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WO1995008571A1 - Somatotropine porcine ayant une stabilite accrue et son procede de production - Google Patents

Somatotropine porcine ayant une stabilite accrue et son procede de production Download PDF

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Publication number
WO1995008571A1
WO1995008571A1 PCT/US1994/010740 US9410740W WO9508571A1 WO 1995008571 A1 WO1995008571 A1 WO 1995008571A1 US 9410740 W US9410740 W US 9410740W WO 9508571 A1 WO9508571 A1 WO 9508571A1
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Prior art keywords
pst
sodium phosphate
porcine somatotropin
glu
eluted
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Inventor
David F. Johnson
Patrick C. Mowery
Vivian Doelling
Ursula Eul
Roger Kriegl, Jr.
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Wyeth Holdings LLC
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American Cyanamid Co
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Priority to AU78771/94A priority Critical patent/AU7877194A/en
Publication of WO1995008571A1 publication Critical patent/WO1995008571A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin

Definitions

  • Porcine somatotropin is a growth-stimulating hormone produced by the anterior pituitary gland in pigs.
  • pST Porcine somatotropin
  • mutant proteins muteins
  • selected amino acids in the primary sequence of the protein were converted into amino acids expected to confer greater solubility and/or thermostability.
  • the current invention pertains to methods of producing a purified preparation of porcine somatotropin (pST) , from which impurities associated with destabilization have been removed.
  • the purification methods include: aggregate depletion; diafiltration of aggregated solutions; gel filtration, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography, displacement chromatography, gel electrophoresis, "salting-out” fractionation, or ion exchange.
  • the method is a two-step isocratic ion exchange process, comprising loading a pST preparation onto an ion exchange column, eluting impurities which contribute to instability with a phosphate salt, and then eluting pST with a phosphate salt.
  • the process generally involves the following steps: equilibration of an ion exchange column containing a positively charged ion exchange resin, with phosphate salt buffers, to a pH and conductivity which are optimum for the separation of pST; loading of a pST preparation onto the column; displacement of the loading buffer with a phosphate salt, until the pH and conductivity are equivalent to equilibration conditions; elution of the impurities; and subsequent elution of pST monomer.
  • the invention additionally pertains to pST preparations produced by the purification methods, and particularly by the two-step isocratic exchange methods.
  • the pST preparations have enhanced stability because of the removal of instability-associated impurities. These pST preparations have increased solubility, which results in increased release of pST from implants for improved therapeutic value and cost-effectiveness of pST treatment.
  • Figure 1 is a line graph representation of the decline in soluble protein content following dissolution of six powdered preparations of the porcine somatotropin (pST) analog glu,glu-pST (in which the cysteines at amino acid positions have been replaced with glutamic acid residues) in phosphate buffered saline (PBS) to 100 mg/mL at 43°C.
  • pST analog preparations designated API and AP2, which yield superimposable results
  • ⁇ preparations AP3, AP4 and AP5, which yield mutually superimposable results
  • (•) preparation AP6.
  • Figure 2 is a line graph representation of the decline in soluble protein content following dissolution of the powdered glu,glu-pST preparation AP6 (O) and its aggregate- depleted derivative AP6-AD (•) in PBS to 100 mg/mL at 43°C.
  • Figure 3 is a line graph representation of the decline in soluble protein content following dissolution of the powdered preparations AP6 (glu,glu-pST; ⁇ ) and AP7 (I122L glu,glu-pST, in which the isoleucine at amino acid position 122 in glu,glu-pST is replaced by leucine; o) , their respective neutralized derivatives AP6-N (O) and AP7-N ( ⁇ ) , and their respective neutralized and aggregate-depleted derivatives AP6-NAD (•) and AP7-NAD (A) , each in PBS to 100 mg/mL at 43°C.
  • Figure 4 is a line graph representation of the decline in soluble protein content following dissolution of each of the following preparations in PBS to 100 mg/mL at 43°C: AP6 (glu,glu-pST; O) , its aggregate-depleted derivative AP6-AD ( ⁇ ) , and its repurified derivative AP6-R (•) .
  • Figure 5 is a line graph representation of results showing that the observed decline in soluble protein content over a 14 day period is independent of the initial concentration at which the following preparations are dissolved in PBS at 39°C: AP6 (glu,glu-pST; O) , its aggregate-depleted derivative AP6-AD (•) , and its repurified derivative AP6-R (O) .
  • Figure 6 comprises a series of histograms, individually labelled as 6A, 6B, 6C, and 6D, depicting validation results for quasielastic light scattering (QELS) studies using profilin and phosphatidylinositol-4,5- biphosphate (PIP2) .
  • QELS quasielastic light scattering
  • 6C Profilin.
  • 6D PIP2.
  • Figure 7 comprises a series of histograms, individually labelled as 7A, 7B, IC, and 7D, depicting QELS results, with or without a two-hour incubation at 39°C to induce aggregation, for glu,glu-pST preparation AP6-N and its aggregate-depleted derivative, AP6-NAD, each at 30 mg/mL in 0.1 M NaCl, 0.01 M phosphate buffer, pH 7.2.
  • Figure 8 comprises a series of histograms, individually labelled as 8A, 8B, 8C, and 8D, depicting QELS results, with or without a two-hour incubation at 39°C to induce aggregation, for I122L glu,glu-pST preparation AP7-N and its aggregate-depleted derivative, AP7-NAD, each at 30 mg/mL in the buffer referred to previously.
  • Figure 9 comprises a series of histograms, individually labelled as 9A, 9B, 9C, and 9D, depicting QELS results, with or without a two-hour incubation at 39°C to induce aggregation, for repurified A6T,S11R glu,glu-pST monomer (glu,glu-pST in which the alanine at amino acid position 6 is replaced with threonine, and the serine at position 11 is replaced with arginine) preparation AP8-R and its corresponding pST destabilizing fraction (PDF) AP8- Rpdf, each at 5 mg/mL in the buffer referred to previously.
  • PDF pST destabilizing fraction
  • Figure 10 is a representation of the reaction by which a cysteine bridge in rpST is converted to lanthionine.
  • the current invention pertains to methods of producing a purified porcine somatotropin (pST) preparation, as well as the resulting purified pST preparation.
  • pST porcine somatotropin
  • a minor component comprising lanthionine pST is identified as being associated with decreased stability in pST preparations.
  • lanthionine pST refers to a pST molecule in which a cystine residue has been converted to a lanthionine residue ("intramolecular” lanthionine pST) and/or two pST molecules which are linked by a lanthionine residue ("intermolecular” lanthionine pST) .
  • elution buffers i.e., sodium chloride, sodium phosphate, or sodium acetate
  • elution studies reveal that variant conditions can cause dissimilar impurities, such as lanthionine pST and pST dimer, to co-elute in the same fraction.
  • lanthionine pST, pST dimer and other minor components co- elute in the earlier portion of the gradient with some buffers, such as sodium phosphate and sodium acetate, whereas with other buffers, such as sodium chloride, lanthionine pST and some minor components elute in the earlier portion whereas dimer and other components co-elute with pST monomer.
  • some buffers such as sodium phosphate and sodium acetate
  • other buffers such as sodium chloride
  • These purification methods include processes such as: aggregate depletion of impurities associated with destabilization; diafiltration of aggregated solutions, during which the aggregate initiators are trapped in solution, while the monomeric pST passes through a semipermeable membrane; gel filtration chromatography; hydrophobic interaction chromatography; reverse phase chromatography to remove the suspected deamidation product and other impurities associated with destabilization; affinity chromatography; displacement chromatography in which aggregated impurities associated with distabilization are removed by filtration through a column; electrophoresis; "salting-out” fractionation in which aggregated impurities associated with destabilization are removed by centrifugation and subsequent removal- of the impurities, which fall out of solution; or ion exchange chromatography (see Current Protocols in Molecular Biology, Ausubel et al. (eds) , John Wiley & Sons (1994)).
  • the purification methods also include combinations of these steps.
  • a two-step elution process is used.
  • an ion exchange column is equilibrated with a phosphate salt buffer to establish a pH and conductivity which are optimal for binding and elution of pST.
  • the ion exchange column comprises a column packed with a positively charged ion exchange resin; for example, diethylaminoethyl-Sepharose (DEAE-Sepharose) can be used.
  • DEAE-Sepharose diethylaminoethyl-Sepharose
  • Any phosphate salt buffer such as sodium phosphate or potassium phosphate, can be used.
  • the pH and conductivity resulting from the equilibration of the column are herein referred to as the "equilibrium pH” and "equilibrium conductivity", and are known together as the "equilibrium conditions”.
  • a pST solution with a pH equal to the equilibration pH and a conductivity less than or equal to the equilibration conductivity of the column is loaded onto the column.
  • the column is then re-equilibrated by displacing the loading buffer with a phosphate salt buffer until the pH and conductivity are equivalent to equilibration conditions.
  • Impurities, such as lanthionine pST, are eluted with the phosphate salt at a molarity that is optimum for their elution.
  • pST monomer is eluted with the phosphate salt at a molarity that is optimum for its elution.
  • a column containing a positively charged ion exchange resin is equilibrated with sodium phosphate buffers, such that the equilibrium pH is approximately 6 to 8, and the equilibrium conductivity is equal to that of approximately 5 mM to 20 mM sodium phosphate.
  • the column is loaded with a pST solution with a pH is equal to the equilibrium pH, and a conductivity is less than or equal to the conductivity of the equilibration conductivity.
  • the loading buffer is displaced with sodium phosphate until the pH and conductivity are equivalent to the equilibration conditions.
  • the lanthionine pST, dimer, and other impurities are eluted with approximately 30 to 60 mM sodium phosphate; and the pST monomer is eluted with approximately 70 to 125 mM sodium phosphate.
  • sodium acetate is used in place of sodium phosphate, with appropriate adjustments to the equilibrium conditions and elution buffer concentrations in order to achieve maximum separation of pST from destabilizing minor components.
  • a DEAE-Sepharose column is equilibrated with sodium phosphate buffers to conditions of pH 7.5 and conductivity equaling that of 10 mM sodium phosphate, pH 7.5.
  • the column is loaded with a pST solution of pH 7.5 and a conductivity less than or equal to the conductivity of the original equilibration conductivity.
  • the conductivity can equal that of 1.9 mM sodium phosphate.
  • the loading buffer is displaced with 10 mM sodium phosphate, pH 7.5, until the pH and conductivity are equivalent to the equilibration conditions.
  • Lanthionine pST, dimer, and other impurities are eluted with 35 mM sodium phosphate, pH 7.5; and the pST monomer is eluted with approximately 70 to 125 mM sodium phosphate, pH 7.5.
  • the purification methods including the processes described above, yield a pST preparation, herein referred to as "process-purified pST” or “process-purified pST preparation", that is substantially free of lanthionine pST and other impurities which are associated with instability.
  • a process-purified pST preparation has greater stability (i.e., a higher percentage of pST remains soluble over time, at high concentrations, or at high temperatures) than pST preparations from which lanthionine pST and other instability-associated impurities have not been removed.
  • the process-purified pST preparations yielded by this process are also encompassed by the current invention.
  • Process-purified pST is useful for treatment of pigs to produce leaner meat, by methods such as incorporation into a sustained release formulation which is administered to pigs, for example.
  • thermostability of preparations of recombinant pST (rpST) and analogs of rpST (herein referred to as pST) which reveals that different preparations exhibit different stability, even though the same protein is present in each preparation.
  • aggregate depletion and repurification studies which identify, isolate and describe a destabilizing factor, described herein as a minor component migrating as a 20 kDa protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) , present in the pST preparations.
  • C183,191E-pST (more often referred to herein as glu,glu-pST) is a mutein in which three additional amino acid residues (methionine, aspartic acid and glutamine) are present at the N-terminus, and the cysteines of the small loop disulfide (amino acid positions 183 and 191) are replaced with glutamic acid residues.
  • sequence of glu,glu-pST is included herein as SEQ ID NO. 1.
  • the alanine at amino acid position 6 is replaced with threonine
  • the serine at position 11 is replaced with arginine, in addition to the changes to the N-terminus and positions 183 and 191.
  • Escherichia coli K12 cells containing expression plasmids for pST or for the analogs are fermented and induced to express the desired protein in inclusion bodies.
  • the protein is purified from the inclusion bodies as follows: the E . coli cells are harvested by centrifugation, and the pelleted cells are suspended in buffer, disrupted by ho ogenization, and centrifuged. The pellet resulting from the cell disruption is suspended in buffer, subjected to lysozyme, and centrifuged. The resultant pellets contain the insoluble pST.
  • the pST is then washed and solubilized in an appropriate solubilizing agent.
  • the solubilized pST is subjected to ultrafiltration and concentration.
  • the proteins are then purified by the following process
  • the protein sample is loaded onto an ion exchange column, such as DEAE- Sepharose, which is equilibrated with 10 mM borate, pH 9.0 (equilibration buffer) .
  • the loaded column is washed with three bed volumes (BV) of equilibration buffer, and the pST is subsequently eluted from the column in substantially pure form with 125 mM NaCl, 10 mM borate, pH 9.0 (elution buffer) .
  • the resultant purified material is concentrated, diafiltrated, lyophilized and formulated for commercial use.
  • Stability is assessed by redissolving the protein in an appropriate buffer under controlled temperature conditions and monitoring, by appropriate techniques, its physical or physicochemical behavior over time at the predetermined temperature.
  • the borate process does not result in satisfactory separation of the desired pST monomer from impurities and pST dimer.
  • two different lots of glu,glu-pST purified by the same procedure are found to possess considerable variation in solution stability, particularly at high concentrations of protein. The observed differences in rpST stability are of sufficient magnitude to impinge on the manufacture of a commercial product. Other lots of glu,glu-pST, purified by quite different procedures, exhibit similar variations.
  • preparations of other pST analogs also show variations in stability, often when purified by the same process, regardless of whether purification is carried out on the laboratory or the large scale. That is, the extent of irreversible aggregation is not reproducible from preparation to preparation of a given rpST analog, despite the fact that the purified powders are expected to contain the same species of protein.
  • This instability-associated minor component possesses an electrophoretic mobility (under reducing conditions) consistent with a 20 kDa polypeptide, in contrast to the 22 kDa mobility of the bulk rpST; it is referred to herein as the L component, the L minor component, the destabilizing minor component, the minor component migrating as a 20 kDa band on SDS-PAGE, or the pST destabilizing component, factor or agent.
  • the L component is present in glu,glu- pST, A6T,S11R glu,glu-pST and I122L glu,glu-pST preparations.
  • the solution stability and aggregate depletion procedures employed previously are combined with a pH neutralization step.
  • the powdered samples employed as starting material are slightly basic because they have been exposed to dilute sodium hydroxide during the original purification of expressed pST from inclusion bodies.
  • PBS phosphate buffered saline
  • the final pH of the resulting pST solution is raised to about 9.0, thus providing an environment favorable to deamidation.
  • Neutralization prior to aggregate depletion and solution stability testing thus allows the investigation of other minor components in pST preparations.
  • the results of Study IV further implicate the L component as a pST destabilizing agent by revealing that levels of this component are elevated in aggregated pST fractions and diminished in aggregate-depleted pST preparations.
  • Study V additionally indicates that although the repurified samples acquire an improved solution stability, they do not have the level of stability provided by a long term (two weeks at 43°C) aggregate depletion treatment of the original samples, suggesting that at least one other component is also involved in destabilizing rpST solutions.
  • the stability of the depleted samples and of protein repurified by DEAE-Sepharose chromatography are therefore distinguishable: in no instance does the stability of the repurified pSTs approach the stability of depleted material.
  • Method Aggregate depletion is carried out by dissolving the protein at 200 to 300 mg/mL in PBS, incubating the solutions at 43°C for equivalent periods of time, recovering the soluble protein and desalting by dialysis against 0.1 M ammonium bicarbonate and then freeze drying. This material is then redissolved in PBS and analyzed for solution stability as above. Samples from these depletion studies are compared for the presence of minor components by reverse phase (RP) HPLC.
  • RP reverse phase
  • AP6-AD AP6-AD
  • the present samples are also submitted for biological potency testing in hypophysectomized rats according to the method of Groesbeck and Parlow (1987) , ENDOCRINOLOGY 120: 2582-2590, as modified by Buckwalter et al . (1992), J. AGRI FOOD CHEM 40 (No. 2): 356-362.
  • preparations of pST at various doses are administered by subcutaneous injection to hypophysectomized albino rats (Sprague Dawley derived; Taconic Farms, Germantown, New York) for ten consecutive days.
  • the preparations increase growth in rats; the increased growth, as measured by total weight gain during the test period, is used to determine the relative activity of somatotropin preparations as compared to a standard.
  • results of the biological potency testing indicate that the depleted material (AP6-AP) has an average potency of 1.55 compared to the analytical standard ASP (assigned a potency of 1.0).
  • the starting material (AP6) in this same assay has an average potency of 1.03, indicating that depletion events do not decrease biological activity.
  • the apparent increase in potency (1.55 vs. 1.03) is therefore considered to result from the elimination of improperly folded forms (which could contribute to solution instability) . Alternatively, it is thought that this result might also arise from a deamidation reaction.
  • Reducing SDS-PAGE analysis confirms that the vast majority of protein species present in the pST powder corresponds to a band having an electrophoretic mobility consistent with a 22 kDa protein, as expected.
  • a minor component migrating as a 20 kDa band (the L component) is also noted.
  • levels of the L component in the powdered pST samples seem to reflect the sequence of stability variations.
  • glu,glu-pST sample AP6 is dissolved to about 200 mg/mL in PBS, pH 7.4, and 100 L is dialyzed for 24 hours against PBS.
  • Another sample, AP7 (I122L glu,glu- pST) , is also treated in this fashion for direct comparison. The final pH of these solutions are 7.25 and 7.24 respectively.
  • their respective protein concentrations are 148 mg/mL for AP6 and 153 mg/mL for AP7.
  • a 10 mL portion of each sample is desalted by dialysis against 0.1 M ammonium bicarbonate and then freeze-dried.
  • the resulting neutralized pST powders are designated, respectively, AP6-N and AP7-N.
  • the neutralization step itself appeared to improve stability: 1.3 fold for AP6-N and 1.1 fold for AP7- N.
  • the RP-HPLC suspected deamidation product does not destabilize pST solutions.
  • the suspected deamidated pST is increased in the more stable forms of the samples (i.e. it is not depleted upon aggregation) , and is not substantially enriched in the aggregated material.
  • SDS-PAGE results for these samples indicate that the minor component migrating as a 20 kDa band (the L component) is more than likely involved in destabilization of pST solutions.
  • the column is equilibrated in 10 mM sodium phosphate containing 50 mM NaCl, at either pH 7.0 or 7.5.
  • the flow rates and superficial linear velocities are the same as detailed above.
  • One gram of pST preparation AP8 is solubilized in the equilibration buffer at either pH 7.0 or 7.5. These solutions are very hazy in appearance, suggesting a lower solubility of the pST in the presence of 50 mM NaCl.
  • the column is washed in the equilibration buffer and the remaining pST is eluted in 150 mM NaCl. At pH 7, the estimated column yield is 76.5%.
  • the load onto the column is 16 mg protein/mL resin.
  • the L component is eluted from the column at a superficial linear velocity of 45.9 cm/hour with 10 bed volumes (BV) of 60 mM NaCl in 10 mM sodium phosphate buffer, pH 7.5.
  • BV bed volumes
  • Purified pST is eluted with 150 mM NaCl in 10 mM sodium phosphate to result in approximately 75% yield (Superose® 12 assay) .
  • the pST of preparation AP8 (500 g) is redissolved in 10 mM sodium phosphate, pH 7.5, at an approximate concentration of 5 mg pST/mL.
  • the pST is rechromatographed using a 32 L Pharmacia column packed with DEAE-Sepharose. The column is equilibrated with 10 mM sodium phosphate, pH 7.5.
  • the fraction including the L component is eluted with 10 BV of the phosphate buffer containing 60 mM NaCl.
  • the main fraction is eluted with 150 mM NaCl in the phosphate buffer.
  • Fractions are collected and analyzed by SDS-PAGE and Superose® 12. Fractions low in dimer and L component are pooled, desalted and lyophilized. Final pST yield is 350 g (70%) .
  • Analysis of the final powder by SDS-PAGE shows the reduction of the minor component migrating as a 20 kDa band to about 0.50% from about 2.0% in the original technical powder.
  • AP8-Rpdf represents the pST destabilizing fraction (PDF) from this repurification. This PDF is subsequently added to a more pure sample of A6T,S11R glu,glu-pST (AP9) , which contains only 0.5% of the L component.
  • the resultant sample, designated AP9-SPpdf is made by mixing 30 mg of AP8-Rpdf with 70 mg of AP9, creating a sample having approximately 6% of the L component of pST. Solution stability, RP-HPLC and SDS-PAGE are carried out as described above.
  • the resultant sample is designated AP6-R.
  • results As shown in Table 4, below, the level of the minor L component migrating as a 20 kDa band on SDS-PAGE is reduced from 2.2% to ⁇ 0.25 % upon repurification.
  • the improvement in the solution stability at 43°C for AP8-R relative to AP8 is about 1.3 fold at day 14.
  • the spiked sample AP9-SPpdf exhibits a considerably reduced solution stability (by about 1.5 fold), when compared to the original material, AP9.
  • sample AP6 The repurification of sample AP6 confirms these results: levels of the minor L component in AP6-R are reduced from 5.08% to 1.1%, while this repurified sample shows about a 1.3 fold improved stability over the starting material (see Figure 4) .
  • AP6-NAD has a potency 1.4 fold that of AP6.
  • the rat assay also indicates that the PDF derived from the repurification of AP6 has only 40% of the activity of its parent sample. This residual activity is possibly due to a pST form that copurifies with the L component upon ion-exchange chromatography.
  • Method pST preparation AP6 (glu,glu-pST) and its repurified product, AP6-R, are compared with the depleted sample AP6-NAD in a concentration-dependent stability assay in PBS.
  • Samples of each preparation are dissolved at various concentrations up to 275 mg/mL and incubated at 39°C for 14 days and analyzed by SE-HPLC and SDS-PAGE as described above. Results
  • repurification of the AP6 sample yields a considerable improvement in stability, but this is still not as good as the stability obtained by aggregate depletion.
  • SDS-PAGE of the depleted and repurified samples indicates that the remaining destabilizing component almost certainly has a molecular weight of 22 kDa, as this is the only significant proteinaceous material remaining in the repurified sample.
  • T m Melt transitions
  • Another physical property of pST analogs which bears on stability is the degree of aggregation in a given protein solution.
  • a method that conveniently assesses this property is dynamic light scattering. This method is also termed photon correlation or quasielastic light scattering (QELS) spectroscopy.
  • QELS quasielastic light scattering
  • an intense, monochromatic light an Argon laser
  • Macromolecules diffuse through the light beam and interact by Brownian motion, causing a fluctuation in the intensity of scattered light.
  • a small cross-section of this beam is isolated optically, and the light intensity fluctuations are measured directly via single photon counting.
  • the translational diffusion constants of macromolecules are measured directly, and their sizes are calculated from models describing molecular diffusion.
  • Study VII utilizes latex spheres as a standard; Study . VIII employs profilin, a 15 kDa protein for which the particle size has been reported recently by an independent laboratory, is employed as a standard.
  • the other population has diameters ranging from about 100 nm to 3,000 nm and represents rpST in the highly aggregated states which lead to its precipitation. Aggregate-depleted samples are devoid of these higher aggregate particles, since the destabilizing factor(s) which initiate aggregate nucleation events have been removed.
  • the results of Study XIII support the view that the L component is responsible for promoting the aggregation phenomena by serving as a platform for one or more types of hydrophobic nucleation events. The appearance of the L component during processing could result from several causes and is attributable in part to a covalent modification of the protein, as described below.
  • the measured Brownian motion (diffusion) of the particles is converted to molecular diameter by assuming that the particles are spherical, and that the size distribution is log normal in nature.
  • Fourth order cumulants analysis (herein referred to as cumulants analysis) is used to calculate a mean diameter for the particles.
  • a nonlinear least squares method of analysis (herein referred to as least squares analysis), such as CONTIN (Provencher et al . (1978), J. CHEM. PHYS. 69: 4273-4276; Provencher (1982) , COMP. PHYS. COMMUN. 27: 213-227), provides graphical representations of the particle diameters.
  • Mode refers to the number of divisions in the size distribution of a sample of particles. An ensemble of particles is bimodal, for example, when their size distribution is divided into two populations.
  • Dispersity refers to the uniformity of diameters of particles. A particular modality of particles is polydisperse, for example, if their diameters are non-uniform. A monomodal distribution of particle sizes can be described by the method of cumulants, whereas multimodal systems require more sophisticated nonlinear least squares analyses.
  • the QELS particle size analyses described herein are conducted using a Brookhaven Instruments BI8000 autocorrelator, a BI200SM goniometer, and a 3 watt Lexel 3000 Argon laser.
  • the sample temperature is controlled using a Neslab recirculating bath which regulates the temperature of a dish of decalin in which the sample tube is immersed.
  • the QELS apparatus is tested with suspensions (in PBS at 24°C) of standard latex spheres having mean diameters of 20, 54, or 104 nm (from Duke Scientific) ; or 300 nm (from National Institute of Standards and Technology (NIST) ) .
  • the temperature of each sample is measured using a thermocouple that is calibrated to a NIST-standardized thermometer.
  • One ⁇ sec sample times using 20 channels, and a sample time mode ratio of 1:1.4 (for the spacing of the time channels) are employed. Data are acquired until the scattering incidents counted are greater than IO 7 , and the difference between the measured and calculated baselines is less than 1%.
  • the spheres are found to be monodisperse.
  • the agreement between the label claim and the experimentally measured diameters for 20, 50, and 100 nm diameter spheres is within the claimed precision.
  • the largest error occurs with the 300 nm spheres. This is in part because the laser used has an operating wavelength of 488 nm. Hence, there is error due to refraction as well as scattering of the beam by these large particles. The observed error is tolerated for present purposes, however, as the presence of large particles in solution is of greater interest than the precise measurement of size.
  • Profilin is known to bind phosphatidylinositol-4, 5-bisphosphate (PIP2) . Data are therefore acquired for profilin, PIP2, and profilin-PIP2 mixtures in PBS. QELS analyses of the samples reported herein are made at 25°C in order to assure uniform quality. Results The mean diameter of profilin is calculated to be 4.9 nm, using cumulants analysis. Least squares analysis reveals that virtually all of the profilin material has a mean diameter of 4 nm, with traces having diameters > 500 nm, representing contaminants.
  • the large diameter particles observed in the profilin also clearly are present, and can be removed by filtering the profilin/PIP2 mixture through a l ⁇ filter.
  • least squares analysis such as by CONTIN, provides a reasonable size analysis for the overwhelmingly dominant 4 nm particles, along with information about the existence of large ( > 0.5 ⁇ ) particles in solution.
  • Glu,glu-pST (AP6 or AP6-R) solutions are adjusted to 5 and 10 mg/mL in 0.1 M NaCl, 10 mM phosphate, pH 7.3. Samples are subjected to QELS at 25°C as described above. Then, 10 mg/mL samples are incubated for 2 hr at 39°C, and QELS data are acquired at the higher temperature. Under these conditions, the sample time is 1 ⁇ sec, and data are acquired using 19 channels with a sample time mode ratio of 1:1.8.
  • the mean particle diameters are calculated using cumulants analysis. At 25°C, the distribution of aggregates of glu,glu-pST is found to be monomodal but polydisperse both at 5 and 10 mg/mL protein. Deviations representing 95% confidence intervals are shown below in Table 5. At either protein concentration, the precision of the measurements is better than 3.5%, using twelve degrees of freedom at 10 mg/mL and fourteen degrees of freedom at 5 mg/mL. Following incubation for two hours at 39°C, the mean diameter is 95.8 + 9.2 nm at 95% confidence for six experiments. The data for this study are summarized in Table 5 (below) , along with the results of Studies VII and VIII.
  • Secondary and tertiary structural CD spectra are acquired using parameters acquired initially from recombinant bovine somatotropin (rbST) .
  • the reference standard is rpST (preparation ASP) instead of rbST.
  • the secondary structural CD spectra are identical for rpST and rbST; however, the tertiary structural spectra differ.
  • the molar dichroism at 284 nm is used as the structural criterion instead of that at 292 nm.
  • results The mean particle diameters are calculated using cumulants analysis.
  • the CD analyses and mean diameters measured at 25°C suggest that the aggregate- depleted proteins are folded in their native configurations, and that their states of aggregation in solution are typical of these analogs (see Table 6, below) .
  • the components which accelerate unfolding of these proteins at 39°C in concentrated solutions appear to be present at concentrations which are too low to perturb the physical properties of the native analogs appreciably at room temperature.
  • Samples having protein concentrations of 30 mg/mL and of 5 mg/mL in PBS are prepared from the neutralized, aggregate-depleted preparations of both glu,glu-pST and I122L glu,glu-pST employed previously. The extent of aggregation at 25°C is measured by QELS for each sample. Then, each is incubated at 39°C for two hours before being analyzed again at this elevated t sterature.
  • results The mean particle diameters are calculated using cumulants analysis, and histograms are generated using least squares analysis. The effects of incubation at 30 mg/mL are shown for glu,glu-pST in Figure 7, and for I122L glu,glu-pST in Figure 8. It is readily apparent that the aggregate depletion treatment removes one or more components, resulting in a significant stabilization of the pST analogs.
  • Mean particle diameters at 39°C drop from 3 ⁇ in AP6-N to 9 nm for the depleted sample AP6-NAD. An improvement in stability at 39°C is also seen in AP7 and its aggregate-depleted derivative, AP7-NAD.
  • the improvement is from 123 nm in AP7 to just 7.5 nm in AP7-NAD.
  • Large aggregates of pST ( > 1 ⁇ ) are seen in the original powders, whereas only small aggregates ( ⁇ 8 nm) remain in the aggregate- depleted pST analogs.
  • results The mean particle diameters are calculated using cumulants analysis, and histograms are generated using least squares analysis.
  • QELS data of the repurified monomer at 25°C, shown in Figure 9 provide a mean diameter for the A6T,S11R pST analog comparable to the value measured for glu,glu-pST itself.
  • the data for the PDF demonstrate that this fraction is more prone to aggregation than the natively folded analog, even at 25°C.
  • 39°C its instability relative to the repurified monomer is even more dramatic.
  • the mean particle diameter in sample AP8-Rpdf is 1 ⁇ , in contrast to the 50 nm observed 5 for AP8-R.
  • AP6-R represents the repurified monomer
  • AP6-Rpdf is the PDF.
  • Sample AP6-Rpdf is only partly soluble at 5 mg/mL protein in PBS, pH 7.4. After this solution is passed through a 0.2 ⁇ filter, the protein concentration is measured using the
  • the spectrophotometric data presented above provide a clearer understanding of the nature of the protein(s) in PDF samples.
  • the properties of PDF differ considerably from those of the corresponding monomeric pST analogs, even at 25°C.
  • the CD spectra at 222 nm indicate that the secondary structures of PDF proteins are identical to that of standard rpST (ASP) .
  • the spectra reflecting tertiary structure (284 nm) are significantly altered for both AP8-Rpdf and AP6-Rpdf.
  • T m values of these PDF samples differ little from those of repurified monomers, although the magnitudes of the increases in fluorescence intensities are much smaller.
  • the behavior of the L component upon ion-exchange chromatography and SDS-PAGE provides indications that it does not comprise simply a folding variant of the protein, since this type of variant would exist in equilibrium with the monomeric pST. Thus, attempts to eliminate it would be futile.
  • studies are conducted to isolate, identify and characterize the pST form present in the L component, and most importantly, to illuminate the mechanism of its formation.
  • the FAB-MS results indicate that the modification involves the elimination of a sulfur atom, and/or intramolecular cross-linking of rpST polypeptides by a thioether bridge rather than a disulfide bridge. That is, FAB-MS results are fully consistent with a model in which a cystine residue in rpST is converted to lanthionine according to the reaction depicted in Figure 10. The reaction proceeds under alkaline, non-reducing conditions, such as those encountered in the original purification of rpST analogs from inclusion bodies.
  • lanthionine residue either intramolecular (within one pST molecule) or intermolecular (i.e., joining two pST molecules) , impedes the reduction of polypeptides or regions thereof in the presence of jS-mercaptoethanol, and thereby prevents the formation of fully extended polypeptides whose electrophoretic mobility can be accurately correlated with molecular weight.
  • Amino acid analysis of the electroeluted proteins under conditions appropriate for detecting low copy residues confirms the presence of a lanthionine derivative of pST in the L component.
  • a 13.5 minute peak, corresponding to lanthionine is observed in the sequence of protein obtained from the L component. This peak is absent in monomeric pST.
  • a 23.5 minute peak, corresponding to cysteine is present in monomeric pST and absent in the sequence of the protein obtained from the L component.
  • lanthionine pST The controlled generation of lanthionine pST is achieved in Study XVI by exposure of purified pST to an oxidative alkaline environment. Both forms of lanthionine pST (i.e., intramolecular and intermolecular) are formed. Subsequently, it is shown that alkaline exposure under reducing conditions suppresses lanthionine formation. These results contribute to the development of new methods for processing rpST from inclusion bodies in such a manner as to avoid or suppress formation of the lanthionine pST. Studv XIV; Isolation and Peptide Sequencing of the Minor Component
  • Method 36 mg of a pST preparation rich in the L component is fractionated by preparative SDS-PAGE on a 6 mm thick gel, using a Hoefer SE 600 electrophoretic separation unit. A portion of the gel is stained to reveal the location of resolved proteins, and the regions migrating at an apparent molecular weight of 20 and 22 kDa are excised. Proteins are electroeluted from the gel matrix in ammonium carbonate sodium dodecyl sulfate buffer, using an elutrap device (Schleicher and Schuell) . In this manner, about 1 mg each of pure proteins from the bands migrating at 20 kDa and 22 kDa are obtained.
  • N-terminal amino acid analysis is performed, and electroeluted proteins are compared with standard pST and the expected gene sequence.
  • Portions of the purified L component protein and monomeric protein are reduced with dithiothreitol, alkylated with iodoacetamide, and subjected to trypsin digestion to provide tryptic peptides for mapping.
  • Trypsin is used at a ratio of 25:1 (pST:trypsin) , and allowed to incubate for 24 hours at 37°C in 2 M urea buffered to pH 8.
  • the resultant tryptic peptides are fractionated on a Vydac C 18 RP-HPLC column, using trifluroacetic acid-acetonitrile as the elution buffer.
  • Electroeluted L component protein, monomeric pST, and standard pST preparations yield N- terminal sequences in agreement with that of the predicted gene product. Tryptic peptide maps of reduced and alkylated L component protein, monomeric pST, and standard pST preparations are related, but reveal a unique peak in the L component protein digest.
  • the sequence corresponding to this peak indicates that it is in fact an equimolar mixture of two peptides, both corresponding to the only cysteine-containing peptides in glu,glu-pST, i.e., those peptides encompassing amino acid residues 55 and 166, respectively (see SEQ ID NO. 1) .
  • the revealing aspect of the pST sequence obtained from tryptic mapping is a gap at the cycles corresponding to these residues.
  • these two peptides when isolated from monomeric pST, elute separately and sequence as modified cysteines at residues 55 and 166, as expected where a disulfide bridge is present in the protein under investigation.
  • Peptide sequencing thus yields data indicative of a nonreducible covalent bond between the amino acids as positions 55 and 166, present in the L component protein but absent in monomeric pST.
  • AP8 and their respective repurified derivatives are analyzed, together with the fractions from repurification that are enriched in the L component (respectively AP6-Rpdf and AP8-Rpdf; subsequently referred to as pST destabilizing fractions or PDF) .
  • the L component represents 35.3% of fraction AP6-Rpdf, and 21.0% of fraction AP8-Rpdf.
  • the electroeluted preparation of pure L component discussed in the preceding study is also analyzed.
  • the atomic weight of a sulfur atom is 32.06 daltons. Accordingly, these data are fully consistent with the elimination of a sulfur atom from the disulfide bond to form a nonreducible lanthionine linkage between residues 55 and 166 in rpST and analogs thereof.
  • Method Standard pST is exposed to pH 12.3 at 20°C for 24 hours, with timepoint samples being withdrawn and prepared for SDS-PAGE at 0, Vz, %, 2, 4, 8, and 24 hours. Samples are compared to standard pST and a PDF fraction by SDS-PAGE under reducing conditions.
  • Titration curve analysis shows that the appropriate pH range for separation is between pH 6 and 8.
  • a column containing a positively charged ion exchange resin is equilibrated with sodium phosphate buffers, such that the equilibrium pH is approximately 6 to 8, and the equilibrium conductivity is equal to that of approximately 5 mM to 20 mM sodium phosphate.
  • the column is loaded with a pST solution with a pH is equal to the equilibrium pH, and a conductivity is less than or equal to the conductivity of the equilibration conductivity.
  • the loading buffer is displaced with sodium phosphate until the pH and conductivity are equivalent to the equilibration conditions.
  • the lanthionine pST, dimer, and other impurities are eluted with approximately 30 to 60 mM sodium phosphate; and the pST monomer is eluted with approximately 70 to 125 mM sodium phosphate. These conditions remove the minor components, including lanthionine pST, and also maintain low levels of dimer during monomer elution.
  • the conditions consist of loading a DEAE-Sepharose column with pST at a concentration of approximately 40 mg pST/mL DEAE-Sepharose, washing the column with three bed volumes of 10 mM sodium phosphate, pH 7.5, eluting the minor components with 35 mM sodium phosphate, pH 7.5, and eluting the pST monomer with approximately 70 to 125 mM sodium phosphate, pH 7.5.
  • a gradient of sodium phosphate is used for elution in place of the isocratic elution with NaCl.
  • a column is loaded with pST at a concentration of 17 mg pST/mL resin and washed as described.
  • the running buffer is 10 mM sodium phosphate, pH 7.5.
  • the protein is eluted with a linear gradient of 10 to 250 mM sodium phosphate, pH 7.5, instead of NaCl, over 30 column volumes. Fractions are analyzed by SDS-PAGE and Superose®2 12. Data indicate that the sodium phosphate gradient appears to provide conditions resulting in the desired separation of components.
  • the L component, monomer and dimer are found. As the gradient increases, levels of monomer increase, but levels of dimer and L component decrease.
  • levels of dimer are found to be less than 5% and in some fractions the level of dimer is below the limit of detection.
  • Main peak fractions are pooled and found to contain 3.4% dimer with levels of L component below 0.5%.
  • a two-step isocratic elution procedure is developed for a large scale purification, using the information acquired from the sodium phosphate gradient experiment.
  • Several experiments are conducted to delineate the conditions for separation.
  • a DEAE-Sepharose column is equilibrated to pH 7.5 with 100 mM sodium phosphate, pH 7.5, followed by equilibration of conductivity with 10 mM sodium phosphate, pH 7.5.
  • Superficial linear velocity is 100 cm/hr throughout.
  • the column is loaded with the 10K concentrate, pH 7.5, at a pST concentration of 5-10 mg/mL and a resin load of 17 mg pST/mL resin.
  • the column is washed with th-s ⁇ ee bed volumes (BV) of 10 mM sodium phosphate, pH 7.5.
  • the fraction containing the L component is eluted with 35 M sodium phosphate, pH 7.5 (conductivity approximately 4.9 mS/cm) .
  • the pST monomer is eluted with 70 mM sodium phosphate, pH 7.5 (conductivity about 9.4 mS/cm) in approximately 7 BV of buffer.
  • Fractions with an A 280 greater than 0.5 are pooled. The pool contains 3.2% dimer and less than 0.5% of L component.
  • the final conductivity after pH adjustment is about 600 ⁇ S/cm, giving a calculated sodium phosphate concentration of 1.9 mM, not 10 mM as had been used previously.
  • the column is loaded at 22 mg pST/mL resin.
  • the elution profile observed in this experiment does not differ from that observed in the laboratory experiments. It appears that the lower phosphate molarity of the loading solution and the higher pST load do not have any significant impact on the elution of components from DEAE-Sepharose.
  • the main peak is eluted with 8.5 BV of 70 mM sodium phosphate, pH 7.5.
  • Fractions are analyzed by Superose® 12 and SDS-PAGE. Results reveal an elution pattern similar to that seen in the lab-scale purifications.
  • the final column yield after combination of the two column pools is approximately 76%. Thus, the results from the laboratory procedure are reproducible in a larger scale.
  • the next run utilizes the same loading and buffer conditions as the control, with the exception that the superficial linear velocity is increased by 50 percent to 150 cm/hr. Fractions are collected during elution and analyzed by SDS-PAGE and Superose® 12. Results indicate that the increase in superficial linear velocity does not affect the elution profile and provides pST with the same yield and quality as that of the control.
  • the pool from the initial fractions of the 35 mM sodium phosphate wash contain 10.2% of the original load and about 4% dimer whereas the pool from the remainder of the peak contains 70.3% of the load and approximately 2% dimer.
  • the unusual elution profile indicates that the maximum pST load under these conditions is 100 mg pST/mL resin, but this load does not give adequate separation.
  • the optimized conditions are verified starting with column load prepared from frozen washed pellet (AP-FWP) .
  • the 10K concentrate is loaded onto a DEAE-Sepharose column at 40 mg pST/mL resin.
  • the column is washed with 3 BV of 10 mM sodium phosphate, pH 7.5.
  • the fraction containing minor components is eluted with 35 mM sodium phosphate, pH 7.5 for 7 BV.
  • the 22 kDa main peak is eluted with 125 mM sodium phosphate.
  • Superficial linear velocity during the run is 150 cm/hr. After analysis of the fractions by Superose® 12 and SDS-PAGE, fractions low in dimer and L component are pooled, desalted and lyophilized.
  • the main peak elutes in 6 bed volumes (BV) .
  • High dimer above 5% is seen in the latter fractions of the main peak, and these fractions are excluded from the pool.
  • These excluded fractions contribute to the low ion-exchange yield of 72% as compared to processes where monomer is eluted with 70 mM sodium phosphate.
  • Some of the fractions could be included without affecting yield or dimer content significantly, but because of the incomplete separation of monomer and dimer under these conditions, it is preferred that Superose® 12 column analysis of each fraction be performed to choose the optimal fractions for Inclusion in the pool.
  • the final powder obtained (AP-I) is submitted for stability testing, and found to have better stability than the standard.
  • Glu Lys Leu Lys Asp Leu Glu Glu Gly lie Gin Ala Leu Met Arg Glu 115 120 125 Leu Glu Asp Gly Ser Pro Arg Ala Gly Gin lie Leu Lys Gin Thr Tyr 130 135 140

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Abstract

L'invention se rapporte à des procédés pour purifier la somatotropine porcine (pST), afin d'en éliminer les constituants qui la déstabilisent, en particulier la molécule de pST lanthionine. Dans un mode de réalisation préféré, on utilise un processus d'échange ionique isocratique en deux étapes. L'invention se rapporte également à des préparations de somatotropine porcine qui sont pratiquement exemptes de la molécule de pST lanthionine et d'autres constituants qui les déstabilisent, ces préparations possédant ainsi une solubilité et une stabilité accrues.
PCT/US1994/010740 1993-09-22 1994-09-21 Somatotropine porcine ayant une stabilite accrue et son procede de production Ceased WO1995008571A1 (fr)

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AU78771/94A AU7877194A (en) 1993-09-22 1994-09-21 Porcine somatotropin having enhanced stability; process for producing

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US12565093A 1993-09-22 1993-09-22
US08/125,650 1993-09-22
US30336394A 1994-09-08 1994-09-08
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011178A1 (fr) * 1995-09-21 1997-03-27 Genentech, Inc. Variants de l'hormone de croissance humaine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0365858A2 (fr) * 1988-10-26 1990-05-02 American Cyanamid Company Procédé pour réduire le contenu agrégat de matériaux semblables à l'hormone de croissance
EP0445099A1 (fr) * 1990-02-28 1991-09-04 Monsanto Company Procédé de préparation de monomères purifiés de somatotropine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0365858A2 (fr) * 1988-10-26 1990-05-02 American Cyanamid Company Procédé pour réduire le contenu agrégat de matériaux semblables à l'hormone de croissance
EP0445099A1 (fr) * 1990-02-28 1991-09-04 Monsanto Company Procédé de préparation de monomères purifiés de somatotropine

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004931A (en) * 1993-05-25 1999-12-21 Genentech, Inc. Method for inhibiting growth hormone action
US6136563A (en) * 1993-05-25 2000-10-24 Genentech, Inc. Human growth hormone variants comprising amino acid substitutions
WO1997011178A1 (fr) * 1995-09-21 1997-03-27 Genentech, Inc. Variants de l'hormone de croissance humaine
US5849535A (en) * 1995-09-21 1998-12-15 Genentech, Inc. Human growth hormone variants
US6057292A (en) * 1995-09-21 2000-05-02 Genentech, Inc. Method for inhibiting growth hormone action

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