WO2016207328A1 - Procédé pour la purification de polypeptides γ-carboxylés - Google Patents
Procédé pour la purification de polypeptides γ-carboxylés Download PDFInfo
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- WO2016207328A1 WO2016207328A1 PCT/EP2016/064634 EP2016064634W WO2016207328A1 WO 2016207328 A1 WO2016207328 A1 WO 2016207328A1 EP 2016064634 W EP2016064634 W EP 2016064634W WO 2016207328 A1 WO2016207328 A1 WO 2016207328A1
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- carboxylated
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- polypeptide
- carboxylated polypeptide
- anion exchange
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- 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
- C07K14/745—Blood coagulation or fibrinolysis factors
Definitions
- the present invention relates to a process for the purification of ⁇ -carboxylated polypeptides, such as Factor VII, and to the manufacturing of a recombinant ⁇ -carboxylated polypeptide employing a respective purification process.
- ⁇ -carboxylated polypeptides such as Factor VII
- ⁇ -Carboxylation is a posttranslational modification of proteins wherein a carboxylic acid functional group is added to a glutamate amino acid residue (Glu) in a protein, forming a v- carboxyglutamate (Gla) residue.
- Glu glutamate amino acid residue
- Gla v- carboxyglutamate residue
- the following human Gla-containing proteins have been characterized to the level of primary structure: the blood coagulation factors II (prothrombin), VII, IX, and X, the anticoagulant proteins C and S, and the factor X-targeting protein Z. Further, ⁇ -carboxylation has been found in the bone Gla protein osteocalcin, the calcification-inhibiting matrix Gla protein (MGP), the cell growth regulating growth arrest specific gene 6 protein (Gas6) and the four transmembrane Gla proteins (TMGPs).
- MGP calcification-inhibiting matrix Gla protein
- Gas6 cell growth regulating growth arrest specific gene 6 protein
- TMGPs transmembrane Gla proteins
- the blood coagulation factors are expressed as a proprotein which is activated by cleavage of the polypeptide chain, resulting in two subunits forming the active factor.
- the presence of the Gla residues in these proteins are essential for their biological (proteolytic) activity.
- Several of the known Gla-containing proteins can be used as therapeutic
- the object of the present invention therefore is to provide a less complex and preferably cost-efficient purification process which results in ⁇ -carboxylated polypeptides such as Factor VII in high yield and purity.
- the present invention relates to a purification process for ⁇ -carboxylated polypeptides such as Factor VII comprising subjecting a liquid containing the ⁇ -carboxylated polypeptide to the following steps:
- step (1 ) affinity chromatography, and (2) multimodal anion exchange chromatography; wherein step (1 ) is performed prior to step (2).
- the present invention pertains to a process for manufacturing a v- carboxylated polypeptide of interest, comprising the following steps:
- step (b) purifying said recombinantly expressed ⁇ -carboxylated polypeptide of interest by subjecting the liquid containing said ⁇ -carboxylated polypeptide obtained in step (a) at least to the steps of:
- step (1 ) affinity chromatography, and (2) multimodal anion exchange chromatography; wherein step (1 ) is performed prior to step (2).
- the present invention pertains to a method of virus inactivation in a biological sample comprising a ⁇ -carboxylated polypeptide of interest, comprising the step of incubating the biological sample at a pH of 4.0 or less for at least 15 min.
- the present invention is directed to a method for activating a blood coagulation factor, in particular Factor VII, comprising the following steps:
- Polypeptide means any naturally occurring or artificially designed polypeptide, including variants thereof. "Variants” may include amino acid additions, deletions and/or substitutions with respect to the naturally occurring polypeptide. Preferably, the variant does not include more than 20 additions, deletions and/or substitutions, preferably not more than 10, particularly preferred not more than 5, in particular not more than two or one amino acid addition, deletion and/or substitution compared to the parent amino acid sequence.
- a polypeptide in particular may be a protein.
- ⁇ -Carboxylated polypeptide refers to a polypeptide which comprises at least one v- carboxylation.
- a ⁇ -carboxylation in particular is a modification of a glutamic acid residue within the polypeptide resulting in a ⁇ -carboxyglutamate residue. This modification may be catalyzed by the enzyme ⁇ -glutamyl carboxylase.
- chromatography as used herein in particular refers to column chromatography. Chromatography comprises the use of a matrix or stationary phase and a mobile phase including, e.g., the sample, loading buffer, wash buffer and elution buffer. In certain embodiments, the matrix is stationary in the column and the mobile phase runs through the column.
- the "liquid containing the ⁇ -carboxylated polypeptide” may be any liquid composition containing ⁇ -carboxylated polypeptide, in particular aqueous compositions. It may be obtained from recombinant expression of the ⁇ -carboxylated polypeptide such as cell culture supernatant, cell culture harvest or other natural source composition.
- the "liquid containing the ⁇ -carboxylated polypeptide” is a cell culture supernatant containing recombinantly produced the ⁇ -carboxylated polypeptide.
- the host cells in particular were removed from the supernatant and preferably the cell culture supernatant was not subjected to any chromatography step prior to the methods according to the present invention.
- ⁇ -carboxylated polypeptides such as Factor VII can be purified directly from cell culture supernatant using only two chromatography steps, affinity chromatography and multimodal anion exchange chromatography, resulting in a highly pure composition of the ⁇ -carboxylated polypeptide.
- the purification process furthermore, gives a high yield of the ⁇ -carboxylated polypeptide and can be performed without any buffer exchange and concentration steps prior to and between the chromatography steps.
- the ⁇ -carboxylated polypeptide, in particular Factor VII can simply be incubated after elution from the second chromatography column in order to activate it.
- a very simple and straightforward purification process with a minimum of method steps is provided.
- the present invention relates to a purification process for ⁇ -carboxylated polypeptides such as Factor VII comprising subjecting a liquid containing the ⁇ -carboxylated polypeptide to the following steps: (1 ) affinity chromatography, and
- step (1 ) multimodal anion exchange chromatography; wherein step (1 ) is performed prior to step (2).
- the purification process may optionally comprise additional steps, e.g. filtration steps such as diafiltration, ultrafiltration or nanofiltration, and/or at least one virus inactivation/depletion step.
- additional steps e.g. filtration steps such as diafiltration, ultrafiltration or nanofiltration, and/or at least one virus inactivation/depletion step.
- the process of the present invention comprises no more than two, in particular no more than one additional chromatography step.
- the purification method of the invention provides the ⁇ -carboxylated polypeptide such as Factor VII in high purity and in solvated monomeric form, which may then be formulated as a pharmaceutical composition.
- the purity in general is above 90%, preferably above 92%, more preferably above 95%, based on total protein.
- the purification method of the invention is easily scalable, even up to industrial size, without major changes in the purification conditions.
- the crude ⁇ -carboxylated polypeptide which forms the starting material for the purification process according to the present invention may be provided in or obtained from liquids of natural sources or by recombinant techniques such as e.g. in cell culture harvests containing the ⁇ -carboxylated polypeptide. ln the steps of chromatography typically commercially available resins are used, preferably polymer-based resins or agarose-based resins.
- the process involves as first chromatographic step a step of affinity chromatography (1 ) (AC).
- the affinity chromatography is used as capture step in which the ⁇ -carboxylated polypeptide is enriched, e.g. from the natural source liquid or the cell culture harvest.
- Affinity chromatography in particular refers to a chromatography step wherein ligands specifically binding to the molecule to be isolated are immobilized on the chromatography matrix.
- the molecule of interest binds to the ligand on the matrix and essentially all other molecules do not bind and are washed out of the chromatography matrix.
- antibodies or antibody fragments directed against the molecule to be isolated are used as ligands.
- specific binding peptides or binding proteins of the molecule to be isolated are used, for example naturally interaction partners thereof.
- the ligand specifically binding to the molecule to be isolated in particular is a peptide, polypeptide or protein.
- the affinity column material is made of a resin to which the ligand binding the ⁇ -carboxylated polypeptide may be attached.
- Typical column materials are agarose and polystyrene.
- the ligand may be attached to the column material via a suitable linker which may in particular covalently link the ligand to the column material.
- the ⁇ -carboxylated polypeptide is Factor VII and the affinity chromatography matrix comprises antibody or antibody fragments specifically binding Factor VII.
- the antibody or antibody fragment may be immobilized to the matrix.
- a particularly preferred affinity matrix for isolation of Factor VII is VllSelect (GE Healthcare).
- the affinity chromatography is usually performed by equilibrating and loading the column, followed by a wash and subsequent elution, each with a buffer preferably adapted to the binding conditions of the immobilized antibody.
- the equilibration, load and wash are preferably carried out by using a mobile phase buffering at neutral pH, for example at or about pH 7 to 8, more preferably at or about 7.5.
- Any buffer suitable for biomolecules may be used, such as e.g. a Tris buffer or a HEPES buffer.
- the liquid containing the ⁇ -carboxylated polypeptide is loaded onto the affinity chromatography column without any preceding buffer exchange step and/or concentration step of the ⁇ -carboxylated polypeptide.
- the liquid containing the ⁇ - carboxylated polypeptide is a biological sample such as a cell culture supernatant in these embodiments.
- loading of the sample is followed by a wash step.
- the wash is performed with the same buffer as equilibration and/or loading of the column.
- the affinity chromatography column may in particular be washed with at least 15 column volumes, in particular at least 20, at least 30 or at least 40 column volumes, preferably at least 50 column volumes, at least 55 column volumes or at least 60 column volumes of wash buffer.
- the ⁇ -carboxylated polypeptide is eluted from the affinity chromatography matrix using an elution buffer with a pH in the range of from 2.0 to 4.0, in particular about 3.0.
- the elution buffer comprises no propanediol, especially no diol or even no alcohol.
- the elution buffer may comprise glycine as the only organic compound.
- the affinity chromatography step can include a virus inactivation step.
- Virus inactivation may be achieved by incubating the ⁇ -carboxylated polypeptide eluted from the matrix at a pH of 4.5 or less, in particular at a pH in the range of from 4.0 to 2.0, such as a pH of about 3.0.
- the ⁇ -carboxylated polypeptide may in particular be incubated in the elution buffer. Incubation may be performed for at least 20 min, in particular at least 30 min, at least 45 min or at least 1 h.
- the composition comprising the v- carboxylated polypeptide preferably is neutralized to a pH in the range of from 5.0 to 9.5, in particular from 6.5 to 9.5, 7.0 to 9.0, or 5.5 to 7.0, such as about 8.0 or about 6.0.
- Neutralization may be achieved by adding a base such as Tris base or NaOH to the composition.
- This virus inactivation step securely depletes Pseudorabies virus (PRV), an enveloped, double-stranded DNA virus. It is preferred that no buffer exchange is performed between steps (1 ) and (2).
- the eluate of the affinity chromatography can directly be loaded onto the multimodal anion exchange chromatography, optionally after the above-described incubation and neutralization. Furthermore, preferably no concentration step is performed between step (1 ) and step (2).
- Multimodal anion exchange chromatography step (2) The process of the present invention also involves a step of multimodal anion exchange chromatography (2) (MM AIX), e.g. for further purifying of the ⁇ -carboxylated polypeptide.
- MM AIX multimodal anion exchange chromatography (2)
- Multimodal anion exchange chromatography in particular refers to a chromatography step wherein a chromatographic matrix is used which has anion exchange activity as well as one or more other binding activities.
- the multimodal anion exchange chromatography matrix binds to the ⁇ -carboxylated polypeptide via ionic interactions and additionally via hydrophobic interactions and/or hydrogen bonding.
- the ligands of the matrix of the multimodal anion exchange chromatography are positively charged and comprise a hydrophobic group and/or a hydroxyl group.
- the matrix in particular comprises a quarternary ammonium ion as positively charged group.
- a suitable example for a hydrophobic group is a phenyl group.
- the matrix of the multimodal anion exchange chromatography comprises ligands comprising the following chemical structure:
- R depicts the attachment point of the ligand to the column material, optionally further comprising a linker.
- Multimodal anion exchange column material is made of a resin to which the above ligand may be attached.
- Typical column materials are agarose and polystyrene.
- a particularly preferred multimodal anion exchange matrix for isolation of ⁇ -carboxylated polypeptides such as Factor VII is Capto adhere ImpRes (GE Healthcare).
- the multimodal anion exchange chromatography is run in the bind- elute mode.
- the composition comprising the ⁇ -carboxylated polypeptide is loaded onto the multimodal anion exchange chromatography column under conditions at which the ⁇ -carboxylated polypeptide binds to the multimodal anion exchange matrix, in particular using a suitable loading buffer.
- the loading buffer comprises a buffering agent such as e.g. Tris or histidine, and is adjusted to a pH of about 5 to about 9, in particular about 6 to about 9, about 7 to about 8.5, or about 5.5 to about 7, such as about 8.0 or about 6.0.
- the multimodal anion exchange chromatography may also be run in the flow-through mode.
- the composition comprising the ⁇ -carboxylated polypeptide is loaded onto the multimodal anion exchange chromatography column under conditions at which the v- carboxylated polypeptide does not bind to the multimodal anion exchange matrix, in particular using a suitable loading buffer.
- the loading buffer comprises calcium ions and/or magnesium ions.
- the loading buffer may comprise at least 1 mM calcium and/or magnesium ions, in particular at least 5 mM, at least 10 mM, at least 20 mM or at least 30 mM calcium and/or magnesium ions.
- the loading buffer may comprise a buffering agent such as e.g. Tris or histidine, and is adjusted to a pH of about 5 to about 9, in particular about 6 to about 9, about 7 to about 8.5, or about 5.5 to about 7, such as about 8.0 or about 6.0.
- Elution of the ⁇ -carboxylated polypeptide from the multimodal anion exchange matrix is in particular effected using an elution buffer comprising calcium ions and/or magnesium ions.
- the elution buffer may comprise at least 5 mM or at least 10 mM calcium and/or magnesium ions, in particular at least 20 mM or at least 30 mM calcium and/or magnesium ions.
- the elution buffer may contain calcium chloride and/or magnesium chloride.
- the elution buffer is identical to the loading buffer except for the additional calcium or magnesium salt present in the elution buffer.
- the elution buffer has a pH of about 5.0 to about 9.0, in particular about 6.5 to about 9.5, about 7.5 to about 9.0, or about 5.5 to about 7.0, such as about 8.0 or about 6.0.
- elution of the ⁇ -carboxylated polypeptide is effected under conditions at which ⁇ - ⁇ -carboxylated forms of the ⁇ -carboxylated polypeptide stay bound to the multimodal anion exchange matrix.
- the process of the present invention may optionally include additional steps known to the person skilled in the art, e.g. filtration steps and/or virus inactivation steps. Preferred additional steps include activation, filtration such as diafiltration, ultrafiltration and nanofiltration, and virus inactivation/depletion.
- the process of the present invention comprises no more than two, in particular no more than one additional chromatography step(s), and especially does not comprise any additional chromatography steps.
- the process according to the invention does not comprise a buffer exchange step directly prior to and/or directly subsequent to the affinity chromatography.
- the process of the present invention comprises the step of clear filtration of the liquid containing the ⁇ -carboxylated polypeptide. This step in particular may be performed as first step, in particular prior to step (1 ) and prior to any other chromatographic step. Furthermore, in specific embodiments the process of the present invention comprises the step of increasing the concentration of the v- carboxylated polypeptide in the liquid. This step may be performed using tangential flow filtration, precipitation, e.g. by ammonium sulfate or ethanol, or chromatography as described below. Activation step
- the composition comprising the ⁇ -carboxylated polypeptide is incubated at moderately basic pH, in particular at a pH of about 7.5 to about 9.0, such as about 8.0, for activating the ⁇ -carboxylated polypeptide.
- the composition in particular comprises calcium ions and/or magnesium ions, especially at least 1 mM, at least 5 mM or at least 10 mM calcium and/or magnesium ions, at least 20 mM or at least 30 mM calcium and/or magnesium ions for example in form of calcium chloride and/or magnesium chloride.
- the composition comprising the ⁇ -carboxylated polypeptide is incubated directly after elution, in particular without any buffer exchange. Especially the ⁇ -carboxylated polypeptide is present in the elution buffer.
- the activation step is preferably used in case the ⁇ -carboxylated polypeptide is a blood coagulation factor such as in particular Factor VII.
- the polypeptide chain of the blood coagulation factor is cleaved, resulting in two separate polypeptide chains which each are a subunit forming the active blood coagulation factor such as Factor Vila.
- the ⁇ -carboxylated polypeptide in the liquid containing said ⁇ -carboxylated polypeptide prior to subjecting it to the affinity chromatography of step (1 ) is in the inactive form.
- the activation step is performed after elution from the affinity chromatography step (1 ) and prior to the multimodal anion exchange chromatography step (2), in particular after the acidic virus inactivation step performed directly after elution from the affinity chromatography step.
- the composition comprising the ⁇ -carboxylated polypeptide may be incubated for at least 1 day, in particular at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 1 week.
- Incubation in particular occurs at ⁇ ⁇ ' ⁇ or less, such as in the range of from 2 ° to 8°C. In particular, incubation is performed until at least 75%, especially at least 80%, at least 85%, at least 90% or at least 95% of the ⁇ - carboxylated polypeptide in the composition is activated.
- the activation is stopped by decreasing the pH of the composition.
- the pH may for example be adjusted to a value in the range of from about 5.0 to about 7.0, such as about 6.0. Decrease of the pH may in particular be achieved by performing a concentration and/or buffer exchange step, e.g. using ultrafiltration and/or diafiltration, as described below.
- the activation may also be stopped by addition of at least 3 mM zinc ions, in particular about 10 mM zinc ions, for example in the form of zinc chloride.
- a method for activating a blood coagulation factor comprising the following steps: (a) providing a sample comprising the blood coagulation factor;
- the features, embodiments and examples as described herein, in particular those described herein for the activation step, also apply likewise to the method for activating a blood coagulation factor.
- the method for activating a blood coagulation factor may be used as activation step in the methods and processes described herein, and/or the purification process for ⁇ -carboxylated polypeptides described herein may be used as the step of purifying the blood coagulation factor in the method for activating a blood coagulation factor.
- the blood coagulation factor is selected from the group consisting of Factor VII, Factor VIII and von Willebrand factor, and in particular is Factor VII.
- Suitable examples of the chromatographic step include anion exchange chromatography and multimodal anion exchange chromatography. Particularly suitable is multimodal anion exchange chromatography as described herein.
- the solid chromatography matrix to which the blood coagulation factor is bound especially is a multimodal anion exchange chromatography matrix, such as those described herein.
- the elution buffer in particular is an elution buffer as described herein with respect to the multimodal anion exchange chromatography and/or has the features as described for the composition in which the ⁇ - carboxylated polypeptide is incubated during the activation step.
- the elution buffer has a pH of about 8 and comprises at least 5 mM or at least 10 mM calcium ions.
- the elution buffer furthermore may comprise a buffering agent and/or an amine compound.
- the amine compound is the buffering agent, such as tris(hydroxymethyl)aminomethane (Tris).
- the process comprises optionally a subsequent concentration and/or buffer exchange step, in particular using ultrafiltration and/or diafiltration, and optionally a virus depletion step, in particular using nanofiltration.
- the composition comprising the ⁇ -carboxylated polypeptide is subjected to an ultrafiltration and/or diafiltration step.
- the ultrafiltration and/or diafiltration is performed in order to increase the concentration of the ⁇ -carboxylated polypeptide in the composition and/or to exchange the other components of the composition, e.g. to perform a buffer exchange.
- the ultrafiltration and/or diafiltration is preferably carried out using a membrane or hollow fiber filter having a cut-off of about 3-30 kDa, most preferably about 10 kDa. It is preferred to perform during ultrafiltration and/or diafiltration a buffer exchange to a pre-formulation buffer.
- the pH of the composition comprising the ⁇ -carboxylated polypeptide is adjusted to moderately acidic, in particular in the range of from about 7.0 to about 5.0, especially about 6.0, in the ultrafiltration and/or diafiltration step.
- the concentration of the ⁇ -carboxylated polypeptide in the composition is increased by the ultrafiltration and/or diafiltration step.
- the concentration of the ⁇ -carboxylated polypeptide may be increased to at least 0.5 g/l, in particular at least 1 .0 g/l, for example to a concentration in the range of from about 0.75 g/l to about 5.0 g/l or from about 0.9 g/l to about 2.0 g/l or from about 1 .0 g/l to about 1 .5 g/l, such as about 1 .2 or 1 .3 g/l.
- sterile filtration steps can be used to remove biological contaminations such as eukaryotic and/or prokaryotic cells, in particular bacteria, and/or viruses.
- these steps are performed at or near the end of the purification process to prevent a further contamination after the sterile filtration step.
- the filter used for sterile filtration preferably has a pore size of 0.22 ⁇ or less, preferably 0.1 ⁇ or less.
- a nanofiltration step may be performed.
- the process comprises a step of nanofiltration, in particular as a virus clearance step; i.e.
- Nanofiltration may be performed at any stage of the purification process. However, it is particularly preferred to carry out nanofiltration after the end of the chromatographic procedure. Nanofiltration may be performed more than one time, for example it may be performed twice. Preferred nanofiltration devices have a pore size of about 15 to 20 nm.
- Another additional step which can be performed in the purification process according to the invention is a virus inactivation step via incubation of the ⁇ -carboxylated polypeptide at a specific pH or in the presence of specific solvents and/or detergents.
- the v- carboxylated polypeptide is incubated at a pH of 4.0 or less, preferably at about pH 3.0.
- the incubation time preferably is at least 15 min, at least 30 min, at least 60 min or at least 80 min.
- Incubation may be performed at low temperature such as ⁇ ⁇ ' ⁇ or less or 4 ⁇ ⁇ or less, or at about room temperature.
- the ⁇ -carboxylated polypeptide may be incubated at a pH of about 3.0 for about 90 min at about room temperature.
- virus inactivation may be achieved by the solvent/detergent method.
- the ⁇ - carboxylated polypeptide may be incubated in the presence of an organic solvent and a non- ionic detergent.
- the organic solvent may for example be a trialkyl phosphate such as tri-(n- butyl) phosphate.
- non-ionic detergents examples include polyethylene glycol p- (1 ,1 ,3,3-tetramethylbutyl)-phenyl ether (Triton X-100) and polyoxyethylene sorbitan monooleate (polysorbate 80, Tween 80).
- Triton X-100 polyethylene glycol p- (1 ,1 ,3,3-tetramethylbutyl)-phenyl ether
- polyoxyethylene sorbitan monooleate polysorbate 80, Tween 80.
- One or both of these virus inactivation steps can be performed at any time during the purification process and preferably is performed after the last chromatography step. In specific embodiments, a virus inactivation step using the solvent/detergent method is performed prior to the affinity chromatography step (1 ).
- a virus inactivation step using the solvent/detergent method is performed prior to the affinity chromatography step (1 ) and a virus inactivation step using acidic pH conditions, especially as described above, is performed after the affinity chromatography step (1 ).
- a virus depletion step using nanofiltration can be performed after the end of the chromatographic procedure.
- the liquid composition resulting from the purification process as described above and containing purified ⁇ -carboxylated polypeptide may be frozen for storage as is, or may be subjected to lyophilization ("freeze-drying") to remove solvent.
- the process according to the invention comprises the step of lyophilization of the composition comprising the ⁇ -carboxylated polypeptide. Additional chromatographic steps
- the process according to the invention comprises one or two further chromatographic steps in addition to steps (1 ) and (2).
- the further chromatographic steps may be performed prior to step (1 ), between steps (1 ) and (2), after step (2) or after the activation step.
- one further chromatographic step is performed after step (2), especially after the activation step.
- This further chromatographic step may be for further decreasing the amount of contamination, such as in particular the amount of nucleic acids such as DNA in the composition comprising the ⁇ -carboxylated polypeptide.
- Certain examples of the further chromatographic step are anion exchange chromatography and hydrophobic interaction chromatography. This step may be performed using matrices, buffers and chromatography parameters as known in the art.
- the process of the present invention comprises an anion exchange chromatography step as further chromatographic step performed after step (2).
- one further chromatographic step is performed before step (1 ).
- This further chromatographic step in particular may be a membrane chromatography of the liquid containing the ⁇ -carboxylated polypeptide before it is loaded onto the affinity chromatography column.
- an anion exchange membrane chromatography can be used. This step may be performed using membrane matrices, buffers and chromatography parameters as known in the art.
- the further chromatographic step performed before step (1 ) is used for increasing the concentration of the ⁇ -carboxylated polypeptide in the liquid.
- Any chromatographic step can be used, in particular those employing matrix material to which the ⁇ -carboxylated polypeptide can bind. This chromatographic step is in particular performed in bind-elute mode.
- the process of the present invention comprises the following steps in the order shown below: (1 ) affinity chromatography,
- the process of the present invention comprises the following steps in the order shown below: (0) optionally clear filtration; (0a) optionally concentration;
- the process of the present invention comprises the following steps in the order shown below:
- the process of the present invention comprises the following steps in the order shown below: (0) optionally clear filtration; (0a) optionally concentration;
- the purification processes described above are preferably performed without including any further chromatography steps and/or ultrafiltration steps and/or diafiltration steps.
- the purification processes described above may further comprise additional steps, in particular one or more of the additional steps described herein, for example those used for removing or inactivating undesired or and/or hazardous substances.
- An advantage of the present invention is that the purification process is highly effective, reduces the number chromatographic steps to a minimum of 2 chromatographic steps, and provides the ⁇ -carboxylated polypeptide in high purity and high yield.
- the process provides a high degree of purity and specific bioactivity of at least 90%, preferably at least 92%, more preferably at least 95% w/w, each based on total protein as measured, for example, by HCP- ELISA, PAGE and/or size exclusion chromatography.
- the purification process according to the invention provides a surprisingly high recovery of the ⁇ -carboxylated polypeptide of interest present in the starting material.
- compositions used in the chromatography steps (1 ) and/or (2), in particular the equilibration buffer, loading buffer, wash buffer and/or elution buffer comprise zinc ions.
- the compositions especially comprise zinc chloride.
- the zinc ion is present in these compositions in particular in a concentration of at least 10 ⁇ , preferably at least 50 ⁇ , more preferably about 100 ⁇ or more.
- the compositions used in steps (1 ) and/or (2), in particular the equilibration buffer, loading buffer, wash buffer and/or elution buffer may contain an antioxidant, such as L-methionine. Alternate antioxidants include t-butyl-4-methoxyphenol, 2,6-bis(1 , 1 -dimethylethyl)-4-methyl phenol; potassium or sodium bimetabisulfite, sodium bisulfite.
- the process may be generally performed at about room temperature, such as a temperature in the range of from ⁇ ⁇ ' ⁇ to 25 q C, or at a temperature of ⁇ ⁇ ' ⁇ or less, in particular in the range of from about 2 ° to about 8°C.
- room temperature such as a temperature in the range of from ⁇ ⁇ ' ⁇ to 25 q C, or at a temperature of ⁇ ⁇ ' ⁇ or less, in particular in the range of from about 2 ° to about 8°C.
- room temperature such as a temperature in the range of from ⁇ ⁇ ' ⁇ to 25 q C, or at a temperature of ⁇ ⁇ ' ⁇ or less, in particular in the range of from about 2 ° to about 8°C.
- a specific temperature is given which for those steps is preferred of said general temperature.
- the loading buffers and elution buffers used in the purification process have a low salt concentration and/or a low ionic strength, for example a salt concentration and/or an ionic strength below 400 mM, such as below 300 mM or below 250 mM.
- the loading buffer and/or the elution buffer used in the multimodal anion exchange chromatography and/or the composition comprising the ⁇ -carboxylated polypeptide which is loaded onto the multimodal anion exchange chromatography may have a low salt concentration and/or a low ionic strength.
- the loading buffer and/or the elution buffer used in the multimodal anion exchange chromatography and/or the composition comprising the ⁇ -carboxylated polypeptide which is loaded onto the multimodal anion exchange chromatography have a salt concentration and/or an ionic strength below 250 mM, such as below 200 mM or below 1 50 mM.
- the loading buffer and elution buffer used in the affinity chromatography may have a low salt concentration and/or a low ionic strength.
- the loading buffer and/or the elution buffer used in the affinity chromatography have a salt concentration and/or an ionic strength below 400 mM, such as below 300 mM or below 250 mM.
- the ionic strength of a composition is calculated by the formula wherein ⁇ is the ionic strength, C, is the concentration of the ion, and Z, is the overall charge of the ion.
- virus inactivation in compositions comprising blood coagulation factor such as Factor VI I was obtained by incubation in the presence of solvents and detergents.
- these proteins were considered to denature at highly acidic conditions.
- the present inventors now found that blood coagulation factor and in particular Factor VI I is stable at the specific conditions used in the virus inactivation process described herein. It was even surprisingly demonstrated that the amount of aggregation decreases with time after incubation under acidic conditions.
- the invention pertains to a method of virus inactivation in a biological sample comprising a ⁇ -carboxylated polypeptide of interest, comprising the step of incubating the biological sample at a pH of 4.5 or less for at least 15 min. Furthermore, the invention pertains to a method of decreasing the aggregation of a ⁇ -carboxylated polypeptide of interest in a composition, comprising the step of incubating the biological sample at a pH of 4.5 or less for at least 15 min.
- the ⁇ -carboxylated polypeptide of interest in particular is a blood coagulation factor in activated or inactive form such as Factor VII.
- the biological sample may be any composition directly or indirectly obtained from a biological source such as a cell culture or the human or animal body. Examples of the biological sample include cell culture harvests, including cell culture supernatants or extracts, and samples of body fluids such as blood samples and plasma samples, prior to any purification as well as compositions obtained therefrom after one or more purification steps.
- the biological sample is a partly or highly purified composition comprising the ⁇ -carboxylated polypeptide of interest obtained by production in a cell culture.
- incubation of the biological sample is performed at a pH in the range of from 4.0 to 2.0, in particular from 3.5 to 2.5, such as a pH of about 3.0. Incubation may be performed for at least 20 min, in particular at least 30 min, at least 45 min or at least 1 h, and/or up to 24 h, up to 12 h, up to 8 h or up to 4 h, in particular up to 3 h, up to 2 h, up to 1 h 30 min or up to 1 h 15 min. Especially, incubation is performed at a pH in the range of from 3.5 to 2.5 for a time in the range of from 45 min to 1 h 15 min or from 45 min to 3 h.
- the method of virus inactivation further comprises the step of stopping the virus inactivation reaction, especially by increasing the pH.
- the pH is adjusted to a pH in the range of from 5.0 to 9.5, in particular from 6.5 to 9.5, 7.0 to 9.0, or 5.5 to 7.0, such as about 8.0 or about 6.0. This may be achieved by adding a base such as Tris base or NaOH to the biological sample.
- the v-carboxylated polypeptide is a polypeptide, in particular a protein, that contains one or more glutamate residues which contain an additional carboxylic acid group.
- the ⁇ -carboxylated polypeptide to be purified is preferably selected from the group consisting of a blood coagulation factor, in particular factor II (prothrombin), factor VII, factor IX and factor X; an anticoagulant protein, in particular anticoagulant protein C, anticoagulant protein S and factor X-targeting protein Z; osteocalcin; calcification-inhibiting matrix Gla protein (MGP); cell growth regulating growth arrest specific gene 6 protein (Gas6); and a transmembrane Gla protein (TMGP).
- the ⁇ -carboxylated polypeptide preferably is a blood coagulation factor, in particular Factor VII, including naturally occurring and recombinant versions thereof as well as any isoforms, variants and analogues thereof.
- the ⁇ -carboxylated polypeptide is produced recombinantly.
- the ⁇ -carboxylated polypeptide is naturally produced and in particular obtained from the human or animal body.
- the isoforms, variants and analogues of the blood coagulation factor exhibit one or more biological activities of the natural blood coagulation factor.
- the ⁇ -carboxylated polypeptide obtained by the purification process may be formulated for any kind of administration, preferably for injection, in particular intravenously such as by intravenous infusion.
- the ⁇ -carboxylated polypeptide formulation may be lyophilized, in which case it is dissolved in water just prior to injection.
- the ⁇ -carboxylated polypeptide formulation may also be a liquid formulation, in which case it can be injected directly, without prior dissolution.
- the formulation may contain known excipients and stabilizers and may additionally comprise antioxidants and/or surfactants.
- the ⁇ -carboxylated polypeptide formulation may be single dose or multiple dose.
- bacteriostatic agent such as, for example, alkylparabene, benzyl alcohol, meta- cresol, thymol or phenol.
- Single dose formulations may also comprise a bacteriostatic agent.
- the ⁇ -carboxylated polypeptide may be formulated with known excipients and stabilizers, for example, sucrose or mannitol. It may also comprise an antioxidant, such as methionine.
- the ⁇ -carboxylated polypeptide of the invention is suitable for use in all treatments where the ⁇ -carboxylated polypeptide is indicated.
- Factor VII is particularly useful in the treatment of hemophilia A, hemophilia B, acquired hemophilia and congenital Factor VII deficiency, as well as for reducing bleeding during and after surgery and to control or prevent hemorrhages.
- recombinant refers to preparations of ⁇ -carboxylated polypeptide such as Factor VII that are produced through the use of recombinant DNA technology.
- a method of expressing a ⁇ -carboxylated polypeptide using recombinant technology is the transfection of a suitable host cell, preferably a eukaryotic host cell, with an expression vector comprising a DNA sequence encoding the ⁇ -carboxylated polypeptide of interest.
- the expression vector carries a strong promoter driving the expression of the ⁇ -carboxylated polypeptide, e.g. CMV or SV40 and a suitable selection marker for selecting host cells that have incorporated the vector.
- the eukaryotic host cell is selected from primate cells, preferably human cells and rodent cells, preferably CHO or BHK cells.
- Suitable human cells for production of ⁇ -carboxylated polypeptide such as Factor VII, including immortalized human blood cells such as myeloid leukemia derived cells, are disclosed for example in WO 2008/028686 A2.
- the purification process according to the invention is useful for purifying natural as well as recombinant ⁇ -carboxylated polypeptide, including isoforms and variants thereof.
- variant preferably encompasses ⁇ -carboxylated polypeptides derived from a natural y- carboxylated polypeptide, such as mutant forms thereof, fusion proteins thereof and/or fragments thereof.
- the ⁇ -carboxylated polypeptide variants and/or isoforms exhibit one or more activities which are qualitatively and/or quantitatively similar or identical to those of the natural ⁇ -carboxylated polypeptide.
- ⁇ -carboxylated polypeptide variant such as “Factor VII variant” is meant to encompass those molecules differing in amino acid sequence, number of y-carboxylation sites (including additional or deleted ⁇ -carboxylation sites) or inter-subunit linkage from human ⁇ -carboxylated polypeptides but exhibiting one or more of its activities.
- the Factor VII variants referred to herein also include long-lasting Factor VII variants, i.e. variants which have a longer circulation half-life in the human body.
- These variants in particular include fusion proteins of Factor VII with other proteins or protein fragments as well as Factor VII proteins to which polymers such as polyethylene glycol are attached.
- the Factor VII is produced recombinantly, either in a serum- containing or in a serum-free medium.
- Factor VII is secreted by the host cells producing it.
- the expression "crude recombinant ⁇ -carboxylated polypeptide” refers to the cell culture supernatant from recombinant cells expressing the ⁇ -carboxylated polypeptide, before it has undergone any chromatographic step. The expression in particular encompasses the raw form of the supernatant (as isolated from cells). Process for manufacturing v-carboxylated polypeptide
- a process for manufacturing a ⁇ -carboxylated polypeptide of interest by performing the process for the purification of a ⁇ -carboxylated polypeptide described herein.
- the ⁇ -carboxylated polypeptide can be obtained from natural sources or recombinantly.
- a process for manufacturing a ⁇ -carboxylated polypeptide of interest comprising the following steps:
- step (b) purifying said recombinantly expressed ⁇ -carboxylated polypeptide of interest by subjecting the liquid containing said ⁇ -carboxylated polypeptide obtained in step (a) at least to the steps of:
- step (1 ) multimodal anion exchange chromatography; wherein step (1 ) is performed prior to step (2).
- the respective manufacturing process leads to the production of very pure ⁇ -carboxylated polypeptides which are in particular suitable for use in pharmaceutical formulations.
- Said manufacturing process preferably comprises at least one or more steps as described above in conjunction with the purification process.
- the respective disclosure also applies to the manufacturing process according to the present invention and it is referred to the above disclosure to avoid repetitions.
- the manufacturing process according to the present invention may comprise a step of formulating the ⁇ -carboxylated polypeptide of interest in form of a pharmaceutical formulation.
- Suitable liquid or lyophilized formulations are known in the prior art and are described above, we refer to the respective disclosure.
- the ⁇ -carboxylated polypeptide of interest that is produced by the manufacturing method according to the present invention is selected from the blood coagulation factors, preferably Factor VII.
- Figure 1 shows a flow chart of the purification process.
- Figure 2 shows gel pictures of gel electrophoreses using non-reducing (A) and reducing (B) conditions. Under non-reducing conditions, Factor VI I monomers, dimers and fragments are visible. Under reducing conditions, the not activated proprotein forming one band at about 50 kDa and the activated (cleaved) Factor Vila forming two bands at about 30 kDa and 20 kDa, respectively, are visible. Lane 1 : Factor VI I after affinity chromatography (step (1 )); lane 2: NovoSeven (commercially available control product of NovoNordisk) ; lane 3: Factor VI I after multimodal anion exchange chromatography and activation (steps 2 and 3) ; lane M: molecular weight marker.
- Figure 3 shows a gel picture of a gel electrophoresis using reducing conditions.
- the not activated proprotein forming one band at about 50 kDa and the activated (cleaved) Factor Vila forming two bands at about 30 kDa and 20 kDa, respectively, are visible.
- Lanes 1 to 5 Factor VI I after incubation at pH 8 for 0, 1 , 2, 4 and 6 days; lane M: molecular weight marker.
- Figure 4 shows the 280nm spectrum of the Ultimate 3000 size exclusion chromatography of a Factor VI I construct incubated for 1 h (black), 3h (dark grey), 5h (light grey), 8h (long dashes), 12h (short dashes) or 24h (dots) at pH 3.
- the graphs were normalized to the monomer peak of the 24h sample. Elution times of the different species: fragments: 9.6 min; monomers: 8.1 min; dimers: 7.2 min; aggregates: 6.3 min.
- Step 1 Affinity chromatography (AC; VI I Select column)
- the crude Factor VI I forming the starting material was derived from cell culture supernatants containing recombinant Factor VII.
- Loading/wash buffer 50 mM Tris, 1 50 mM NaCI, 1 00 ⁇ ZnCI 2 , pH 7.5
- Elution buffer 50 mM glycine, 100 ⁇ ZnCI 2 , pH 3.0
- the Vl lSelect column (GE Healthcare) was equilibrated with loading buffer.
- the cell culture supernatant containing Factor VI I was loaded onto the column. After loading, unbound material is washed out for about 60 CV (column volumes) using wash buffer.
- Factor VII is eluted applying the elution buffer.
- the fractions comprising Factor VI I are pooled and incubated for 1 h for virus inactivation. The composition is then neutralized to pH 8.0.
- a composition comprising Factor VII is obtained, wherein at least 85% of the Factor VI I protein is monomeric (see Figure 1 , lane 1 ).
- the remaining Factor VI I proteins form aggregates or dimers.
- the amount of host cell proteins (HCP) is at about 300 to 600 ppm and human serum albumin (HSA) is at about 10 to 30 ppm.
- Step 2 Multimodal anion exchange chromatography (MM AIX; Caoto adhere ImpRes column)
- Loading/wash buffer 25 mM Tris, 1 00 ⁇ ZnCI 2 , pH 8.0
- Elution buffer 25 mM Tris, 30 mM CaCI 2 , 100 ⁇ ZnCI 2 , pH 8.0
- the neutralized composition obtained from step 1 was loaded onto the Capto adhere ImpRes column (GE Healthcare), equilibrated with loading buffer. After washing, ⁇ -carboxylated Factor VI I is eluted via pseudo-affinity elution using calcium ions.
- the composition containing Factor VI I obtained from the multimodal anion exchange chromatography contains at least 98% of the Factor VI I in monomeric form (see Figure 1 , lane 3).
- HCP is at about 20 to 30 ppm and HSA is below the detection minimum.
- the yield of Factor VII after the multimodal anion exchange chromatography is about 40% to 50% compared to the amount of Factor VII in the cell culture supernatant.
- the pooled eluate of the multimodal anion exchange chromatography is incubated at 2 to S ⁇ for several days. After 4 days, essentially all Factor VII was cleaved, forming the activated protein consisting of a light chain and a heavy chain (see Figure 2).
- Step 4 Diafiltration (membrane having a cut-off of 10 kDa)
- the composition containing Factor VII obtained after activation step 3 was then subjected to diafiltration.
- the buffer is exchanged to preformulation buffer and Factor VII is adjusted to the desired concentration.
- the pH is adjusted to 6.0 in order to slow down or stop the activation of Factor VII.
- Step 5 Formulation of the final composition
- the product from the diafiltration step was directly applied to a 20 nm nanofiltration device to remove virus particles. Then the composition is lyophilized to obtain the final product.
- Example 2 A ⁇ -carboxylated Factor VII construct was incubated at pH 3 in the elution buffer of the affinity chromatography column (50 mM glycine, 100 ⁇ ZnCI 2 , pH 3.0) for different time intervals. Then the sample was neutralized with 1 /9 volume 200 mM histidine (pH 7.5) to a final pH of about 6. After incubation for 1 h, 3h, 5h, 8h, 12h or 24h and subsequent neutralization, the amounts of fragments, monomer, dimer and aggregate were determined by SEC (see Figure 4): Table 3
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Abstract
La présente invention concerne un procédé pour la purification d'un polypeptide γ-carboxylé de type facteur VII, qui consiste à soumettre un liquide contenant ledit polypeptide γ-carboxylé à des étapes de chromatographie d'affinité et de chromatographie d'échange d'anions multimodale. L'invention concerne également un procédé de production pour l'obtention du polypeptide γ-carboxylé d'intérêt.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020102505A1 (fr) * | 2018-11-15 | 2020-05-22 | Bayer Healthcare Llc | Procédés d'inactivation virale pour la fabrication continue d'anticorps |
RU2807176C2 (ru) * | 2018-11-15 | 2023-11-10 | БАЙЕР ХелсКер ЛЛСи | Способы инактивации вируса для непрерывного получения антител |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006067230A1 (fr) * | 2004-12-23 | 2006-06-29 | Novo Nordisk Health Care Ag | Reduction du contenu en contaminants proteiques de compositions renfermant une proteine d'interet dependante de la vitamine k |
WO2007026000A2 (fr) * | 2005-08-31 | 2007-03-08 | Novo Nordisk Health Care Ag | Anticorps specifiques de fvii et leur utilisation |
WO2008028686A2 (fr) | 2006-09-10 | 2008-03-13 | Glycotope Gmbh | Système de production à haut rendement, entièrement humain, pour anticorps et protéines améliorés |
WO2009024620A2 (fr) * | 2007-08-23 | 2009-02-26 | Octapharma Ag | Procédé pour l'isolement et la purification d'une protéine cible exempte d'une protéine prion (prpsc) |
WO2009156430A1 (fr) * | 2008-06-24 | 2009-12-30 | Octapharma Ag | Procédé de purification du facteur de coagulation viii |
WO2010063717A1 (fr) * | 2008-12-02 | 2010-06-10 | Novo Nordisk A/S | Purification de polypeptides |
WO2012051147A1 (fr) * | 2010-10-11 | 2012-04-19 | Abbott Laboratories | Procédés de purification de protéines |
WO2014004103A1 (fr) * | 2012-06-29 | 2014-01-03 | Emd Millipore Corporation | Méthodes permettant d'inactiver des virus lors d'un procédé de purification de protéines |
-
2016
- 2016-06-24 WO PCT/EP2016/064634 patent/WO2016207328A1/fr active Application Filing
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006067230A1 (fr) * | 2004-12-23 | 2006-06-29 | Novo Nordisk Health Care Ag | Reduction du contenu en contaminants proteiques de compositions renfermant une proteine d'interet dependante de la vitamine k |
WO2007026000A2 (fr) * | 2005-08-31 | 2007-03-08 | Novo Nordisk Health Care Ag | Anticorps specifiques de fvii et leur utilisation |
WO2008028686A2 (fr) | 2006-09-10 | 2008-03-13 | Glycotope Gmbh | Système de production à haut rendement, entièrement humain, pour anticorps et protéines améliorés |
WO2009024620A2 (fr) * | 2007-08-23 | 2009-02-26 | Octapharma Ag | Procédé pour l'isolement et la purification d'une protéine cible exempte d'une protéine prion (prpsc) |
WO2009156430A1 (fr) * | 2008-06-24 | 2009-12-30 | Octapharma Ag | Procédé de purification du facteur de coagulation viii |
WO2010063717A1 (fr) * | 2008-12-02 | 2010-06-10 | Novo Nordisk A/S | Purification de polypeptides |
WO2012051147A1 (fr) * | 2010-10-11 | 2012-04-19 | Abbott Laboratories | Procédés de purification de protéines |
WO2014004103A1 (fr) * | 2012-06-29 | 2014-01-03 | Emd Millipore Corporation | Méthodes permettant d'inactiver des virus lors d'un procédé de purification de protéines |
Non-Patent Citations (4)
Title |
---|
BROZE G J ET AL: "HUMAN FACTOR VII", METHODS IN ENZYMOLOGY, ACADEMIC PRESS, US, vol. 80, 1 January 1981 (1981-01-01), pages 228 - 237, XP009062920, ISSN: 0076-6879, DOI: 10.1016/S0076-6879(81)80021-3 * |
JOHNSTON A ET AL: "LOW PH, CAPRYLATE INCUBATION AS A SECOND VIRAL INACTIVATION STEP IN THE MANUFACTURE OF ALBUMIN PARAMETRIC AND VALIDATION STUDIES", BIOLOGICALS, ACADEMIC PRESS LTD., LONDON, GB, vol. 31, no. 3, 1 September 2003 (2003-09-01), pages 213 - 221, XP001149490, ISSN: 1045-1056, DOI: 10.1016/S1045-1056(03)00062-9 * |
THIM L ET AL: "Amino acid sequence and posttranslational modifications of human factor VII-a from plasma and transfected baby hamster kidney cells", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 27, no. 20, 1 January 1988 (1988-01-01), pages 7785 - 7793, XP002203678, ISSN: 0006-2960, DOI: 10.1021/BI00420A030 * |
TOMOKIYO K ET AL: "Large-scale production and properties of human plasma-derived activated Factor VII concentrate", VOX SANGUINIS, S. KARGER AG, BASEL, CH, vol. 84, no. 1, 1 January 2003 (2003-01-01), pages 54 - 64, XP002286502, ISSN: 0042-9007, DOI: 10.1046/J.1423-0410.2003.00247.X * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020102505A1 (fr) * | 2018-11-15 | 2020-05-22 | Bayer Healthcare Llc | Procédés d'inactivation virale pour la fabrication continue d'anticorps |
CN113015795A (zh) * | 2018-11-15 | 2021-06-22 | 拜耳医药保健有限责任公司 | 用于连续制备抗体的病毒灭活方法 |
US20220002679A1 (en) * | 2018-11-15 | 2022-01-06 | Bayer Healthcare Llc | Viral Inactivation Methods for Continuous Manufacturing of Antibodies |
RU2807176C2 (ru) * | 2018-11-15 | 2023-11-10 | БАЙЕР ХелсКер ЛЛСи | Способы инактивации вируса для непрерывного получения антител |
US12123028B2 (en) * | 2019-11-14 | 2024-10-22 | Bayer Healthcare Llc | Viral inactivation methods for continuous manufacturing of antibodies |
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