48 or embodiment 49, wherein the assay uses effector cells from at least 6 healthy donors.

51. The composition of embodiment 48 or embodiment 49, wherein the assay utilizes effector cells from at least 10 healthy donors.

52. The composition of any one of embodiments 48-51, wherein said daclizumab is DAC HYP.

53. A composition useful for preparing a pharmaceutical formulation of daclizumab, comprising about 150-190mg/mL of daclizumab and an amount of excipient such that dilution of the composition with a dilution buffer results in a diluted composition comprising about 85-165mg/mL of daclizumab and having an osmolality in the range of about 267-327mOsm/kg and a pH at 25 ℃ in the range of about pH 5.8-6.2, wherein at least about 95% of the daclizumab is in monomeric form, as determined by size exclusion chromatography.

54. The composition of embodiment 53, comprising an amount of excipient such that, upon dilution with a dilution buffer, the diluted composition comprises about 85-115mg/mL of daclizumab.

55. The composition of embodiment 53, comprising an amount of excipient such that, upon dilution with a dilution buffer, the diluted composition comprises about 150 ± 15mg/mL of daclizumab.

56. A composition comprising about 4 to 15mg/mL of daclizumab, wherein 0.1% or less of the daclizumab is in the form of aggregates.

57. The composition of embodiment 56, obtained by purifying a daclizumab composition comprising about 4 to 15mg/mL daclizumab, wherein up to 2.5% of the daclizumab is in the form of aggregates by column chromatography on a weak cation exchange resin.

58. The composition of embodiment 57, wherein the weak cation exchange resin is CM-650M.

59. The composition of embodiment 58, wherein the CM-650M resin is equilibrated with an equilibration buffer comprising about 20mM sodium citrate, pH 4.4-4.6, and the daclizumab is eluted with an elution buffer comprising about 20mM sodium citrate and about 75mM sodium sulfate, pH 4.4-4.6.

60. The composition of embodiment 59, wherein the chromatography is performed in a cylindrical column using a resin bed having a height of about 10-30cm or about 17-19cm, and the daclizumab is eluted at a temperature in the range of about 4-22 ℃ or about 18-22 ℃ and a flow rate in the range of about 50-200cm/hr or 90-110 cm/hr.

61. A composition suitable for administration to a human comprising about 85-165mg/mL of daclizumab and about 0.02-0.04% (w/v) polysorbate 80, wherein the composition has an osmolality within the range of about 267-327mOsm/kg and a pH within the range of about pH 5.8-6.2 at 25 ℃, at least about 95% of the daclizumab is in monomeric form as determined by size exclusion chromatography.

62. The composition of embodiment 61, wherein at least about 99% of the daclizumab is in monomeric form as determined by size exclusion chromatography.

63. The composition of embodiment 61, comprising about 85-115mg/mL of daclizumab.

64. The composition of embodiment 63, consisting essentially of about 100mg/mL of daclizumab, about 40mM sodium succinate, about 100mM sodium chloride, and about 0.03% (w/v) polysorbate 80, and having a pH of about 6.0 at 25 ℃.

65. The composition of embodiment 61, comprising about 135-165mg/mL of daclizumab.

66. The composition of embodiment 65, consisting essentially of about 150mg/mL of daclizumab, about 40mM sodium succinate, about 100mM sodium chloride, and about 0.03% (w/v) polysorbate 80, and having a pH of about 6.0 at 25 ℃.

67. The composition of embodiment 65, obtained by a process comprising concentrating a daclizumab composition comprising about 4 to 15mg/mL of daclizumab in a suitable buffer by ultrafiltration to achieve a concentration of daclizumab within the range of about 85-180mg/mL and optionally subjecting the concentrated composition to a dilution step with a dilution buffer.

68. A pharmaceutical composition suitable for subcutaneous administration comprising about 85-165mg/mL of daclizumab, wherein the percentage of daclizumab in the form of aggregates after storage for a period of about 12 months at a temperature in the range of about 2-8 ℃ is no more than about 3%.

69. The pharmaceutical composition of embodiment 68, comprising about 85-115mg/mL of daclizumab.

70. The pharmaceutical composition of embodiment 68, comprising about 135-165mg/mL of daclizumab.

71. The pharmaceutical composition of embodiment 69 or embodiment 70, wherein the percentage of daclizumab in the form of aggregates after storage for a period of about 12 months at a temperature within the range of about 2-8 ℃ is no more than about 2%.

72. The pharmaceutical composition of embodiment 69 or embodiment 70, wherein the percentage of daclizumab in the form of aggregates after storage for a period of about 18 months at a temperature within the range of about 2-8 ℃ is no more than about 3%.

73. A process for harvesting a recombinant protein from a cell culture comprising the steps of:

(i) adjusting the pH of the cell culture expressing and secreting the recombinant protein to a pH in the range of about pH 4.5-5.5;

(ii) incubating the pH-adjusted cell culture at a temperature in the range of about 4 to 15 ℃ for about 30-90 minutes; and

(iii) the incubated pH-adjusted cell culture was centrifuged to remove cell debris.

74. A process for producing a purified daclizumab composition, comprising the steps of:

(i) absorbing the daclizumab from the crude daclizumab preparation onto an affinity chromatography resin;

(ii) washing the affinity resin with a washing buffer to remove contaminants;

(iii) eluting the absorbed daclizumab with an elution buffer;

(iv) inactivating the virus in the eluate by adjusting the pH to a pH in the range of about pH 3-4 and incubating the pH adjusted eluate at a specific temperature for a period of time sufficient to inactivate the virus;

(v) neutralizing the virus-inactivated eluate to a pH in the range of about pH 7.7-7.9 (measured at 25 ℃);

(vi) passing the neutralized eluate through a strong anion exchange chromatography resin;

(vii) (vii) adsorbing the daclizumab in the eluate of step (vi) onto a weak cation exchange chromatography resin; and

(viii) and eluting the adsorbed daclizumab from the weak cation exchange chromatography resin.

75. The process of embodiment 74, wherein said crude preparation of daclizumab is harvested from a cell culture.

76. The process of embodiment 74, wherein said crude preparation of daclizumab is obtained by culturing host cells 7A11-5H7-14-43 under conditions in which daclizumab is secreted into the culture medium and harvesting the secreted daclizumab.

77. The process of embodiment 76, wherein the daclizumab is harvested using the method of embodiment 73.

78. The process of embodiment 74, further comprising the step of:

(ix) (viii) filtering the eluted daclizumab composition of step (viii) to remove virus; and

(x) Concentrating the filtered solution by ultrafiltration to obtain a purified daclizumab composition comprising about 85-180mg/mL of daclizumab.

79. The process of embodiment 78, further comprising the step of diluting the purified daclizumab composition with a dilution buffer to obtain a composition comprising about 85-165mg/mL daclizumab and about 0.02-0.04% (w/v) polysorbate 80, wherein the composition has an osmolality within the range of about 267-327mOsm/kg and a pH at 25 ℃ within the range of about pH 5.8-6.2, at least about 95% of the daclizumab being in monomeric form as determined by size exclusion chromatography.

80. The process of embodiment 79, wherein the obtained composition has less than 50ppm of host cell proteins from a recombinant source of daclizumab, less than 10ppm of protein A, and no more than 3% of the daclizumab in the composition is in the form of aggregates.

81. Basal medium PFBM 2.

82. Feed medium PFFM 3.

83. A daclizumab composition obtained by a process comprising the step of culturing the host cell of any one of embodiments 1-12 under conditions in which daclizumab is secreted into the culture medium.

84. The daclizumab composition of embodiment 83, wherein the process further comprises the step of isolating the secreted daclizumab from the cell culture medium.

85. A buffer useful for sterilizing a protein A affinity chromatography resin comprising about 100mM sodium citrate, about 10-30mM NaOH, and about 0.5-3% (v/v) benzyl alcohol.

86. A method of sterilizing a protein a affinity chromatography column comprising washing the column with the sterilization buffer of embodiment 85 at a flow rate and for a time sufficient to sterilize the column.

87. The method of embodiment 86, wherein the column is washed with about 1.8 column volumes of the sterilization buffer at a flow rate of about 150cm/hr, the washed column is incubated without flow for about 30-45 minutes, followed by equilibration with equilibration buffer.

88. The method of embodiment 87, wherein the equilibration buffer comprises about 20mM sodium citrate and 150mM NaCl, and has a pH of about pH 7 (at 25 ℃).

89. A method of treating a patient suffering from multiple sclerosis comprising administering to the patient DAC HYP in an amount sufficient to provide a therapeutic benefit.

90. The method of embodiment 89, wherein said DAC HYP composition is administered intravenously.

91. The method of embodiment 90, wherein said DAC HYP composition is administered in an amount corresponding to about 0.8-0.9mg/kg DAC HYP.

92. The method of embodiment 91, wherein said DAC HYP composition is administered in an amount corresponding to about 1mg/kg DAC HYP.

93. The method of any one of embodiments 89-92, wherein said DAC HYP is administered once a week for a period of at least 6 weeks, at least 12 weeks, at least 24 weeks.

94. The method of any one of embodiments 89-93, wherein said DAC HYP is administered as a monotherapy.

95. The method of embodiment 94, wherein said patient has failed to respond to or has terminated previous interferon-beta treatment.

96. The method of any one of embodiments 89-93, wherein said DAC HYP adjuvant β -interferon is administered.

97. The method of embodiment 89, wherein said DAC HYP composition is administered subcutaneously.

98. The method of embodiment 97, wherein said DAC HYP composition is administered in an amount corresponding to about 1mg/kg DAC HYP.

99. The method of embodiment 98, wherein said DAC HYP composition is administered once every 2 weeks.

100. The method of embodiment 99, wherein said DAC HYP composition is administered for a total period of about 24 weeks.

101. The method of embodiment 97, wherein said DAC HYP composition is administered in an amount corresponding to about 2mg/kg DAC HYP.

102. The method of embodiment 101, wherein said DAC HYP composition is administered once every 4 weeks.

103. The method of embodiment 102, wherein said DAC HYP composition is administered for a total period of about 24 weeks.

104. The method of embodiment 103, wherein said DAC HYP composition is administered in an amount corresponding to 75mg to 300mg DAC HYP.

105. The method of embodiment 104, wherein said DAC HYP composition is administered in an amount corresponding to 150 mg.

106. The method of embodiment 104, wherein said DAC HYP composition is administered in an amount corresponding to 300 mg.

107. The method of any one of embodiments 103-106, wherein the DAC HYP composition is administered every 4 weeks.

108. The method of embodiment 107, wherein said DAC HYP composition is administered for a total period of at least 48 weeks.

109. The method of any one of embodiments 103-108, wherein the DAC HYP is administered as a monotherapy.

110. The method of embodiment 109, wherein the patient has failed to respond to or has terminated previous interferon-beta treatment.

111. The method of any one of embodiments 103-108, wherein the DAC HYP is administered adjunctively with interferon beta.

112. A recombinant protein from a cell culture obtained or obtainable by a process comprising the steps of:

(i) adjusting the pH of the cell culture expressing and secreting the recombinant protein to a pH in the range of about pH 4.5-5.5;

(ii) incubating the pH-adjusted cell culture at a temperature in the range of about 4 to 15 ℃ for about 30-90 minutes; and

(iii) the incubated pH-adjusted cell culture was centrifuged to remove cell debris.

113. A purified daclizumab composition obtained or obtainable by a process comprising the steps of:

(i) absorbing the daclizumab from the crude daclizumab preparation onto an affinity chromatography resin;

(ii) washing the affinity chromatography resin with a washing buffer to remove contaminants;

(iii) eluting the absorbed daclizumab with an elution buffer;

(iv) inactivating the virus in the eluate by adjusting the pH to a pH in the range of about pH 3-4 and incubating the pH adjusted eluate at a specific temperature for a period of time sufficient to inactivate the virus;

(v) neutralizing the virus-inactivated eluate to a pH in the range of about pH 7.7-7.9 (measured at 25 ℃);

(vi) passing the neutralized eluate through a strong anion exchange chromatography resin;

(vii) (vii) adsorbing the daclizumab in the eluate of step (vi) onto a weak cation exchange chromatography resin; and

(viii) and eluting the adsorbed daclizumab from the weak cation exchange chromatography resin.

114. A purified daclizumab composition obtained or obtainable by the process of any one of embodiments 74-80.

All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

While particular embodiments have been shown and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims (33)

1. A modified NS0 cell adapted to be grown in serum and cholesterol free medium and engineered to express a recombinant protein, said cell being capable of achieving a volumetric productivity of more than 100 mg/L/day of recombinant protein in 100L culture in a 10 day fed batch process when grown in serum and cholesterol free medium.

2. The modified NS0 cell of claim 1, capable of achieving volumetric productivity in excess of:

(a) 100 mg/L/day of recombinant protein in 1,000L culture in a 10 day batch feed process when grown in medium without cholesterol and animal-derived components;

(b) 100 mg/L/day of recombinant protein in 16,000L culture in a 10 day batch feed process when grown in medium without cholesterol and animal-derived components;

(c) 200 mg/L/day of recombinant protein in at least 100L of culture in a 13 day batch feed process;

(d) 200 mg/L/day of recombinant protein in 1,000L culture in a 13 day fed batch process when grown in cholesterol-free medium;

(e) 100 mg/L/day recombinant protein in 16,000L culture in a 10 day batch feed process when grown in serum and cholesterol free medium.

3. The modified NS0 cell of claim 1, wherein the fed batch process comprises adding feed medium according to the following schedule, wherein the added volume represents a percentage of the initial cell culture volume:

sky Volume of addition 0 0 1 0 2 4 3 7.8 4 7.8 5 7.8 6 11 7 13 8 15 9 15

10 0

4. The modified NS0 cell of claim 1, stably transfected with a nucleic acid useful for expressing an anti-CD 25 monoclonal antibody, optionally wherein the anti-CD 25 monoclonal antibody comprises a light chain variable region comprising a sequence corresponding to SEQ id no: 2V in positions 21-233_LThe strands and sequences correspond to SEQ ID NO: 4 VH chain of positions 20-465.

5. A method of producing a recombinant protein comprising culturing the modified NS0 cell of claim 1:

(a) under conditions capable of producing at least 100 mg/L/day recombinant protein in 100L, 1,000L, or 16,000L culture in a 10 day batch feed process or at least 200 mg/L/day recombinant protein in 100L, 1,000L, or 16,000L culture in a 13 day batch feed process;

(b) (ii) absence of serum and cholesterol, and optionally tropolone and hydrocortisone;

(c) in a basal and/or feed medium containing 10-35g/L glucose; or

(d) In a basal medium comprising 15g/L glucose and/or a feed medium comprising 28g/L glucose, optionally wherein the basal medium consists of components of PFBM2 ± 10% or PFFM3 ± 10%, and wherein the cells are cultured in the basal medium for 1-3 days followed by 10-13 days in the feed medium.

6. A vector useful for recombinant expression of a protein of interest comprising a weak promoter driving expression of a selectable marker operable in mammalian cells and a strong promoter driving expression of the protein of interest, optionally wherein the vector is pabx.gpt or phat.igg1.rg.de and/or the protein of interest is (a) a therapeutic antibody; (b) anti-CD 25 antibodies; (c) an anti-CD 25 antibody comprising the daclizumab (daclizumab) CDRs; or (d) daclizumab.

7. A method of obtaining a mammalian host cell with high volumetric productivity of a target protein comprising transfecting the cell with the vector of claim 6 and selecting cells capable of producing at least 100 mg/L/day of the target protein in a 100L, 1,000L or 16,000L culture in a 10 day batch feed process or at least 200 mg/L/day of the recombinant protein in a 100L, 1,000L or 16,000L culture in a 13 day batch feed process.

8. A composition comprising daclizumab, wherein daclizumab is characterized by the presence of a pE/Q heavy chain N-linked isoform and/or a Q/VHS heavy chain N-terminal isoform, optionally wherein the pE/Q heavy chain N-terminal isoform comprises about 3-17%, about 3-15%, about 6-15%, about 5-12%, or about 7-12% of the daclizumab, or optionally wherein the Q/VHS heavy chain N-terminal isoform comprises about 1-15% or about 3-12% of the daclizumab.

9. The composition of claim 8, wherein the daclizumab heavy chain is present in the following N-terminal isoform:

(a)

same type Incidence rate pE/pE 25％-50％ pE/Q 3％-15％ pE/VHS 25％-48％ Q/VHS 1％-15％ VHS/VHS 0.5％-25％

Or (b)

Same type Incidence rate pE/pE 31-46％ pE/Q 5％-12％ pE/VHS 31％-42％ Q/VHS 3％-12％ VHS/VHS 1％-17％

10. The composition of claim 8, wherein said daclizumab is characterized by a cation exchange chromatography equivalent pattern substantially similar to figure 18, optionally wherein said daclizumab is DAC HYP.

11. A composition comprising daclizumab, wherein said daclizumab is characterized by an N-linked glycosylation HPLC profile having two major peaks, one corresponding to oligosaccharide G0-GlcNAc and one corresponding to oligosaccharide G0, wherein the AUC of the combination of the two peaks comprises about 75-100%, about 80-100%, about 85-100%, or about 88-99.5% of the total AUC of all peaks, optionally wherein:

(a) (ii) the AUC of the G0-GlcNAc peak is about 5-20%, about 5-18%, about 7-15%, or about 6-16% of the total AUC of all peaks, the AUC of the G0 peak is about 70-99.2%, about 75-92%, about 75-90%, about 78-90%, or about 81-88% of the total AUC of all peaks, and optionally wherein the N-linked glycosylation profile has less than about 3% Man5 or less than about 0.5% G2, Man6, and/or Man 7;

(b) the N-linked glycosylation profile contains a third peak corresponding to sialylated oligosaccharides whose AUC constitutes 1% or less of the total AUC of all peaks, or wherein the N-linked glycosylation profile contains a third peak corresponding to oligosaccharide G1, whose AUC of the G1 peak constitutes less than about 10%, less than about 5%, less than about 4%, less than about 3%, or about 1-5%, or about 1-4%, or about 1-3% of the total AUC of all peaks;

(c) the N-linked glycosylation profile contains peaks corresponding to Man5, Man6, and Man7 glycoforms, which together are less than about 6% of the total AUC; or

(d) The daclizumab has an N-linked glycosylation HPLC profile substantially similar to that of figure 19 or figure 21 below.

12. A composition comprising daclizumab that exhibits (a) an ADCC average cytotoxicity of less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% or (b) an ADCC average cytotoxicity of 5-30%, 10-25% or 15-25% as measured in an in vitro cell assay using effector cells from at least 3 healthy donors and Kit225K6 as target cells, a concentration of daclizumab being 1 μ g/mL, a ratio of effector cells to target cells of about 25: 1, optionally wherein the test uses effector cells from at least 6 or at least 10 healthy donors.

13. The composition of claim 12, wherein said daclizumab is DAC HYP.

14. A composition useful for preparing a daclizumab pharmaceutical formulation comprising about 150-190mg/mL daclizumab and an amount of excipient such that dilution of the composition with a dilution buffer results in a diluted composition comprising about 85-165mg/mL or 85-115mg/mL or 150 ± 15mg/mL daclizumab and having an osmolality in the range of about 267-327mOsm/kg and a pH at 25 ℃ in the range of about pH 5.8-6.2, wherein at least about 95% of the daclizumab is in monomeric form, as measured by size exclusion chromatography.

15. A composition comprising:

(a) about 4 to 15mg/mL of daclizumab, wherein 0.1% or less of the daclizumab is in the form of aggregates, optionally wherein the composition is obtained by purifying a daclizumab composition comprising about 4 to 15mg/mL of daclizumab, wherein up to 2.5% of the daclizumab is in the form of aggregates, by column chromatography on a weak cation exchange resin; or

(b) About 85-165mg/mL or about 85-115mg/mL or about 135-165mg/mL daclizumab; and about 0.02-0.04% (w/v) polysorbate 80, wherein the composition has an osmolality in the range of about 267-327mOsm/kg and a pH at 25 ℃ in the range of about pH 5.8-6.2, and at least about 95% or at least about 99% of the daclizumab is in monomeric form, as measured by size exclusion chromatography, wherein the composition is suitable for administration to a human, optionally wherein the composition consists essentially of about 100mg/mL or about 150mg/mL daclizumab, about 40mM sodium succinate, about 100mM sodium chloride, and about 0.03% (w/v) polysorbate 80, and has a pH at 25 ℃ of about 6.0.

16. A pharmaceutical composition suitable for subcutaneous administration comprising about 85-165mg/mL, about 85-115mg/mL, or about 135-165mg/mL of daclizumab, wherein the percentage of daclizumab in the form of aggregates after storage for a period of about 12 months at a temperature in the range of about 2-8 ℃ is no more than about 2% or about 3%, or after storage for a period of about 18 months at a temperature in the range of about 2-8 ℃.

17. A method of harvesting a recombinant protein from a cell culture comprising the steps of:

(i) adjusting the pH of the cell culture expressing and secreting the recombinant protein to a pH in the range of about pH 4.5-5.5;

(ii) incubating the pH-adjusted cell culture at a temperature in the range of about 4 to 15 ℃ for about 30-90 minutes; and are

(iii) The incubated pH-adjusted cell culture was centrifuged to remove cell debris.

18. A method of producing a purified daclizumab composition, comprising the steps of:

(i) absorbing the daclizumab from the crude daclizumab preparation onto an affinity chromatography resin;

(ii) washing the affinity resin with a washing buffer to remove contaminants;

(iii) eluting the absorbed daclizumab with an elution buffer;

(iv) inactivating the virus in the eluate by adjusting the pH to a pH in the range of about pH 3-4 and incubating the pH adjusted eluate at a specific temperature for a period of time sufficient to inactivate the virus;

(v) neutralizing the virus-inactivated eluate to a pH in the range of about pH 7.7-7.9 (measured at 25 ℃) or to a pH in the range of about pH 7.7-8.5 (measured at 25 ℃);

(vi) passing the neutralized eluate through a strong anion exchange chromatography resin;

(vii) (vii) adsorbing the daclizumab in the eluate of step (vi) onto a weak cation exchange chromatography resin; and

(viii) eluting the absorbed daclizumab from the weak cation exchange chromatography resin; optionally, comprising the steps of:

(ix) (viii) filtering the eluted daclizumab composition of step (viii) to remove virus; and

(x) Concentrating the filtered solution by ultrafiltration to obtain a purified daclizumab composition comprising about 85-180mg/mL of daclizumab.

19. The method of claim 18, wherein the crude preparation of daclizumab is harvested from a cell culture, optionally using the method of claim 17.

20. The method of claim 18, wherein steps (i) through (x) are performed, further comprising the step of diluting the purified daclizumab composition with a dilution buffer to obtain a composition comprising about 85-165mg/mL daclizumab and about 0.02-0.04% (w/v) polysorbate 80, wherein the composition has an osmolality in the range of about 267-327mOsm/kg and a pH at 25 ℃ in the range of about pH 5.8-6.2, and at least about 95% of the daclizumab is in monomeric form, as measured by size exclusion chromatography, optionally wherein the obtained composition has less than 50ppm host cell protein from a recombinant source of daclizumab, less than 10ppm protein a, and no more than 3% of the daclizumab in the composition is in aggregate form.

21. A culture medium which is a basal medium PFBM2 or a feed medium PFFM 3.

22. A daclizumab composition obtained by a method comprising the step of culturing a host cell according to claim 1 under conditions in which daclizumab is secreted into the culture medium, optionally wherein the method further comprises the step of isolating the secreted daclizumab from the cell culture medium.

23. A buffer for disinfecting a protein A affinity chromatography resin comprising about 100-500mM sodium citrate, about 10-30mM NaOH, and about 0.5-3% (v/v) benzyl alcohol.

24. A method of sterilizing a protein a affinity chromatography column comprising washing the column with the sterilization buffer of claim 23 at a flow rate and for a time sufficient to sterilize the column, optionally wherein the column is washed with about 1.8 column volumes of the sterilization buffer at a flow rate of about 150cm/hr, and the washed column is incubated stagnant for about 30-45 minutes, followed by equilibration with equilibration buffer.

25. A method of treating a patient suffering from multiple sclerosis comprising administering to the patient DAC HYP in an amount sufficient to provide a therapeutic benefit.

26. The method of claim 25, wherein the DAC HYP composition is administered:

(a) intravenously;

(b) in an amount corresponding to about 0.8-0.9mg/kg or about 1mg/kg DAC HYP;

(c) once per week for a period of at least 6 weeks, at least 12 weeks, at least 24 weeks;

(d) as a monotherapy; or

(e) Any combination according to (a) to (d).

27. The method of claim 26, wherein DAC HYP is administered as a monotherapy and the patient has failed to respond to or has terminated prior interferon-beta treatment.

28. The method of claim 26, wherein DAC HYP adjuvanted interferon beta is administered.

29. The method of claim 25, wherein the DAC HYP composition is administered:

(a) subcutaneous injection;

(b) in an amount corresponding to about 1mg/kg DAC HYP, optionally once every 2 weeks, or in an amount corresponding to about 2mg/kg DAC HYP, optionally once every 4 weeks;

(c) for a total of about 24 weeks; or

(d) Any combination according to (a) to (c).

30. The method of claim 29, wherein said DAC HYP composition is administered in an amount corresponding to 75mg to 300mg DAC HYP or 150mg or 300 mg.

31. The method of claim 30, wherein said DAC HYP composition is administered once every 4 weeks, optionally for a total of at least 48 week periods.

32. The method of claim 30, wherein the DAC HYP is administered as a monotherapy, optionally wherein the patient has failed to respond to or has terminated prior interferon-beta treatment.

33. The method of claim 30, wherein said DAC HYP is administered adjunctively with interferon beta.