HK1231928B - Overexpression of n-glycosylation pathway regulators to modulate glycosylation of recombinant proteins - Google Patents

Description

Overexpression of N-glycosylation pathway regulatory genes to regulate the glycosylation of recombinant proteins

Field of the Invention

The present invention generally relates to methods for modulating one or more properties of a recombinant protein produced by a cell culture, including a mammalian cell culture, such as a CHO cell culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/933,192, filed January 29, 2014, which is incorporated herein by reference.

Background of the Invention

Glycosylation is a common post-translational modification in mammalian cells; normal human immunoglobulins and therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary (CHO) cells are glycoproteins. The pharmacokinetic properties and effector functions of therapeutic mAbs can be affected by glycosylation. Terminal sugars such as fucose and galactose can affect antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC; Wright, A. and S.L. Morrison, Trends Biotechnol (1997) 15:26-32). High mannans can increase the serum clearance of certain mAbs, thereby potentially affecting efficacy (Goetze et al., (2011) Glycobiology 21:949-59). Alternatively, high mannose glycoforms can increase antibody affinity for FcγIII receptors, thereby increasing the ADCC activity of certain antibodies (Yu et al. (2012) MAb 4:475-87). Therefore, for each recombinant mAb, certain glycosylation characteristics need to be maintained that best support the mAb's therapeutic potential.

Methods for manipulating the high mannose glycoform content of proteins in cell culture include altering culture medium composition, osmolality, pH, temperature, etc. (Yu et al., supra, Pacis et al., supra, Chee Furng Wong et al. (2005) Biotechnol Bioeng 89: 164-177; Ahn et al. (2008) Biotechnol Bioeng 101: 1234-44). The effectiveness of these methods is specific to the cell line, molecule type, and culture medium environment, and is often achieved through trial and error. Alternatively, these methods may also alter antibody productivity, cell culture behavior, and other antibody quality attributes.

There remains a need to identify mechanisms that can regulate the content of high-mannose glycoforms (particularly Mannose 5) on mAbs without compromising CHO production culture performance and antibody yield. Such an approach would be beneficial in process development for therapeutic proteins. The present invention provides a method for regulating the content of high-mannose glycoforms by manipulating the expression levels of proteins involved in the N-glycosylation pathway.

SUMMARY OF THE INVENTION

The present invention provides a method for regulating the high mannose glycoform content of a recombinant protein during a mammalian cell culture process, comprising transforming a host cell to overexpress a protein involved in the N-glycosylation pathway. The present invention also provides a method for reducing the high mannose glycoform content of a recombinant protein during a mammalian cell culture process. In one embodiment, the protein is N-acetyl-glucosaminyltransferase-1 (encoded by Mgat1); in another embodiment of the present invention, the protein is N-acetyl-glucosaminyltransferase-2 (encoded by Mgat2). In yet another embodiment of the present invention, the protein is UDP-galactose transporter (encoded by Slc35a2).

Additional embodiments of the present invention include transforming host cells to overexpress two or more proteins involved in the N-glycosylation pathway, including combinations of the above proteins. In one embodiment, the host cell is transformed with Mgat1 and Mgat2; in other embodiments, the host cell is transformed with Mgat1 and Slc35a2, with Mgat2 and Slc35a2, or with Mgat1, Mgat2, and Slc35a2.

The present invention also provides for the transfection of a host cell line that has previously been transfected to express a recombinant protein. In one embodiment, the recombinant protein is a protein comprising an antibody Fc region. Yet another embodiment includes a host cell expressing a recombinant protein selected from the group consisting of an Fc fusion protein, an antibody, an immunoglobulin, and a peptibody.

In yet another embodiment, host cells are first transfected to overexpress one or more of Mgat1, Mgat2, and Slc35a2, and then transfected to express a recombinant protein. In one embodiment, the recombinant protein is a protein comprising an antibody Fc region. Yet another embodiment includes expression of a recombinant protein selected from the group consisting of an Fc fusion protein, an antibody, an immunoglobulin, and a peptibody.

Optionally, the present invention further comprises the step of harvesting the recombinant protein produced by the cell culture.In yet another embodiment, the recombinant protein produced by the host cells is purified and formulated into a pharmaceutically acceptable preparation.

In yet another embodiment, the high mannose glycoform content of the recombinant protein is reduced compared to a recombinant protein produced from a culture in which the cells have not been manipulated by transfection to overexpress proteins involved in N-linked glycosylation. In one embodiment, the high mannose species is mannose 5 (Man5). In another embodiment, the high mannose species is mannose 6 (Man6), mannose 7 (Man7), mannose 8 (including mannose 8a and 8b; Man8a and 8b) or mannose 9 (Man9). In yet another embodiment, the high mannose species comprises a mixture of Man5, Man6, Man7, Man8a, Man8b and/or Man9.

The present invention provides yet another embodiment in which the content of high mannose glycoforms in a recombinant protein is reduced. In yet another embodiment, the content of high mannose glycoforms in the recombinant protein is less than or equal to 5%. In another embodiment, the content of high mannose glycoforms in the recombinant protein is less than or equal to 10%. In yet another embodiment, the content of high mannose glycoforms in the recombinant protein produced by the cell culture of the present invention is less than 6%, 7%, 8%, 9%, or 10%. In yet another embodiment, the content of high mannose glycoforms in the recombinant protein produced by the cell culture of the present invention is less than 0.5%, 1%, 2%, 3%, 4%, or 5%. Yet another embodiment includes a high mannose glycoform content of less than 12%, less than 15%, less than 20%, or less than 30%, 40%, or 50%.

Additional embodiments include the use of batch or fed-batch cultures and the use of perfusion cultures. In one embodiment, the culture is perfused using alternating tangential flow (ATF).

In conjunction with any embodiment of the invention described herein, an antifoaming agent may also be added to the culture vessel as needed. Alternatively or additionally, 1 M sodium carbonate or another suitable base is used to maintain the pH at the desired set point.

As described herein, in one aspect of the invention, cell cultures can be maintained by perfusion. In one embodiment, perfusion begins at or about day 1 to at or about day 9 of the cell culture. In a related embodiment, perfusion begins at or about day 3 to at or about day 7 of the cell culture. In one embodiment, perfusion begins when the cells have reached the production phase. In yet another embodiment of the invention, perfusion is accomplished by alternating tangential flow. In a related embodiment, perfusion is accomplished by alternating tangential flow using an ultrafilter or microfilter.

In yet another embodiment of the present invention, continuous perfusion is provided; in yet another embodiment, the perfusion rate is constant. In one embodiment of the present invention, perfusion is provided at a rate of less than or equal to 1.0 working volume per day. In a related embodiment, perfusion is performed during the cell culture at a rate that increases from 0.25 working volumes per day to 1.0 working volume per day during the production phase. In another related embodiment, perfusion is performed on day 9 to day 11 of the cell culture at a rate that reaches 1.0 working volume per day. In another related embodiment, perfusion is performed on day 10 of the cell culture at a rate that reaches 1.0 working volume per day.

In one embodiment, the cell culture receives a perfusion cell culture medium feed prior to day 3-7 of culture.

In another aspect of the invention, the cell culture is maintained by batch feeding. In one embodiment of a fed-batch culture, the culture is fed three times during the production period. In yet another embodiment, the culture is fed on a day between the second and fourth day, a day between the fifth and seventh day, and a day between the eighth and tenth day. Another embodiment provides a fed-batch method in which the culture is fed four times during the production period. In yet another embodiment, the culture is fed on a day between the second and fourth day, a day between the fifth and sixth day, a day between the seventh and eighth day, and a day between the eighth and tenth day or later.

According to one embodiment of the present invention, the mammalian cell culture is established by inoculating a bioreactor with at least 0.5 x ¹⁰ cells/mL to 3.0 x ¹⁰ cells/mL in a serum-free medium. In an alternative or further embodiment, the mammalian cell culture is established by inoculating a bioreactor with at least 0.5 x ¹⁰ cells/mL to 1.5 x ¹⁰ cells/mL in a serum-free medium.

The present invention may also include a temperature change during the culture period. In one embodiment, the temperature change is between 36°C and 31°C. In one embodiment, the present invention also includes a temperature change between 36°C and 33°C. In a related embodiment, the temperature change occurs at the transition between the growth phase and the production phase. In a related embodiment, the temperature change occurs during the production phase.

In another embodiment, the present invention further comprises inducing cell growth arrest by L-asparagine starvation followed by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less. In another embodiment, the present invention further comprises inducing cell growth arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less. In a related embodiment, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 5 mM. In a related embodiment, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 4.0 mM. In a related embodiment, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 3.0 mM. In a related embodiment, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 2.0 mM. In a related embodiment, the concentration of L-asparagine in the serum-free perfusion medium is less than or equal to 1.0 mM. In a related embodiment, the concentration of L-asparagine in the serum-free perfusion medium is 0 mM. In a related embodiment, the L-asparagine concentration of the cell culture medium is monitored before or during L-asparagine starvation.

In yet another embodiment, the invention comprises a packed cell volume of less than or equal to 35% during the production phase. In a related embodiment, the packed cell volume is less than or equal to 35%. In a related embodiment, the packed cell volume is less than or equal to 30%.

In a related embodiment, the viable cell density of the mammalian cell culture at a cell packing density of less than or equal to 35% is between 10 x 10 ⁶ viable cells/ml and 80 x 10 ⁶ viable cells/ml. In another embodiment, the viable cell density of the mammalian cell culture is between 20 x 10 ⁶ viable cells/ml and 30 x 10 ⁶ viable cells/ml.

In yet another embodiment, the bioreactor has a capacity of at least 500 L. In yet another embodiment, the bioreactor has a capacity of at least 500 L to 2000 L. In yet another embodiment, the bioreactor has a capacity of at least 1000 L to 2000 L.

In yet another embodiment, the mammalian cell is a Chinese Hamster Ovary (CHO) cell.In yet another embodiment, the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein or a cytokine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the clonal variability in doubling time during passage of the cell lines used in the first set of fed-batch cultures in Example 2. In this example, cells from a cell line expressing MAb B were transformed to overexpress Mgat1, Mgat2, and/or Slc35A2. M1M2 represents a cell line overexpressing Mgat1 and Mgat2 (individual clones represented by solid circles); M1M2S represents a cell line overexpressing Mgat1, Mgat2, and Slc35a2 (individual clones represented by open triangles); and S represents a cell line overexpressing Slc35A2 (individual clones represented by solid squares). Control cells (individual clones represented by open squares) were transformed with empty vector. The central box covers the first quartile (Q1) to the third quartile (Q3), and the height of the box is the interquartile range (IQR). The band within the box is the median. If no value is greater than Q3 + 1.5 IQR, the upper whisker extends from Q3 to the maximum value or maximum value below Q3 + 1.5 IQR. If no value is less than Q1 - 1.5 IQR, the lower whisker extends from Q1 to the minimum value or minimum value above Q1 - 1.5 IQR.

FIG2 illustrates the clonal variability in doubling time during passage of the cell lines used for the second set of fed-batch cells in Example 2. In this example, cells from a cell line expressing MAb B were transformed to overexpress Mgat1, Mgat2, and/or Slc35A2. M1M2 indicates a cell line overexpressing Mgat1 and Mgat2; M1M2S indicates a cell line overexpressing Mgat1, Mgat2, and Slc35a2; and S indicates a cell line overexpressing Slc35A2. Control cells were transformed with an empty vector. Individual clones are shown as described for FIG1; the parameters for each box are the same as those described for FIG1.

Figure 3 shows the growth shown in terms of viable cell density compared to the control on day 10 of the second fed-batch experiment described in Example 2 for all overexpressing cell lines (M1M2, M1M2S, and S). The individual values in each box again illustrate the observed clonal variability. As described for Figure 1, individual clones are represented; the parameters of each box are the same as described for Figure 1.

Figure 4 presents a comparison of clonal variability in the titers of antibodies produced during the second fed-batch experiment described in Example 2. Individual clones are represented as described for Figure 1; the parameters of each box are as described for Figure 1.

Figure 5 presents the clonal variability in the specific productivity of antibodies produced during the second fed-batch experiment described in Example 2. Individual clones are represented as described for Figure 1; the parameters of each box are as described for Figure 1.

Figure 6 provides an indication of the clonal variability in the HM percentage of antibodies produced during the second fed-batch experiment described in Example 2. Individual clones are represented as described for Figure 1; the parameters for each box are as described for Figure 1.

Detailed Description of the Invention

Although the terms used in this application are standard in the art, the definition of certain terms is provided herein to ensure that the claims are clear and unambiguous. Units, prefixes and symbols can be represented in the form accepted by their SI (International System of Units). The numerical ranges listed herein include the numbers that define the range, and include and support each integer within the defined range. Unless otherwise indicated, the methods and techniques described herein are generally based on the conventional methods described in the various general references and more specific references as known in the art and as cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992) and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

The disclosed methods are applicable to adherent or suspension cultures grown in stirred tank reactors (including traditional batch cell culture and fed-batch cell culture, which may, but need not, include spin filters), perfusion systems (including alternating tangential flow ("ATF") cultures, acoustic perfusion systems, depth filter perfusion systems, and others), hollow fiber bioreactors (HFBs, which in some cases can be used for perfusion processes), and various other cell culture methods (see, e.g., Tao et al., (2003) Biotechnol. Bioeng. 82:751-65; Kuystermans and Al-Rubeai, (2011) “Bioreactor Systems for Producing Antibodies from Mammalian Cells” in Antibody Expression and Production, Cell Engineering 7:25-52, Al-Rubeai (ed) Springer; Catapano et al., (2009) “ Bioreactor Design and Scale-Up” in Cell Biotechnology, incorporated herein by reference in their entirety). and Tissue Reaction Engineering: Principles and Practice , Eibl et al. (eds. Springer-Verlag).

All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference.What is described in one embodiment of the invention may be combined with other embodiments of the invention.

definition

As used herein, the terms "a" and "an" mean one or more unless specifically indicated otherwise. Furthermore, unless the context otherwise requires, singular terms will include the plural, and plural terms will include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

The present disclosure provides methods for regulating the properties of cell cultures expressing "target proteins"; "target proteins" include naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that do not exist in nature and have been designed and/or created by humans). Target proteins may be, but need not be, proteins that are known or suspected to be therapeutically relevant. Specific examples of target proteins include antigen binding proteins (as described or defined herein), peptibodies (i.e., peptides fused directly or indirectly to the Fc domain of other molecules such as antibodies, wherein the peptide portion specifically binds to the desired target; for example, as described in U.S. Patent Application Publication No. US2006/0140934, which is incorporated herein by reference in its entirety, the peptides may be fused to the Fc region or embedded in an Fc-loop or modified Fc molecule), fusion proteins (e.g., Fc fusion proteins, in which the Fc fragment is fused to a protein or peptide (including peptibodies)), cytokines, growth factors, hormones, and other naturally occurring secreted proteins, and mutant forms of naturally occurring proteins.

The term "antigen binding protein" is used in its broad sense and means a protein comprising a portion that binds to an antigen or target and optionally comprising a framework or framework portion that allows the antigen binding portion to adopt a configuration that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab fragments; F(ab') ₂ fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof. Antigen binding proteins can include, for example, alternative protein frameworks or artificial frameworks with transplanted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived frameworks comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein; and fully synthetic frameworks comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53 (1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20 : 639-654 (2004). In addition, peptide antibody mimetics ("PAMs") can be used, as can antibody mimetic frameworks that utilize fibronectin components as frameworks.

Antigen binding proteins can have the structure of, for example, naturally occurring immunoglobulins. "Immunoglobulins" are tetrameric molecules. In naturally occurring immunoglobulins, each tetramer is primarily composed of two identical polypeptide chain pairs, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50 kDa-70 kDa). The amino terminal portion of each chain includes a variable region with about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains are classified as kappa light chains and lambda light chains. Heavy chains are classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively.

Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain can be performed according to the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, (1991). If desired, the CDRs may also be redefined according to an alternative nomenclature scheme, such as that of Chothia (see, Chothia and Lesk, (1987) J. Mol. Biol. 196 :901-917; Chothia et al., (1989) Nature 342 :878-883 or Honegger and Pluckthun, (2001) J. Mol. Biol. 309 :657-670).

In the context of the present disclosure, an antigen binding protein is said to "specifically bind" or "selectively bind" to its target antigen when the dissociation number ( _KD ) ≤ ^10-8 M. An antibody specifically binds the antigen with "high affinity" when _KD ≤ 5 x ^10-9 M, and specifically binds the antigen with "very high affinity" when _KD ≤ 5 x ^10-10 M.

Unless otherwise indicated, the term "antibody" includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or to antigen-binding regions thereof that compete with intact antibodies for specific binding. Furthermore, unless otherwise indicated, the term "antibody" refers to intact immunoglobulins or antigen-binding portions thereof that compete with intact antibodies for specific binding. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies and can form elements of the target protein. Antigen-binding portions particularly include Fab, Fab', F(ab') ₂ , Fv, domain antibodies (dAbs), fragments comprising complementary determining regions (CDRs), single-chain antibodies (scFvs), chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a polypeptide.

A Fab fragment is a monovalent fragment having _VL , _VH , _CL , and _CH1 domains; a F(ab') ₂ is a bivalent fragment having two Fab fragments connected by a disulfide bond at the hinge region; an Fd fragment has the _VH and _CH1 domains; an Fv fragment has the _VL and _VH domains of a single arm of an antibody; and a dAb fragment has a _VH domain, a _VL domain, or an antigen-binding fragment of a _VH or _VL domain (U.S. Patent Nos. 6,846,634; 6,696,245; U.S. Application Publication Nos. 05/0202512; 04/0202995; 04/0038291; 04/0009507; 03/0039958; Ward et al. (1989) Nature 341:544-546).

Single-chain antibodies (scFv) are antibodies in which the _VL and _VH regions are connected by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, where the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are bivalent antibodies comprising two polypeptide chains, each comprising a _VH domain and a _VL domain connected by a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6444-48; and Poljak et al., (1994) Structure 2: 1121-23). If the two polypeptide chains of a diabody are identical, then the diabody produced by pairing them will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to produce diabodies with two different antigen-binding sites. Similarly, triabodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which may be the same or different.

One or more CDRs can be covalently or non-covalently incorporated into a molecule to make the molecule an antigen binding protein. An antigen binding protein can incorporate one or more CDRs as part of a larger polypeptide chain, can covalently link one or more CDRs to another polypeptide chain, or can incorporate one or more CDRs non-covalently. CDRs allow an antigen binding protein to specifically bind to a particular target antigen.

An antigen binding protein may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites.

For the purposes of clarity, and as described herein, it should be noted that antigen binding proteins can, but need not, be human (e.g., human antibodies), and in some cases will include non-human proteins such as rat proteins or mouse proteins, and in other cases, antigen binding proteins may include hybrids of human and non-human proteins (e.g., humanized antibodies).

The target protein may include human antibodies. The term "human antibody" includes all antibodies having one or more variable regions and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable domains and constant domains are derived from human immunoglobulin sequences (fully human antibodies). Such antibodies can be prepared in a variety of ways (including by immunization with the target antigen of mice), and the mice, such as mice derived from UltiMab ^™ or systems, are genetically modified to express antibodies derived from human heavy chain encoding genes and/or light chain encoding genes. Phage-based methods can also be used.

Alternatively, target protein can include humanized antibodies." humanized antibodies " have a sequence different from the sequence of the antibody derived from non-human species because of one or more amino acid replacements, deletions and/or additions, so that compared with non-human species antibodies, humanized antibodies are unlikely to induce an immune response and/or induce a less severe immune response when used to human subjects. In one embodiment, certain amino acids in the framework domains and constant domains of the heavy chain and/or light chain of non-human species antibodies are mutated to produce humanized antibodies. In another embodiment, one or more constant domains from human antibodies are fused with one or more variable domains of non-human species. The example of how to make humanized antibodies can be found in U.S. Patent numbers 6,054,297, 5,886,152 and 5,877,293.

As used herein, an "Fc" region comprises two heavy chain fragments comprising the _CH2 and _CH3 domains of an antibody. The two heavy chain fragments are bound together by two or more disulfide bonds and by hydrophobic interactions of the _CH3 domains. Target proteins comprising an Fc region, including antigen binding proteins and Fc fusion proteins, form another aspect of the present disclosure.

A "hemibody" is an immunologically functional immunoglobulin construct comprising a complete heavy chain, a complete light chain, and a second heavy chain Fc region paired with the Fc region of the complete heavy chain. A linker may, but need not, be used to connect the heavy chain Fc region and the second heavy chain Fc region. In specific embodiments, a half antibody is a monovalent form of an antigen-binding protein disclosed herein. In other embodiments, a charged residue pair may be used to associate one Fc region with a second Fc region. In the context of the present disclosure, a half antibody may be a protein of interest.

The term "host cell" means a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes progeny of a parent cell, whether or not the progeny are identical to the original parent cell in morphology or genetic makeup, as long as the gene of interest is present. A cell culture may comprise one or more host cells.

The term "hybridoma" means a cell or progeny of a cell produced by the fusion of an immortalized cell with an antibody-producing cell. The hybridoma produced is an immortalized cell that produces an antibody. The individual cells used to create hybridomas can be from any mammalian source, including but not limited to hamsters, rats, pigs, rabbits, sheep, goats and humans. The term also includes a trio of hybridoma cell lines produced when heterohybrid myeloma fusions are fused. The trio of hybridoma cell lines is the product of the fusion between a human cell and a mouse myeloma cell line, which is subsequently fused with a plasma cell. The term is intended to include any immortalized hybrid cell line that produces an antibody, such as, for example, a quadroma (see, e.g., Milstein et al., (1983) Nature, 537: 3053).

The terms "culture" and "cell culture" are used interchangeably and refer to a cell population maintained in a culture medium under conditions suitable for the survival and/or growth of the cell population. It will be clear to those skilled in the art that these terms refer to a combination comprising a cell population and the culture medium in which the population is suspended.

The terms "polypeptide" and "protein" (e.g., as used in the context of a protein or polypeptide of interest) are used interchangeably herein to refer to polymers of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms may also include amino acid polymers that have been modified (e.g., by the addition of carbohydrate residues to form a glycoprotein) or phosphorylated. Polypeptides and proteins can be produced by naturally occurring and non-recombinant cells, or they can be produced by genetically engineered or recombinant cells. Polypeptides and proteins can comprise molecules having the amino acid sequence of a naturally occurring protein, or molecules having the native sequence of one or more amino acids deleted, added and/or substituted.

The terms "polypeptide" and "protein" include molecules containing only naturally occurring amino acids, as well as molecules containing non-naturally occurring amino acids. Examples of non-naturally occurring amino acids (which may be substituted for any naturally occurring amino acid present in any sequence disclosed herein, as desired) include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In accordance with standard usage and convention, in polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction.

A non-limiting list of examples of non-naturally occurring amino acids that can be inserted into a protein or polypeptide sequence or replace a wild-type residue in a protein or polypeptide sequence includes β-amino acids, homoamino acids, cyclic amino acids, and amino acids with derivatized side chains. Examples include (in L- or D-form; as abbreviated in parentheses): citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn), Nα-methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Nα-methylarginine (NMeR), Nα-methylleucine (Na-MeL or NMeL), N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1, 2, 3 , 4-tetrahydroisoquinoline (Tic), octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (IgI), p-indolephenylalanine (pI-Phe), p-aminophenylalanine (4AmP or 4-amino-Phe), 4-guanidinophenylalanine (Guf), glycyllysine (abbreviated "K(Nε-glycyl)" or "K(gly)" or "K(gly)"), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu) , carboxyproline (hydroxypro), p-carboxy-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methylvaline (NMeVal), N-α-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or diphenylalanine; 4Bip), α-amino-isopropylaminopropionic acid (ISO-1, 1, 2-diaminopropionic acid), 4 ... butyric acid (Aib), β-alanine, β-alanine, pipecolic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-amino-O-phthalic acid (4APA), and other similar amino acids and derivative forms of any of those amino acids explicitly listed.

"Cell culture" or "culturing" refers to the growth and proliferation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or when attached to a solid substrate. A fluidized bed bioreactor, hollow fiber bioreactor, roller flask, shake flask, or stirred tank bioreactor with or without microcarriers can be used. In one embodiment, a 500 L to 2000 L bioreactor is used. In one embodiment, a 1000 L to 2000 L bioreactor is used.

The term "cell culture medium" (also referred to as "culture medium," "cell culture media," "tissue culture medium") refers to any nutrient solution used to grow cells, such as animal or mammalian cells, and which typically provides at least one or more components from the following: an energy source (usually in the form of carbohydrates such as glucose); one or more of all the essential amino acids, and typically the 20 basic amino acids, plus cysteine; vitamins and/or other organic compounds, usually required at low concentrations; lipids or free fatty acids; and trace elements, such as inorganic compounds or naturally occurring elements, usually required at very low concentrations, often in the micromolar range.

Depending on the needs of the cells to be cultured and/or the desired cell culture parameters, the nutrient solution may optionally be supplemented with additional optional components to optimize cell growth, such as hormones and other growth factors, for example, insulin, transferrin, epidermal growth factor, serum, etc.; salts, for example, calcium, magnesium and phosphates and buffers, for example, HEPES; nucleosides and bases, for example, adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, for example, hydrolyzed animal or plant proteins (peptone or peptone mixtures, which can be obtained from animal by-products, refined gelatin or plant matter); antibiotics, for example, gentamicin; cytoprotectants or surfactants, such as F68 (also known as F68 and P188; a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (polypropylene oxide) flanked by two hydrophilic chains of polyethylene oxide (polyethylene oxide); polyamines, for example, putrescine, spermidine and spermine (see, for example, WIPO Publication No. WO 2008/154014) and pyruvate (see, e.g., U.S. Patent No. 8,053,238).

Cell culture media include those commonly employed and/or known for use in any cell culture method, such as, but not limited to, batch culture, extended batch culture, fed-batch culture, and/or perfusion culture or continuous culture of cells.

"Basal" (or batch) cell culture medium refers to a cell culture medium that is typically used to initiate cell culture and is sufficient to fully support the cell culture.

"Growth" cell culture medium refers to a cell culture medium that is typically used in cell culture during the exponential growth phase (growth phase) and is sufficient to fully support cell culture during this phase. Growth cell culture medium may also contain a selection agent that confers resistance or viability to a selectable marker incorporated into the host cell line. Such selection agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, bleomycin, methionine sulfoximine, methotrexate, glutamine-free cell culture medium, cell culture medium lacking glycine, hypoxanthine, and thymidine, or cell culture medium lacking thymidine alone.

"Production" cell culture medium refers to a cell culture medium that is typically used in the transition period of cell culture when exponential growth is coming to an end and protein production takes over the "transition" phase and/or "product" phase and is sufficient to fully maintain the desired cell density, cell viability and/or product titer during this phase.

"Perfusion" cell culture medium refers to a cell culture medium that is typically used in cell cultures maintained by perfusion or continuous culture methods and is sufficient to fully support the cell culture during this process. Perfusion cell culture medium formulations can be richer or more concentrated than basal cell culture medium formulations to accommodate methods used to remove spent culture medium. Perfusion cell culture medium can be used during both the growth phase and the production phase.

The concentrated cell culture medium can contain some or all of the nutrients required to maintain the cell culture; in particular, the concentrated culture medium can contain nutrients identified or known to be consumed during the production phase of the cell culture. The concentrated culture medium can be based on almost any cell culture medium formulation. Such concentrated feed medium can contain, for example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even 1000X their normal amounts of some or all of the components of the cell culture medium.

Components used to prepare cell culture media can be completely ground into a powdered medium preparation; partially ground with a liquid supplement and added to the cell culture medium as needed; or added to the cell culture medium in its entirety in liquid form.

Cell culture can also be supplemented with independent concentrated feeds of specific nutrients that may be difficult to prepare or that are rapidly depleted in cell culture. Such nutrients can be amino acids, such as tyrosine, cysteine and/or cystine (see, for example, WIPO Publication No. 2012/145682). In one embodiment, independently to a cell culture grown in a cell culture medium containing tyrosine, a concentrated tyrosine solution of feed is added so that the concentration of tyrosine in the cell culture is no more than 8mM. In another embodiment, independently to a cell culture grown in a cell culture medium lacking tyrosine, cystine or cysteine, a concentrated solution of feed tyrosine and cystine is added. The independent feed can be started before the production phase or at the beginning of the production phase. The independent feed can be realized by the batch feed of the cell culture medium on the same or different day as the concentrated feed medium. The independent feed can also be perfused on the same or different day as the perfusion medium.

"Serum-free" refers to cell culture media that do not contain animal serum, such as fetal bovine serum. In other cases, various tissue culture media (including defined media) are commercially available. For example, any one or combination of the following cell culture media can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Eagle's Medium, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as the EX-CELL ^™ 300 series (JRH Biosciences, Lenexa, Kansas). Serum-free versions of such media are also available. Depending on the needs of the cells to be cultured and/or the desired cell culture parameters, the cell culture media can be supplemented with additional or increased concentrations of components, such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc.

The term "bioreactor" means any container for the growth of cell cultures. The cell cultures of the present disclosure can be grown in a bioreactor, which can be selected based on the application of the target protein produced by the cells grown in the bioreactor. The bioreactor can be of any size, as long as it is useful for cell culture; typically, the size of the bioreactor is suitable for the volume of the cell culture grown therein. Typically, the bioreactor will be at least 1 liter and can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1500, 2000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more or any volume in between. The internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during the culture period. Those skilled in the art will know and will be able to select a suitable bioreactor for practicing the present invention based on relevant considerations.

"Cell density" refers to the number of cells in a given volume of culture medium. "Viable cell density" refers to the number of viable cells in a given volume of culture medium as determined by a standard viability assay, such as the trypan blue dye exclusion method.

The term "cell viability" means the ability of cells in culture to survive under a given set of culture conditions or experimental variables. The term also refers to the fraction of cells that are alive at a particular time relative to the total number of cells (live and dead) in the culture at that time.

"Packed cell volume" (PCV), also known as "percent packed cell volume" (%PCV), is the ratio of the volume occupied by cells to the total volume of the cell culture, expressed as a percentage (see, Stettler et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33). Packed cell volume is a function of cell density and cell diameter; an increase in packed cell volume can be caused by an increase in cell density or cell diameter, or both. Packed cell volume is a measure of the solids content in a cell culture. Solids are removed during harvest and downstream purification. More solids means more effort to separate the solid material from the desired product during harvest and downstream purification steps. In addition, the desired product can be trapped in the solids and lost during the harvest process, resulting in reduced product yield. Because host cells vary in size and cell cultures can also contain dead and dying cells and other cell debris, packed cell volume is a more accurate way to describe the solids content within a cell culture than cell density or viable cell density. For example, a 2000 L culture with a cell density of 50 x ¹⁰⁶ cells/ml will have very different packed cell volumes depending on cell size. In addition, some cells will increase in size while in a growth-arrested state, thus increasing biomass due to the increase in cell size, making it likely that the packed cell volumes before and after growth arrest will be different.

"Growth arrest," also known as "cell growth arrest," is the moment when cells stop increasing in number or when the cell cycle no longer progresses. Growth arrest can be monitored by measuring the viable cell density of a cell culture. Some cells in a growth-arrested state may increase in size but not in number, so the packed cell volume of a growth-arrested culture may increase. If the cells are not in declining health, growth arrest can be reversed to some extent by reversing the conditions that caused it.

The term "titer" refers to the total amount of a polypeptide or protein of interest (which may be naturally occurring or recombinant) produced by a cell culture in a given volume of culture medium. The titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other volumetric measure) of culture medium. The "cumulative titer" is the titer produced by the cells during the culture process and can be determined, for example, by measuring daily titers and using these values to calculate the cumulative titer.

The term "fed-batch culture" refers to a form of suspension culture and means a method of culturing cells in which additional components are provided to the culture one or more times following the start of the culture process. The components provided typically include nutritional supplements for the cells that have been depleted during the culture process. Additionally or alternatively, the additional components may include supplementary components (e.g., cell cycle inhibitory compounds). The fed-batch culture is typically stopped at some point, and the cells and/or components in the culture medium are harvested and optionally purified.

The terms "integrated viable cell density" or "IVCD" are used interchangeably and mean the average viable cell density during a culture multiplied by the amount of time the culture has been run.

The "cumulative viable cell density" (CVCD) was calculated by multiplying the mean viable cell density (VCD) between two time points by the duration between the two time points. The CVCD was the area under the curve formed by plotting VCD against time.

Description of the cell culture process

During recombinant protein production, it is desirable to have a controllable system that allows cells to grow to a desired density and then shift the physiological state of the cells to a high-productivity state where the cells utilize energy and substrates to produce the target recombinant protein rather than forming more cells in a growth-arrested state. Various methods exist for achieving this goal and include temperature shifts and amino acid starvation as well as the use of cell cycle inhibitors or other molecules that can arrest cell growth without causing cell death.

The production of recombinant protein begins with setting up a mammalian cell production cell culture expressing the protein in a culture dish, culture bottle, culture tube, bioreactor or other applicable container. Typically, a smaller production bioreactor is used. In one embodiment, the bioreactor is 500L to 2000L. In another embodiment, a 1000L-2000L bioreactor is used. The seed cell density used to inoculate the bioreactor can have a positive impact on the recombinant protein level produced. In one embodiment, at least 0.5x10 in serum-free medium is used; ^individual viable cells/mL is to and exceeds 3.0x10; ^individual viable cells/mL is inoculated into the bioreactor. In another embodiment, 1.0x10 is inoculated into ^individual viable cells/mL.

Mammalian cells then undergo an exponential growth phase. Cell culture can be maintained in the absence of supplementary feed until the desired cell density is reached. In one embodiment, cell culture is maintained for up to three days with or without supplementary feed. In another embodiment, the culture can be inoculated at the desired cell density to begin the production phase without a brief growth phase. In any embodiment herein, the conversion from growth phase to production phase can also be initiated by any of the aforementioned methods.

At the transition between the growth phase and the production phase and during the production phase, the percent packed cell volume (%PCV) is equal to or less than 35%. The desired packed cell volume maintained during the production phase is equal to or less than 35%. In one embodiment, the packed cell volume is equal to or less than 30%. In another embodiment, the packed cell volume is equal to or less than 20%. In yet another embodiment, the packed cell volume is equal to or less than 15%. In yet another embodiment, the packed cell volume is equal to or less than 10%.

The transition between the vegetative phase and the production phase and the required viable cell density maintained during the production phase can be different according to the project. It can be determined based on the equivalent packed cell volume from historical data. In one embodiment, the viable cell density is at least about 10x10 ⁶ individual viable cells/mL to 80x10 ⁶ individual viable cells/mL. In one embodiment, the viable cell density is at least about 10x10 ⁶ individual viable cells/mL to 70x10 ⁶ individual viable cells/mL. In one embodiment, the viable cell density is at least about 10x10 ⁶ individual viable cells/mL to 60x10 ⁶ individual viable cells/mL. In one embodiment, the viable cell density is at least about 10x10 ⁶ individual viable cells/mL to 50x10 ⁶ individual viable cells/mL. In one embodiment, the viable cell density is at least about 10x10 ⁶ individual viable cells/mL to 40x10 ⁶ individual viable cells/mL. In another embodiment, the viable cell density is at least about 10x10 ⁶ viable cells/mL to 30x10 ⁶ viable cells/mL. In another embodiment, the viable cell density is at least about 10x10 ⁶ viable cells/mL to 20x10 ⁶ viable cells/mL. In another embodiment, the viable cell density is at least about 20x10 ⁶ viable cells/mL to 30x10 ⁶ viable cells/mL. In another embodiment, the viable cell density is at least about 20x10 ⁶ viable cells/mL to at least about 25x10 ⁶ viable cells/mL, or at least about 20x10 ⁶ viable cells/mL.

Lower packed cell volumes during the production phase help alleviate the dissolved oxygen sparging problem that can hinder higher cell density perfusion cultures. Lower packed cell volumes also allow for smaller culture medium volumes, which allow for the use of smaller culture medium storage containers and can be combined with lower flow rates. Compared to higher cell biomass cultures, smaller packed cell volumes also have less impact on harvesting and downstream processing. All of this reduces the costs associated with producing recombinant protein therapeutics.

Three methods are usually used in the commercial process for producing recombinant proteins by mammalian cell culture: batch culture, fed-batch culture and perfusion culture. Batch culture is a discontinuous method for growing cells in a fixed volume culture medium for a short period of time and then all in the crops. The culture grown using batch method experiences an increase in cell density until reaching maximum cell density, and subsequently, along with the horizontal accumulation of culture medium components consumption and metabolic byproducts (such as lactate and ammonia), viable cell density declines. Conventionally, the results occur when reaching maximum cell density (depending on culture medium formulations, cell lines, etc., generally ^5x106 cells/mL- ^10x106 cells/mL) moment. Batch method is the simplest culture method, but viable cell density is subject to the limitation of nutrient availability, and once cell is in maximum density, culture declines and production reduces. There is no ability to extend the production phase, because waste accumulation and nutrient depletion have caused culture decline rapidly, (usually approximately 3 days to 7 days).

Fed-batch culture improves batch process by providing perfusion or continuous culture medium feed to supplement those culture medium components consumed. Because fed-batch culture always receives additional nutrients in the whole operation process, they have the potentiality of reaching higher cell density (depending on culture medium formulation, cell line, etc., > ^10x106 cells/ml to ^30x106 cells/ml) and increased product titer when compared with batch process. Different from batch process, biphasic culture can be set up and maintained by manipulating feed strategy and culture medium formulation, to distinguish the cell proliferation period reaching required cell density (vegetative phase) and suspended or slow cell growth period (production phase). Like this, compared with batch culture, fed-batch culture has the potentiality of reaching higher product titer. Usually, batch process is used in the vegetative phase and fed-batch process is used in the production phase, but fed-batch feeding strategy can be used in the whole process. However, different from batch process, bioreactor volume is the limiting factor limiting feed amount. In addition, as with batch methods, accumulation of metabolic byproducts can lead to culture decline, which limits the duration of the production phase, which is approximately 1.5 to 3 weeks. Fed-batch culture is discontinuous, and harvesting typically occurs when metabolic byproduct levels or culture viability reach a predetermined level. When compared to batch cultures without fed-batch production, fed-batch cultures can produce larger amounts of recombinant protein. See, for example, U.S. Patent No. 5,672,502.

Perfusion provides the potential improvement that is better than batch process and fed-batch process by adding fresh culture medium and removing spent culture medium simultaneously.Typical large-scale commercial cell culture strategy strives to reach almost one-third to more than 1/2 reactor volume for biomass high cell density, ^60x106 cell/mL-90 (+) ^x106 cell/mL.With perfusion culture, reached＞ ^1x108 individual cell/mL's limiting cell density, and even predicted higher density.Typical perfusion culture starts from the batch culture startup that continues one or two days, subsequently in the whole growth phase of cultivation and production phase, in culture, continuously, progressively and/or intermittently add fresh feed culture medium and remove spent culture medium simultaneously and retain cell and other high molecular weight compound such as protein (based on filter molecular weight cut-off value).The various methods such as sedimentation, centrifugation or filtration can be used to remove spent culture medium, maintain cell density simultaneously.The perfusion flow rate of a part of working volume every day to many multiple working volumes every day has been reported.

An advantage of perfusion methods is that production cultures can be maintained for longer periods than batch or fed-batch methods. However, increased medium preparation, use, storage, and handling are required to support long-term perfusion cultures, particularly those with high cell densities, which also require even more nutrients, and all of this drives production costs even higher compared to batch or fed-batch methods. In addition, higher cell densities can cause problems during production, such as maintaining dissolved oxygen levels and having increased outgassing, which involves providing more oxygen and removing more carbon dioxide, which will lead to more foaming and the need for changes in defoamer strategies; as well as problems during harvesting and downstream processing, where the effort required to remove excess cell material can result in product loss, negating the benefit of increased titer due to increased cell clumps.

Also provided are large-scale cell culture strategies that combine fed-batch feeding during the growth phase followed by continuous perfusion during the production phase.The methods target maintaining the production phase of the cell culture at less than or equal to 35% packed cell volume.

In one embodiment, a fed-batch culture with perfusion feeding is used to maintain the cell culture during the growth phase. Perfusion feeding can then be used during the production phase. In one embodiment, perfusion begins when the cells have reached the production phase. In another embodiment, perfusion begins between day 3 or about day 3 and day 9 or about day 9 of the cell culture. In another embodiment, perfusion begins between day 5 or about day 5 and day 7 or about day 7 of the cell culture.

The use of perfusion feeding during the growth phase allows the cells to transition into the production phase, resulting in less reliance on temperature fluctuations as a means of initiating and controlling the production phase, however, a temperature fluctuation of 36°C to 31°C can occur between the growth phase and the production phase. In one embodiment, the fluctuation is from 36°C to 33°C. In another embodiment, the initiation of growth arrest of cells in fed-batch culture can be initiated by exposing the fed-batch culture to a cell cycle inhibitor. In another embodiment, the initiation of growth arrest of cells in fed-batch culture can be achieved by perfusing with a serum-free perfusion medium containing a cell cycle inhibitor.

As described herein, the bioreactor can be inoculated with at least 0.5 x 10 ⁶ viable cells/mL and up to and exceeding 3.0 x 10 ⁶ viable cells/mL in serum-free culture medium, for example 1.0 x 10 6 viable cells/mL.

Perfusion culture is a culture in which a cell culture receives fresh perfusion feed medium while the spent medium is removed. Perfusion can be continuous, stepwise, intermittent, or any combination of any of these. The perfusion rate can be less than one working volume per day to many working volumes per day. The cells remain in the culture and the spent medium removed contains essentially no cells or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture can also be retained in the culture. Perfusion can be achieved by several means including centrifugation, sedimentation, or filtration, see, for example, Voisard et al., (2003), Biotechnology and Bioengineering 82: 751-65. An example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping the culture medium through a hollow fiber filter assembly. See, for example, U.S. Patent No. 6,544,424; Furey (2002) Gen. Eng. News. 22 (7), 62-63.

"Perfusion flow rate" is the amount of culture medium passed through (added and removed from) a bioreactor in a given time, typically expressed as a fraction or multiple of the working volume. "Working volume" refers to the amount of bioreactor volume used for cell culture. In one embodiment, the perfusion flow rate is one working volume per day or less. The perfusion feed medium can be formulated to maximize perfusion nutrient concentration and minimize perfusion rate.

The cell culture can be supplemented with a concentrated feed medium containing components such as nutrients and amino acids consumed during the production phase of the cell culture. The concentrated feed medium can be based on almost any cell culture medium formulation. Such a concentrated feed medium can contain, for example, about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X or even 1000X of the majority of the components of the cell culture medium in their normal amounts. Concentrated feed medium is often used in fed-batch culture processes.

The method according to the present invention can be used to improve the production of recombinant proteins in a multi-stage culture process. In a multi-stage process, cells are cultured in two or more different stages. For example, cells can first be cultivated under the environmental conditions that maximize cell proliferation and vigor with one or more growth phases, and then transferred to the production phase under the conditions that maximize protein production. In the commercial process for producing proteins by mammalian cells, before final production is cultivated, there are usually multiple (for example, at least about 2, 3, 4, 5, 6, 7, 8, 9 or 10) growth phases present in different culture vessels.

One or more transitional periods can precede or separate the growth phase and the production phase. In a multistage process, the method according to the present invention can be adopted at least during the growth phase and the production phase of the final production phase of commercial cell culture, yet it can also be adopted in the pre-growth phase. The production phase can be carried out on a large scale. The volume of at least about 100, 500, 1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters can be used for large-scale processes. In one embodiment, production is carried out in a 500L, 1000L and/or 2000L bioreactor.

The growth phase can exist at a higher temperature than the production phase. For example, the growth phase can exist at a first temperature of about 35°C to about 38°C, and the production phase can exist at a second temperature of about 29°C to 37°C, optionally about 30°C to about 36°C or about 30°C to about 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylenebisacetamide (HMBA), can be added while, before, and/or after the temperature change. If inducers are added after the temperature change, they can be added one to five days after the temperature change, optionally one to two days after the temperature change. When the cells produce the desired protein, the cell culture can be maintained for several days or even weeks.

Any analytical technique known in the art can be used to monitor and evaluate samples from cell cultures. Various parameters including recombinant protein and culture medium quality and characteristics can be monitored during the culture period. Samples can be intermittently obtained and monitored at a desired frequency, including continuous monitoring in real time or near real time.

Typically, the cell culture before the final production culture (N-x to N-1) is used to produce the seed cells that will be used to inoculate the production bioreactor (N-1 culture). The seed cell density can have a positive impact on the recombinant protein level produced. Product levels tend to rise along with increasing seed density. The improvement in titer is not only subject to higher seed density, but is also likely to be subject to the influence of the metabolic state and cell cycle state of the cells in production.

Seed cells can be produced by any culture method. One such method is perfusion culture using alternating tangential flow filtration. Alternating tangential flow filtration can be used to operate the N-1 bioreactor to provide high-density cells to inoculate the production bioreactor. The N-1 stage can be used to grow cells to a density of >90x ¹⁰⁶ cells/mL. The N-1 bioreactor can be used to produce perfusion seed cultures or can be used as rolling seed stock cultures, which can be maintained to inoculate multiple production bioreactors with high seed cell density. The growth phase duration of production can range from 7 to 14 days and can be designed to maintain exponential growth of cells before inoculating the production bioreactor. Optimize perfusion rate, culture medium formulation, and timing to allow cells to grow and deliver them to the production bioreactor in a state that is most beneficial to optimizing their production. For inoculating the production bioreactor, a seed cell density of > ^15x106 cells/mL can be achieved. Higher seed cell densities during inoculation can reduce or even eliminate the time required to reach the desired production density.

The present invention has found particular utility in regulating the presence and/or amount of glycosylation of recombinant proteins. The cell lines (also referred to as "host cells") used in the present invention are genetically engineered to express polypeptides of commercial or scientific interest. Cell lines are typically derived from lineages derived from primary cultures that can be maintained in culture for an indefinite period of time. Genetically engineered cell lines involve transfection, transformation, or transduction of the cells with recombinant polynucleotide molecules and/or additional alteration (e.g., by homologous recombination and gene activation or fusion of recombinant and non-recombinant cells) to cause the host cells to express the desired recombinant polypeptide. Methods and vectors for genetically engineering cells and/or cell lines to express a polypeptide of interest are well known to those skilled in the art; for example, Current Protocols in Molecular Biology , Ausubel et al., eds. (Wiley and Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); and Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69, describe various techniques.

Animal cell lines are derived from progenitor cells that are derived from multicellular animals. A type of animal cell line is a mammalian cell line. Various mammalian cell lines suitable for growing in culture are purchased from the American Germplasm Collection Center (Manassas, Va.) and commercial suppliers. Examples of cell lines commonly used in industry include VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells are widely used to produce complex recombinant proteins, such as cytokines, coagulation factors, and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988), J. Biol Chem 263:6352-6362; McKinnon et al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990), J. Immunol. 145:3011-3016). Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77: 4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because an efficient DHFR selectable and amplifiable gene expression system allows high-level recombinant protein expression in these cells (Kaufman RJ (1990), Meth Enzymol 185: 537-566). In addition, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and the proteins recombinantly expressed in them have been extensively characterized and have been approved by regulatory agencies for use in clinical commercial production.

On the other hand, the present invention provides host cells into which recombinant expression vectors have been introduced. The host cell can be any prokaryotic cell (e.g., Escherichia coli (E. coli)) or eukaryotic cell (e.g., yeast, insect or mammalian cells (e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Many transfection methods are known in the art and include the use of lipids (e.g., ), calcium phosphate, cationic polymers, DEAE-dextran, activated dendrimers, and magnetic beads. Additional transfection methods utilize instrument-based technology. Examples include electroporation, biolistic techniques, microinjection, and laser transfection/light injection, which uses light (e.g., laser) to introduce nucleic acid into host cells.

For the stable transfection of mammalian cells, it is well known that, according to the expression vector and transfection technique used, only a fraction of cells can be integrated into their genome by foreign DNA. In order to identify and select these integrants, usually the gene encoding selective marker (for example, for antibiotic resistance) is introduced into the host cell together with the target gene. Preferred selective markers include those selective markers that give resistance to drugs such as G418, hygromycin and methotrexate. In alternative methods, the cell (for example, the cell having been incorporated into the selective marker gene will survive, and other cells die) of the nucleic acid stably transfected can be identified by drug selection.

Target protein

The methods of the present invention can be used to cultivate cells expressing a target recombinant protein. The expressed recombinant protein can be secreted into the culture medium from which it can be recovered and/or collected. In addition, proteins from such cultures or components (e.g., from the culture medium) can be purified or partially purified using known methods and products obtained from commercial suppliers. The purified protein can then be "formulated" (meaning buffer exchange), sterilized, packaged in batches, and/or packaged for end users. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th edition, 1995, Mack Publishing Company, Easton, PA.

Examples of polypeptides that can be produced by the methods of the present invention include proteins comprising an amino acid sequence that is identical or substantially similar to all or part of one of the following proteins: tumor necrosis factor (TNF), flt3 ligand (WO 94/28391), erythropoeitin, thrombopoeitin, calcitonin, IL-2, angiopoietin-2 (Maisonpierre et al. (1997), Science 277(5322):55-60), receptor activator of NF-κB ligand (RANKL, WO 01/36637), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 01/36637), and TNF-related apoptosis-inducing ligand (TRAIL, WO 01/36637). 97/01633), thymic stromal lymphopoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF, Australian Patent No. 588819), mast cell growth factor, stem cell growth factor (U.S. Patent No. 6,204,363), epidermal growth factor, keratinocyte growth factor, megakaryote growth and development factor, RANTES, human fibrinogen-like 2 protein (FGL2; NCBI Accession No. NM_00682; Rüegg and Pytela (1995), Gene 160:257-62), growth hormone, insulin, insulinotropic, insulin-like growth factor, parathyroid hormone, interferons including alpha-interferon, gamma-interferon and consensus interferon (U.S. Patent Nos. 4,695,623 and 4,897471), nerve growth factor, brain-derived neurotrophic factor, synaptotagmin-like protein (SLP 1-5), neurotrophin-3, glucagon, interleukin, colony stimulating factor, lymphotoxin-B, leukemia inhibitory factor and oncostatin-M. Descriptions of proteins that can be produced according to the methods of the present invention can be found, for example, in Human Cytokines: Handbook for Basic and Clinical Research, complete volume (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge, MA, 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993); and The Cytokine Handbook, Volumes 1 and 2 (Thompson and Lotze, eds., Academic Press, San Diego, CA, 2003).

In addition, the methods of the present invention can be used to produce proteins comprising all or part of the amino acid sequence of a receptor for any of the above-mentioned proteins, antagonists of such receptors or any of the above-mentioned proteins, and/or proteins that are substantially similar to such receptors or antagonists. These receptors and antagonists include: two forms of tumor necrosis factor receptor (TNFR, also known as p55 and p75, U.S. Patent No. 5,395,760 and U.S. Patent No. 5,610,279), interleukin-1 (IL-1) receptor (type I and type II; European Patent No. 0460846, U.S. Patent No. 4,968,607 and U.S. Patent No. 5,767,064), IL-1 receptor antagonist (U.S. Patent No. 6,337,072), IL-1 antagonist or inhibitor (U.S. Patent Nos. 5,981,713, 6,096,728 and 5,075,222), IL-2 receptor, IL-4 receptor (European Patent No. 0367, 566 and U.S. Pat. No. 5,856,296), IL-15 receptor, IL-17 receptor, IL-18 receptor, Fc receptor, granulocyte-macrophage colony-stimulating factor receptor, granulocyte colony-stimulating factor receptor, receptor for oncostatin-M and receptor for leukemia inhibitory factor, receptor activator of NF-κB (RANK, WO 01/36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No. 6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3 and 4), and receptors containing a death domain, such as Fas or apoptosis-inducing receptor (AIR).

Other proteins that can be produced using the present invention include proteins comprising all or part of the amino acid sequence of differentiation antigens (also referred to as CD proteins) or their ligands or proteins that are substantially similar to either of these two proteins. Such antigens are disclosed in Leukocyte Typing VI (Proceedings of the VIth International Workshop and Conference, Kishimoto, Kikutani et al., ed., Kobe, Japan, 1996). Similar CD proteins were disclosed in subsequent seminars. Examples of such antigens include CD22, CD27, CD30, CD39, CD40, and other ligands (CD27 ligand, CD30 ligand, etc.). Several CD antigens are members of the TNF receptor family, which also includes 41BB and OX40. Ligands are typically members of the TNF family, as are 41BB ligands and OX40 ligands.

The present invention can also be used to produce enzymatically active proteins or their ligands. Examples include proteins comprising all or part of one of the following proteins or their ligands, or proteins that are substantially similar to either of these: members of the disintegrin and metalloproteinase domain families, including TNF-α converting enzyme, various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein A-I, globin, IL-2 antagonist, alpha-1 antitrypsin, ligands for any of the above enzymes, and numerous other enzymes and their ligands.

Examples of antibodies that can be produced include, but are not limited to, those that recognize any one or a combination of proteins including, but not limited to, the proteins listed above and/or the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-1 receptor, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor subunit, FGL2, PDGF-β and its analogs (see, U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF, TGF-β2, TGF-β1, EGF receptor (see, U.S. Pat. No. 6,235,883), VEGF receptor, hepatocyte growth factor, osteoprotegerin ligand, interferon gamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1 and zTNF4; see, Do and Chen-Kiang (2002), Cytokine Growth Factor Rev. .13(1):19-25), C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene product expressed in association with lung cancer), HER-2, HER-3, tumor-associated glycoprotein TAG-72, SK-1 antigen, tumor-associated epitopes present at elevated levels in the serum of patients with colon cancer and/or pancreatic cancer, in breast, colon, squamous cell, prostate, pancreatic, lung and/or renal cancer cells and/or melanoma, Cancer-associated epitopes or proteins expressed on glioma or neuroblastoma cells, tumor necrosis center, integrin α4β7, integrin VLA-4, integrins (including integrins containing α4β7), TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α, adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukocyte integrin adhesin, platelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa tissue factor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic T lymphocyte-associated antigen), Fc-gamma-1 receptor, HLA-DR 10β, HLA-DR antigen, sclerostin, L-selectin, respiratory syncytial virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcus mutans, and Staphlycoccus aureus.

Specific examples of known antibodies that can be produced using the methods of the present invention include, but are not limited to, adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab, bapinezumab, basiliximab, belimumab, brianuzumab, brodalumab, canakinumab, becerolizumab, cetuximab, conatumumab, denosumab, eculizumab, etrolizumab, evolocumab, gemtuzumab ozogamicin, golimumab, and nab. olimumab), ibritumomab tiuxetan, labetuzumab, mapatumumab, matuzumab, mepolizumab, motuzumab, muromonab-CD3, natalizumab, palivizumab, ofatumumab, omalizumab, oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab, ranibizumab, rituximab, rovizumab, tocitumomab, tositumomab, trastuzumab, ustekinumab, vedolizomab, zalumab, and zalumab.

The present invention can also be used to produce recombinant fusion proteins comprising, for example, any of the above-mentioned proteins. For example, the methods of the present invention can be used to produce recombinant fusion proteins comprising one of the above-mentioned proteins plus a multimerization domain such as a leucine zipper, coiled-coil, Fc portion of an immunoglobulin, or a substantially similar protein. See, for example, WO94/10308; Lovejoy et al. (1993), Science 259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury et al. (1994), Nature 371:80-83; Harbury et al. (1999), Structure 7:255-64. Specifically included in such recombinant fusion proteins are proteins in which a portion of a receptor is fused to the Fc portion of an antibody such as etanercept (p75 TNFR:Fc), abatacept, and belatacept (CTLA4:Fc).

The scope of the present invention is not limited by the specific embodiments described herein, which are intended as illustrations of individual aspects of the present invention, and functionally equivalent methods and components constitute aspects of the present invention. Indeed, various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Example

Example 1

A Chinese hamster ovary (CHO) cell line expressing a recombinant human antibody (MAb A) that previously demonstrated low levels of high mannose (HM) expression was used for siRNA experiments. The cell line was obtained using a DHFR-based selection clone; for routine culture, cells were suspended in selective medium containing methotrexate (MTX). Cultures were maintained in vented 125 mL or 250 mL Erlenmeyer shake flasks (Corning LifeSciences, Lowell, MA) or vented 50 mL spin tubes (TPP, Trasadingen, Switzerland) at 36°C, 5% _CO2 , and 85% relative humidity. The Erlenmeyer flasks were shaken at 120 rpm with a 25 mm orbital diameter in a large-capacity automated _CO2 incubator (Thermo Fisher Scientific, Waltham, MA), and the spin tubes were shaken at 225 rpm with a 50 mm orbital diameter in a large-capacity ISF4-X incubator (Kuhner AG, Basel, Switzerland).

Eight different 19-mer siRNAs were tested for Mgat1, Mgat2, and Slc35a2. siRNAs were transiently transfected into the MAb A cell line using RNAiMAX (Invitrogen; Life Technologies; a lipid-based transfection reagent that complexes with nucleic acids and facilitates transfection of cells with nucleic acids) according to the manufacturer's protocol. Briefly, cells were seeded at 2×10 ⁵ cells per well on Corning six-well plates using 500 μl of culture medium on the day of transfection. For transfection, 10 pmol of siRNA was complexed with 1.5 μl of Lipofectamine RNAiMAX in 100 μl of Opti-Mem I medium and incubated at room temperature for 10 minutes. The RNAi-Lipofectamine RNAiMax reagent complex was added to each well. The plates were incubated at 36°C in a CO ₂ incubator for 3 days. The cells were lysed at 50°C for 1 hour using 1X lysis buffer (Affymetrix Inc., Sanata Clara, CA). Lysates were used for mRNA expression analysis by multiplexed assay using the FLEXMAP system (Luminex, Austin TX; protein and genomic multiplexed bead array assay).

For mRNA expression analysis, the QuantiGene Plex 2.0 reagent system (Affymetrix Inc., Santa Clara, CA) was used. Briefly, 1X lysis buffer (QS0100) (Affymetrix Inc., Santa Clara, CA) supplemented with protein kinase K (stock concentration 50 mg/mL) was used to lyse cell pellets from 5 x 10 ⁵ living cells and incubated at 50° C. for 1 hour. Cell lysates were stored at −80° C. until ready for use. Gene-specific probe sets (Affymetrix, Inc. Santa Clara, CA) targeting Mgat1, Mgat2, and Slc35a3 as well as normalization genes were used. Frozen lysates were thawed and processed using Affymetrix's standard protocol for mRNA expression analysis.

Data derived from measuring the median reporter gene fluorescence from 100 beads per well per gene were determined and expressed as median fluorescence intensity (MFI). Background signal was determined in the absence of target mRNA from a blank sample and subtracted from the signal obtained in the presence of target mRNA. Fluorescence intensities of target genes were normalized to two housekeeping genes: GusB and TBP.

The assay intensity for each target RNA was assessed by determining the limit of detection (LOD), defined as the target concentration at which the signal is three standard deviations above background. The coefficient of variation (CV) measures the precision of the assay and is the ratio of the standard deviation to the mean.

All samples were run in triplicate, and lysis buffer was used as a blank. Samples were analyzed using a Luminex Bio-Plex 3D microplate reader, and data were acquired using Bio-Plex Data Manager 5.0 (Bio-Rad Laboratories, Hercules, CA).

: For each gene, selection reaches two kinds of siRNA that＞85% knocks out without any remarkable off-target effect.Then selected siRNA is transfected in MAb A cell line, and carries out 10 days fed-batch antibody production operation.In fed-batch production research, cell is seeded in production culture medium with 3.5x10 ⁵ cells/mL.3mL working volume is used for 24 deep well plates (Axygen Scientific, Union City, CA), or 25mL working volume is used for the shake flask that 125mL is emptied.At the 3rd day, the 6th day and the 8th day, the rapid perfusion feed of the initial culture volume of culture feed supplement 7% is carried out.At the 3rd day, the 6th day and the 8th day, the target of glucose feed supplement is to 10g/L.At the 10th day of production operation, gather in the crops and centrifugal conditioned medium. Samples were also taken on days 3, 6, 8, and 10 for growth, viability, and metabolic data, and on days 6, 8, and 10 for titer and HM analysis.

Cell pellet samples were removed from culture on days 3, 6, 8, and 10 for mRNA expression analysis; the results are shown in Table 1 below.

Table 1: Reduction of Mgat1, Mgat2, and Slc35a2 levels by siRNA treatment

mRNA expression analysis showed that Mgat1 and Mgat2 transcript levels decreased by >50% over the 10-day culture operation. Slc35a2 expression levels decreased by >50% from day 3 to day 6, while only a 7%-14% decrease was seen by day 8 and day 10.

Antibody titer and %HM were measured on the tenth day of fed batch. Titer was measured by using Waters UPLC using affilidin A POROS PA ID sensor box. Caliper GX II HM was tested (Caliper Life Sciences Inc., PerkinElmer company) or UPLC HILIC (hydrophilic interaction chromatography) (Waters Acquity UPLC equipped with UPLC Fluorescence (FLR) detector used with Acquity UPLC BEH Glycan Column) was used to measure high mannose content. Table 2 below shows the result (results are expressed as mean value plus/minus standard deviation).

Table 2: Titers and %HM of cell lines treated with siRNA

sample Potency (g/L) %HM No siRNA 0.75±0.09 0.81±0.07 siRNA MGAT1 0.73±0.05 72.42±3.28 siRNA MGAT2 0.87±0.27 1.98±0.31 siRNA Slc35A2 0.57±0.36 1.56±0.29

Analysis of these results showed that HM levels were reduced by 70% in cells treated with Mgat1 siRNA, whereas knockdown of Mgat2 or Slc35a2 did not significantly affect HM. However, by day 10, Slc35a2 levels had recovered to 90% of control values, making it impossible to exclude a role for this gene in regulating HM for this experiment. No significant changes in the titer of the antibodies produced were observed with siRNA treatment. Furthermore, siRNA treatment did not appear to affect productivity or cell viability, suggesting that the elevated HM levels observed in the presence of reduced Mgat1 mRNA expression are likely directly related to reduced Mgat1 activity.

Example 2

A Chinese hamster ovary (CHO) cell line expressing a recombinant human antibody (MAb B) that has previously shown high levels (i.e., >10%) of high-mannose glycans was used for transfection experiments. The cell line was derived using DHFR-based selective cloning; for routine culture, cells were suspended in selective medium containing MTX. Cultures were maintained in vented 125 mL or 250 mL Erlenmeyer shake flasks (Corning Life Sciences, Lowell, MA) or vented 50 mL spin tubes (TPP, Trasadingen, Switzerland) essentially as described previously.

MAb B cells were transfected with any of the following vectors: a null expression vector control, a bicistronic expression vector containing Mgat1 and Mgat2 linked with furin pep2A (M1M2), a vector containing Slc35a2 (S), or a co-transfected Mgat1, Mgat2, and Slc35a2 vector (M1M2S). After the cells recovered to greater than 80% viability in selective medium, they were single-cell cloned using flow cytometry. For those cell lines derived from single cells, the expression level of the target gene was analyzed. For each of the four different vectors, the expression of the target gene in configurations of greater than 40 clones was analyzed and based on this analysis, twenty overexpression cell lines and ten control cell lines were selected for further characterization in two independent 10-day fed-batch production runs performed essentially as described previously.

At the start of the first fed-batch production, on average, the overexpressing cell lines had higher doubling times (>25 hours) and lower PDL compared to the controls with higher PDL (<25 hours) ( FIG1 ). Therefore, a second 10-day fed-batch experiment was performed for a smaller subset of clones that all showed similar doubling times ( FIG2 ).

Statistical analysis of mRNA expression in selected clones from the second fed-batch experiment confirmed that the fold change in overexpression compared to the control was significant. The Mgat2 gene had a greater fold increase in expression than the Mgat1 gene. Table 3 below shows the results.

Table 3: Average fold increase in Mgat1, Mgat2 and Slc35a2 transcript levels for three groups of overexpressing clones.

sample Mgat1 Mgat2 Slc35A2 M1M2 6.24 26.29 0.99 M1M2S 1.45 7.76 37.28 S 0.83 0.99 27.1

To further investigate the induced overexpression levels, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to quantify the protein levels of Mgat1 and Mgat2. A control cell line transfected with an empty vector (EV) and two lines (B1 and B2) overexpressing Mgat1 and Mgat2 were analyzed. Table 4 below shows the results. Relative protein expression was measured in parts per million (ppm).

Table 4: Normalized mRNA and protein levels in the control cell line and two cell lines overexpressing Mgat1 and Mgat2 at day 10

As shown in Table 4, EVs exhibited basal expression levels of Mgat1 and Mgat2, whereas expression levels of these proteins were significantly elevated in the B1 and B2 cell lines. Furthermore, Mgat2 showed elevated expression levels compared to Mgat1 in both B1 and B2 cell lines. These data correlated well with those observed for mRNA expression levels of Mgat1 and Mgat2 at day 10.

In contrast, the expression level of the housekeeping control protein (GAPDH; data not shown) remained constant for the three cell lines on days 8 and 10 (two days analyzed for protein expression).

Figures 3, 4, and 5 show that growth and specific productivity were similar between the groups; however, titers were significantly increased for clones overexpressing Mgat1 and 2 compared to the other two groups. Cell lines overexpressing Mgat1 and Mgat2 (M1M2) and those overexpressing all three genes (M1M2S) showed a reduction in high mannose levels compared to control cells (70% and 29%, respectively) (Figure 6). However, cell lines overexpressing Slc35a2 (S) did not show statistically significant changes when compared to controls (Figure 6). Because the role of the UDP-galactose transporter (protein encoded by Slc35A2) is to transport nucleotide sugar substrates (including UDP-GlcNAc) to the Golgi lumen, if UDP-GlcNAc levels are restrictive, elevated Slc35a2 levels will not affect subsequent glycan processing.

The glycoform characteristics of secreted recombinant MAb B were evaluated for each overexpressing cell line (M1M2, M1M2S and S). Compared to the control cell line, the M1M2 cell line showed a significant reduction in all HM classes such as M5, M6, M7 and M8b. The results are shown in Table 5 below.

Table 5: Glycosylation profile of antibodies produced in cell lines overexpressing Mgat1, Mgat2 and/or Slc35a2 at day 10 of fed-batch

In the control empty vector cell line, A2G0F was the predominant species among the eight complex glycan species evaluated (49.17%), followed by A2G1F (16.46%) and other complex glycoforms. Similar trends were seen for the percentages of the different species for M1M2 and M1M2S. However, in the case of the M1M2 cell line, the amount of the A2G0F glycoform increased significantly by 27% compared to the control (significance value p = 0.0076). This suggests that with the expression of Mgat2, there is an efficient conversion of the hybrid glycan (A1G0M5) to A2G0, and therefore more substrate is available for fucosyltransferase 8 (Fut8) to produce more A2G0F product compared to the control cell line. Although overexpression of Slc35a2 did not appear to cause a significant increase in complex glycan levels in this experiment, these results suggest that overexpression of Mgat1 and Mgat2 can increase the conversion of HM glycans to complex glycoforms, thereby reducing HM levels.

Example 3

CHO host cell lines were transfected with either Mgat1 or Mgat2 alone, or co-transfected with Mgat1 and Mgat2 expression vectors. After these cells recovered to greater than 80% viability in selective medium, they were single-cell cloned using flow cytometry. A total of 291 clones were analyzed for expression of the Mgat1 and Mgat2 genes. Of these, 48 clones expressing Mgat1 and Mgat2 levels above those detected in recombinant CHO cell lines expressing a human monoclonal antibody (MAb A) with previously low levels (i.e., <5%) of high mannose-type glycans were selected based on good growth and viability.

All 48 clones were grown for at least 60 PDL (population doublings) and analyzed for Mgat 1 and Mgat 2 mRNA expression levels at three different time points during this time course. Sixteen clones were selected based on the stable expression levels of their respective mRNAs. To further assess the transfection ability of these clones, all 48 clones were transiently transfected with a vector containing green fluorescent protein (GFP) in a protein fragment complementation assay essentially as described by Remy and Michnick (1999), Proc. Natl. Acad. Sci., 96: 5394-5399.

Seven clones exhibiting the highest transfection efficiency were selected for further analysis.Table 6 shows the Mgat1 and Mgat2 mRNA fold changes for the top seven clones relative to control CHO.

Table 6: Average fold increase in transcript levels of Mgat1 and Mgat2

A monoclonal antibody (MAb C) that has previously demonstrated high levels (i.e., >15%) of high mannose-type glycans was used for transfection in engineered host cells overexpressing Mgat1 and/or Mgat2 or control non-engineered CHO host cells. As previously described, stable cell populations were created and selected for further characterization in a 10-day fed-batch production run. The glycoform properties and titer of secreted recombinant MAb C were assessed for each overexpressing cell line. Significantly lower levels of high mannans were detected in host 38C2 for MAb C compared to the control host, without compromising productivity (i.e., titer). Table 7 shows the results; values reflect day 10 titers and glycan levels obtained from the fed-batch production trials.

Table 7: Titer and glycosylation profile of non-amplified clones expressing MAb C

Similarly, 150nM and 300nM amplification groups were formed and analyzed in a 10-day fed-batch production experiment. In the case of the 150nM group, all overexpressing host cells showed significantly reduced high mannose % compared to the control host cells, without affecting titer. Tables 8 and 9 show the results.

Table 8: Potency and glycosylation characteristics of amplified clones expressing MAb C at 150 nM

Table 8 continued

*Indicates significant P value

As shown in Table 9, with 300 nM amplification, significantly reduced levels of high mannan levels were detected for five populations (61A9, 45F2, 63C5, 38C2, and 2B8).

Table 9: Potency and glycosylation characteristics of 300 nM amplified MAb C

Table 9 continued

*Indicates significant P value

These results indicate that host cells transformed to overexpress Mgat1 and/or Mgat2 can be used to produce recombinant proteins with increased conversion of HM glycans to complex glycoforms, and thus reduce HM levels.

Claims (30)

1. A method for regulating high mannose content of recombinant proteins during mammalian cell culture, comprising transfecting mammalian host cells to overexpress proteins involved in the N-glycosylation pathway, wherein the proteins involved in the N-glycosylation pathway are selected from Mgat1 and/or Mgat2; and Mgat1, Mgat2 and Slc35a2. 2. The method of claim 1, wherein the host cell has been previously transfected to express the recombinant protein and subsequently transfected to express a protein involved in the N-glycosylation pathway. 3. The method of claim 1, wherein the host cell is first transfected with a protein involved in the N-glycosylation pathway, and then transfected to express the recombinant protein. 4. The method according to claim 1 or 2, wherein the recombinant protein is a protein containing an antibody Fc region. 5. The method according to claim 1 or 2, wherein the recombinant protein is an Fc fusion protein. 6. The method according to claim 1 or 2, wherein the recombinant protein is an antibody. 7. The method according to claim 1 or 2, wherein the recombinant protein is an immunoglobulin. 8. The method according to claim 1 or 2, wherein the recombinant protein is a peptide body. 9. The method of claim 1 or 2, wherein the high mannose content of the recombinant protein expressed in cells transfected to overexpress proteins involved in the N-glycosylation pathway is reduced compared to the recombinant protein produced from cultures whose cells have not been manipulated by transfection to overexpress proteins involved in the N-glycosylation pathway. 10. The method according to claim 1 or 2, wherein the high-mannose glycotype is selected from mannose 5 (Man5); mannose 6 (Man6); mannose 7 (Man7); mannose 8 (Man8); or mannose 9 (Man9); and combinations thereof. 11. The method according to claim 9, wherein the high mannose content of the recombinant protein is controlled to be less than or equal to 10%. 12. The method according to claim 9, wherein the high mannose content of the recombinant protein is controlled to be less than or equal to 5%. 13. The method according to claim 1 or 2, wherein the mammalian cell culture process utilizes fed-batch culture, perfusion culture, or a combination thereof. 14. The method of claim 13, wherein an alternating tangential flow (ATF) perfusion culture is used. 15. The method of claim 14, wherein perfusion begins from day 1 to day 9 of cell culture. 16. The method of claim 14, wherein perfusion begins from day 3 to day 7 of cell culture. 17. The method according to any one of claims 14-16, wherein perfusion begins when the cells have reached the production stage. 18. The method of claim 17, wherein the perfusion is accomplished by using an ultrafiltration or microfiltration device with alternating tangential flow. 19. The method of claim 13, wherein the cell culture is maintained by feeding in batches. 20. The method of claim 19, wherein the culture is fed three times during production. 21. The method of claim 20, wherein the culture is fed on a day between the second and fourth day, a day between the fifth and seventh day, and a day between the eighth and tenth day. 22. The method of claim 19, wherein the culture is fed four times during production. 23. The method of claim 22, wherein the culture is fed on a day between the second and fourth day, a day between the fifth and sixth day, a day between the seventh and eighth day, and a day between the eighth and tenth day or later. 24. The method of claim 13, wherein the regulated high mannose content is reduced compared to mammalian host cells not manipulated by transfection to overexpress proteins involved in the N-glycosylation pathway. 25. The method of claim 1 or 2, wherein the mammalian cell culture is established by inoculating a bioreactor with at least 0.5 x ^10⁶ cells/mL to 3.0 x ^10⁶ cells/mL in a serum-free medium. 26. The method according to claim 1 or 2, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. 27. The method of claim 25, wherein the method reduces the high mannose content of the recombinant protein. 28. The method according to claim 1 or 2, wherein the method further comprises a harvesting step for the recombinant protein. 29. The method of claim 28, wherein the harvested recombinant protein is purified and formulated into a pharmaceutically acceptable formulation. 30. The purified and formulated recombinant protein obtained by the method of claim 29.