The present disclosure relates to methods of detecting and/or quantifying host cell protein impurities during the production of a product, e.g., a recombinant protein, e.g., an antibody.

BACKGROUND

Host cell protein (HCP) is an unwanted complex mixture of host proteins which may present in the final product after various manufacturing process. Those HCPs can pose risks to, inter alia, product efficacy and patient safety. Therefore, there is a need for methods for detecting and quantifying HCPs in final biopharmaceutical products.

SUMMARY

The present disclosure provides, inter alia, for a highly sensitive method for the detection of HCPs. In one aspect, disclosed herein are methods of detecting, monitoring, identifying, or quantifying a GS-CHO host cell protein (HCP) in a sample comprising a recombinant polypeptide, the method comprising: a) providing or obtaining a sample comprising a recombinant polypeptide; b) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCPs; e) detecting the presence of GS-CHO HCP bound to the antibody using a detection means for the detectable antibody; and f) optionally quantifying the level of GS-CHO HCP detected; g) optionally identifying one of more of the GS-CHO HCP detected; and h) optionally quantifying one or more of the GS-CHO HCP identified.

In some embodiments, recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin. In some embodiments, the recombinant polypeptide is a therapeutic polypeptide. In some embodiments, the recombinant polypeptide is one disclosed in Table 1. In some embodiments, the recombinant polypeptide is one disclosed in Table 2. In some embodiments, the recombinant polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a therapeutic antibody.

In some embodiments, the sample is derived from a downstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from an upstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from a final product of the recombinant polypeptide. In some embodiments the method comprises, two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the

recombinant polypeptide. In some embodiments, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is amplified by a fluorimetric reagent. In some embodiments, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, the present disclosure provides methods of manufacturing a recombinant polypeptide drug product, the method comprising: a) providing or obtaining a sample of a recombinant polypeptide preparation; b) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; e) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and f) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP is below a preselected reference level, thereby manufacturing a recombinant polypeptide drug product.

In some embodiments, the processing comprises one or more of: formulating the polypeptide preparation; processing the polypeptide preparation into a drug product; combining the polypeptide preparation with a second component, e.g., an excipient or buffer; changing the concentration of the polypeptide in the preparation; lyophilizing the polypeptide preparation; combining a first and second aliquot of the polypeptide to provide a third, larger, aliquot;

dividing the polypeptide preparation into smaller aliquots; disposing the polypeptide preparation into a container, e.g., a gas or liquid tight container; packaging the polypeptide preparation; associating a container comprising the polypeptide preparation with a label (e.g., labeling); shipping or moving the polypeptide preparation to a different location.

In some embodiments, the processing step comprises combining the polypeptide preparation with an excipient or buffer. In some embodiments, the reference level is about lOOppm (e.g., less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1 ppm). In some embodiments, the reference level is between lppm and lOOOppm. In some embodiments, the reference level is at least lppm, lOpppm, 50ppm, lOOppm, 500ppm, or lOOOppm. In some embodiments, the reference level is no greater than lppm, 5ppm, lOpppm, 20ppm, 30ppm, 40ppm, 50ppm, lOOppm, 500ppm, or lOOOppm. In some embodiments, the level of GS-CHO HCP below the reference level is a specification for commercial release of the drug product. In some

embodiments, in the level of GS-CHO HCP below the reference level is a specification for commercial release of a drug product under Section 351(a) of the Public Health Service (PHS) Act. In some embodiments, the level of GS-CHO HSP is acquired for one, two, or more samples or batches.

In some embodiments, recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin. In some embodiments, the recombinant polypeptide is a therapeutic polypeptide. In some embodiments, the recombinant polypeptide is one disclosed in Table 1. In some embodiments, the recombinant polypeptide is one disclosed in Table 2. In some embodiments, the recombinant

polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a therapeutic antibody.

In some embodiments, the sample is derived from a downstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from an upstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from a final product of the recombinant polypeptide. In some embodiments the method comprises, two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the

recombinant polypeptide. In some embodiments, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is amplified by a fiuorimetric reagent. In some embodiments, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, the present disclosure provides methods of manufacturing a recombinant polypeptide drug product, the method comprising: a) culturing a GS-CHO cell cultured under conditions suitable for production, e.g., expression and secretion, of a recombinant polypeptide, b) separating the secreted recombinant polypeptide from the host cell; c) subjected the secreted recombinant polypeptide to one or more purification steps to produce a recombinant polypeptide preparation; d) taking a sample of a recombinant polypeptide preparation; e) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; f) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; g) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; andh) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP is below a preselected reference level, thereby manufacturing a recombinant polypeptide drug product.

In some embodiments, the processing comprises one or more of: formulating the polypeptide preparation; processing the polypeptide preparation into a drug product; combining the polypeptide preparation with a second component, e.g., an excipient or buffer; changing the concentration of the polypeptide in the preparation; lyophilizing the polypeptide preparation; combining a first and second aliquot of the polypeptide to provide a third, larger, aliquot; dividing the polypeptide preparation into smaller aliquots; disposing the polypeptide preparation into a container, e.g., a gas or liquid tight container; packaging the polypeptide preparation; associating a container comprising the polypeptide preparation with a label (e.g., labeling); shipping or moving the polypeptide preparation to a different location.

In some embodiments, the processing step comprises combining the polypeptide preparation with an excipient or buffer. In some embodiments, the reference level is about lOOppm (e.g., less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1 ppm). In some embodiments, the reference level is between lppm and lOOOppm. In some embodiments, the reference level is at least lppm, lOpppm, 50ppm, lOOppm, 500ppm, or lOOOppm. In some embodiments, the reference level is no greater than lppm, 5ppm, lOpppm, 20ppm, 30ppm, 40ppm, 50ppm, lOOppm, 500ppm, or lOOOppm. In some embodiments, the level of GS-CHO HCP below the reference level is a specification for commercial release of the drug product. In some

embodiments, in the level of GS-CHO HCP below the reference level is a specification for commercial release of a drug product under Section 351(a) of the Public Health Service (PHS) Act. In some embodiments, the level of GS-CHO HSP is acquired for one, two, or more samples or batches.

In some embodiments, recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin. In some embodiments, the recombinant polypeptide is a therapeutic polypeptide. In some embodiments, the recombinant polypeptide is one disclosed in Table 1. In some embodiments, the recombinant polypeptide is one disclosed in Table 2. In some embodiments, the recombinant polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a therapeutic antibody.

In some embodiments, the sample is derived from a downstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from an upstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from a final product of the recombinant polypeptide. In some embodiments the method comprises, two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the

recombinant polypeptide. In some embodiments, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is amplified by a fluorimetric reagent. In some embodiments, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, the present disclosure provides methods of manufacturing a recombinant polypeptide drug product, the method comprising: a) providing or obtaining a sample of a recombinant polypeptide preparation from culture of a GS-CHO cell; b) acquiring a value for the GS-CHO host cell protein (HCP) in the sample, wherein the value is a function of, is

proportional to, or was obtained by, the binding of a polyclonal GS-CHO antibody to the sample, thereby manufacturing a recombinant polypeptide drug product. In some embodiments, the method comprises: evaluating the acquired value, e.g., by comparing the acquired value with a reference value. In some embodiments, the method comprises: responsive to the evaluation, classifying, selecting, accepting, releasing, processing into a drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into commerce, selling or offering for sale the preparation. In some embodiments, the value was determined by a method described herein.

In some embodiments, recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin. In some embodiments, the recombinant polypeptide is a therapeutic polypeptide. In some embodiments, the recombinant polypeptide is one disclosed in Table 1. In some embodiments, the recombinant polypeptide is one disclosed in Table 2. In some embodiments, the recombinant polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a therapeutic antibody.

In some embodiments, the sample is derived from a downstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from an upstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from a final product of the recombinant polypeptide.

In some embodiments the method comprises, two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the

recombinant polypeptide. In some embodiments, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is amplified by a fiuorimetric reagent. In some embodiments, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, the present disclosure provides methods of evaluating a process of manufacturing a recombinant polypeptide in a GS-CHO host cell, the method comprising: a) culturing a GS-CHO cell cultured under conditions suitable for production, e.g., expression and secretion, of a recombinant polypeptide, b) separating the secreted recombinant polypeptide from the host cell to provide a first polypeptide preparation; c) optionally taking a sample of the first polypeptide preparation; d) subjecting the first polypeptide preparation to a purification step to produce a second polypeptide preparation; e) taking a sample of the second polypeptide preparation; f) optionally subjecting the second polypeptide preparation to a second, third, fourth, fifth, or sixth purification step to provide subsequent polypeptide preparations; g) optionally taking a sample of any subsequent polypeptide preparations provided in step f); h) contacting and incubating each of the samples taken with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; i) separating each of the samples from the immobilized antibody-GS-CHO HCP complex; j) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; k) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and 1) based on said quantification, making a determination about the process, thereby evaluating a process of manufacturing a recombinant polypeptide.

In some embodiments, the method comprises comparing the level of GS-CHO HCP to a reference level, e.g., wherein if the level of GS-CHO HCP is below the reference level validating the process for use in the production of the recombinant polypeptide.

In some embodiments, the reference level is: i) between lppm and lOOOppm, ii) at least lppm, lOpppm, 50ppm, lOOppm, 500ppm, or lOOOppm, or iii) no greater than lppm, 5ppm, lOpppm, 20ppm, 30ppm, 40ppm, 50ppm, lOOppm, 500ppm, or lOOOppm.

In some embodiments, recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin. In some embodiments, the recombinant polypeptide is a therapeutic polypeptide. In some embodiments, the recombinant polypeptide is one disclosed in Table 1. In some embodiments, the recombinant polypeptide is one disclosed in Table 2. In some embodiments, the recombinant polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a therapeutic antibody.

In some embodiments, the sample is derived from a downstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from an upstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from a final product of the recombinant polypeptide. In some embodiments the method comprises, two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the

recombinant polypeptide. In some embodiments, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is amplified by a fiuorimetric reagent. In some embodiments, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, the present disclosure provides methods of evaluating a process of purifying a recombinant polypeptide, the method comprising: a) subjecting the first polypeptide preparation to a purification step to produce a second polypeptide preparation; b) taking a sample of the second polypeptide preparation; c) optionally subjecting the second polypeptide preparation to a second, third, fourth, fifth, or sixth purification step to provide subsequent polypeptide preparations; d) optionally taking a sample of any subsequent polypeptide preparations provided in step c); e) contacting and incubating each of the samples taken with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; f) separating each of the samples from the immobilized antibody-GS-CHO HCP complex; g) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; h) quantifying the level of GS-

CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and i) based on said quantification, making a determination about the process, thereby evaluating a process of purifying a recombinant therapeutic.

In some embodiments, the method comprises: comparing the level of GS-CHO HCP to a reference level. In some embodiments, the if the level of GS-CHO HCP is below the reference level validating the process for use in the production of the recombinant polypeptide. In some embodiments, the reference level is between lppm and lOOOppm. In some embodiments, the reference level is at least lppm, lOpppm, 50ppm, lOOppm, 500ppm, or lOOOppm. In some embodiments, the reference level is no greater than lppm, 5ppm, lOpppm, 20ppm, 30ppm, 40ppm, 50ppm, lOOppm, 500ppm, or lOOOppm.

In some embodiments, the purification step comprises one or more or any combination of size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), reverse phase chromatography (RPC), immobilized metal chelate chromatography, ammonium sulfate precipitation, thiophilic adsorption, protein A chromatography, protein G chromatography, protein L chromatography, and affinity

chromatography.

In some embodiments, the purification step comprises one or more affinity

chromatography steps. In some embodiments, the purification step comprises Protein A chromatography and one or more affinity chromatography steps. In some embodiments, the purification step comprises: protein A chromatography and CNBr chromatography. In some embodiments, the purification step comprises: protein A chromatography and subsequent CNBr chromatography. In some embodiments, the purification step comprises: protein A

chromatography and NHS chromatography. In some embodiments, the purification step comprises: protein A chromatography and subsequent NHS chromatography.

In some embodiments, the purification step comprises: i) Protein A chromatography and one or more affinity chromatography steps, ii) protein A chromatography and CNBr

chromatography, e.g., protein A chromatography and subsequent CNBr chromatography, or iii) protein A chromatography and NHS chromatography, e.g., protein A chromatography and subsequent NHS chromatography.

In some embodiments, recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin. In some embodiments, the recombinant polypeptide is a therapeutic polypeptide. In some embodiments, the recombinant polypeptide is one disclosed in Table 1. In some embodiments, the recombinant polypeptide is one disclosed in Table 2. In some embodiments, the recombinant polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a therapeutic antibody.

In some embodiments, the sample is derived from a downstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from an upstream processing step in a production process of the recombinant polypeptide. In some embodiments, the sample is derived from a final product of the recombinant polypeptide. In some embodiments the method comprises, two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the

recombinant polypeptide. In some embodiments, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is amplified by a fiuorimetric reagent. In some embodiments, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, provided herein are kits comprising: a) a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support; b) optionally a detectable antibody; c) optionally a wash buffer; and d) optionally a detecting reagent. In some embodiments, the detecting reagent is the same as the capture reagent. In some embodiments, the detecting reagent is the same as the capture reagent only modified to be amplified by a fiuorimetric reagent. In some embodiments, the detecting antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, provided herein are purified preparations of polyclonal anti-GS-CHO antibody. In some embodiments, the the anti-GS-CHO antibody is a sheep antibody. In some embodiments, the anti-GS-CHO antibody is a goat, horse, rabbit, bovine, murine, hamster, or human antibody. In some embodiments, the preparation is substantially free from non-HCP specific IgGs. In some embodiments, the preparation contains no more than 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% non-HCP specific IgGs. In some embodiments, the

preparation contains less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% non-HCP specific IgGs. In some embodiments, the purified by a method comprising: protein A chromatography, and one or more affinity chromatography steps (e.g., CNBr chromatography, NHS chromatography).

In one aspect, provided herein are reaction mixtures comprising a polyclonal preparation (e.g., a polyclonal preparation described herein) of anti-GS-CHO antibody and a recombinant polypeptide selected from Table 1 or Table 2. In some embodiments, the reaction mixture comprises an antibody that binds to the anti-GS-CHO antibody.

In one aspect, provided herein are methods of determining whether a batch or lot of a recombinant polypeptide drug product meets or satisfies a release specification, wherein the release specification is a preselected reference level of GS-CHO HCP the method comprising: a) providing or obtaining a sample of a recombinant polypeptide preparation; b) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; e) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and f) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP meets, satisfies, or is below the release specification, thereby determining whether a batch or lot of a recombinant polypeptide drug product meets or satisfies a release specification. In some embodiments, processing comprises: classifying, selecting, accepting, releasing, processing into a drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into commerce, selling or offering for sale the preparation. In some embodiments, the value was determined by a method described herein.

In one aspect, disclosed herein are methods of making a polyclonal anti-HCP antibody preparation, the method comprising: a) acquiring a sample comprising antibodies, e.g., antisera, from an animal (e.g., a sheep, rabbit, mouse, rat, hamster, goat, etc.) immunized with HCP of a host cell (e.g., a cell line, e.g., a mammalian, rodent, or insect cell or cell line, e.g., a CHO, SP2, NSO host cell); and b) separating anti-HCP antibody from the sample, e.g., by contacting the sample with an HCP-affinity reagent, thereby producing a polyclonal anti-HCP antibody preparation.

In some embodiments, step b) comprises: b. l) separating antibodies, e.g., IgG antibodies, from the sample to provide an antibody preparation; and b.2) separating anti-HCP antibody from the antibody preparation. In some embodiments, separating antibodies comprises contacting the antibodies with an affinity reagent, e.g., protein A. In some embodiments, separating antibodies comprises purifying a polyclonal anti-HCP antibody from the antisera through a purification method comprising protein A chromatography. In some embodiments, separating anti-HCP antibody from the antibody preparation comprises contacting the antibody preparation with an HCP-affinity reagent. In some embodiments, the HCP-affinity reagent comprises HCP coupled to a substrate, e.g., an insoluble or solid substrate, e.g., an agarose bead, e.g., Sepharase, e.g., cyanogen bromide (CNBr) or N-hydroxysuccinimide (NHS) chromatography) derivatized substrate.

In some embodiments, the purification removes at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs. In some embodiments, the purification removes at least 99% non-HCP specific IgGs. In some embodiments, the purification removes more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs. In some embodiments, the purification removes more than 99% non-HCP specific IgGs. In some embodiments, the polyclonal antibody preparation is substantially free from non-HCP specific IgGs. In some embodiments, the polyclonal antibody preparation contains no more than 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% non-HCP specific IgGs. In some embodiments, the polyclonal antibody preparation contains less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% non-HCP specific IgGs.

In one aspect, disclosed herein are methods of making a polyclonal anti-HCP antibody preparation for use in an ELISA for detection of HCPs in a recombinant polypeptide preparation, the method comprising: a) acquiring a sample comprising antibodies, e.g., antisera, from an animal (e.g., a sheep, rabbit, mouse, rat, hamster, goat, etc.) immunized with HCP of a host cell (e.g., a cell line, e.g., a mammalian, rodent, or insect cell or cell line, e.g., a CHO, SP2, NSO host cell); b) separating antibodies from the antisera with by contacting the antisera with a protein A affinity reagent to provide an antibody preparation; c) separating anti-HCP antibody from the antibody preparation by contacting the antibody preparation with an HCP-affinity reagent, thereby producing a polyclonal anti-HCP antibody preparation.

In some embodiments, the purification removes at least 50%, 60%, 70%, 80%, 90%,

95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs. In some embodiments, the purification removes at least 99% non-HCP specific IgGs. In some embodiments, the purification removes more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs. In some embodiments, the purification removes more than 99% non-HCP specific IgGs. In some embodiments, the polyclonal antibody preparation is substantially free from non-HCP specific IgGs. In some embodiments, the polyclonal antibody preparation contains no more than 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% non-HCP specific IgGs. In some embodiments, the polyclonal antibody preparation contains less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% non-HCP specific IgGs.

In one aspect, disclosed herein are methods of detecting, monitoring, identifying, or quantifying a host cell protein (HCP) (e.g., a CHO HCP) in a sample comprising a recombinant polypeptide, the method comprising: a) providing or obtaining a sample comprising a recombinant polypeptide; b) contacting and incubating the sample with a polyclonal anti-HCP antibody (e.g., an anti-CHO HCP antibody) immobilized to a solid support to form an immobilized antibody-HCP complex, wherein the polyclonal antibody has been produced by any of the methods described herein; c) separating the sample from the immobilized antibody-HCP complex; d) contacting the immobilized antibody-HCP complex with a detectable antibody that binds to GS-CHO HCPs; e) detecting the presence of HCP bound to the antibody using a detection means for the detectable antibody; and f) optionally quantifying the level of HCP detected; g) optionally identifying one of more of the HCP detected; and h) optionally quantifying one or more of the HCP identified.

In one aspect, disclosed herein are methods of detecting, monitoring, identifying, or quantifying a GS-CHO host cell protein (HCP) in a sample comprising a recombinant polypeptide, e.g., therapeutic polypeptide, the method comprising: a) providing or obtaining a sample comprising a recombinant polypeptide; b) incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a support, e.g., an insoluble or solid support, to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable reagent, e.g., an antibody that binds to GS-CHO HCPs; e) detecting the presence of GS-CHO HCP bound to the antibody using a detection means for the detectable antibody; and f) optionally quantifying the level of GS-CHO HCP detected; g) optionally identifying one of more of the GS-CHO HCP detected; and h) optionally quantifying one or more of the GS-CHO HCP identified.

In some embodiments, i) the recombinant polypeptide is a homopolymeric or

heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin, or ii) the recombinant polypeptide is one disclosed in Tables 1 or 2.

In some embodiments, the recombinant polypeptide is an antibody, e.g., a monoclonal antibody, e.g., a therapeutic antibody.

In some embodiments, i) the sample is derived from a downstream processing step in a production process of the recombinant polypeptide, ii) the sample is derived from an upstream processing step in a production process of the recombinant polypeptide, iii) the sample is derived from a final product of the recombinant polypeptide, or iv) the method comprises two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the recombinant polypeptide.

In some embodiments, the detectable antibody is: i) directly detectable, ii) amplified by a fluorimetric reagent, or iii) biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, disclosed herein are methods manufacturing a recombinant polypeptide, e.g., therapeutic polypeptide, drug product, the method comprising: a) providing or obtaining a sample of a recombinant polypeptide preparation; b) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; e) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and f) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP is below a preselected reference level, optionally wherein processing comprises one or more of: formulating the polypeptide preparation; processing the polypeptide preparation into a drug product;

combining the polypeptide preparation with a second component, e.g., an excipient or buffer; changing the concentration of the polypeptide in the preparation; lyophilizing the polypeptide preparation; combining a first and second aliquot of the polypeptide to provide a third, larger, aliquot; dividing the polypeptide preparation into smaller aliquots; disposing the polypeptide preparation into a container, e.g., a gas or liquid tight container; packaging the polypeptide preparation; associating a container comprising the polypeptide preparation with a label (e.g., labeling); shipping or moving the polypeptide preparation to a different location, thereby manufacturing a recombinant polypeptide drug product.

In one aspect, disclosed herein are methods of manufacturing a recombinant polypeptide, e.g., therapeutic polypeptide, drug product, the method comprising: a) culturing a GS-CHO cell cultured under conditions suitable for production, e.g., expression and secretion, of a

recombinant polypeptide, b) separating the secreted recombinant polypeptide from the host cell; c) subjected the secreted recombinant polypeptide to one or more purification steps to produce a recombinant polypeptide preparation; d) taking a sample of a recombinant polypeptide preparation; e) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; f) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; g) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and h) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP is below a preselected reference level, optionally wherein the processing comprises one or more of: formulating the polypeptide preparation; processing the polypeptide preparation into a drug product; combining the polypeptide preparation with a second component, e.g., an excipient or buffer; changing the concentration of the polypeptide in the preparation; lyophilizing the polypeptide preparation; combining a first and second aliquot of the polypeptide to provide a third, larger, aliquot;

dividing the polypeptide preparation into smaller aliquots; disposing the polypeptide preparation into a container, e.g., a gas or liquid tight container; packaging the polypeptide preparation; associating a container comprising the polypeptide preparation with a label (e.g., labeling); shipping or moving the polypeptide preparation to a different location, thereby manufacturing a recombinant polypeptide drug product.

In some embodiments, the reference level is: i) about lOOppm (e.g., less than 100, 90, 80,

70, 60, 50, 40, 30, 20, 10, 5, 1 ppm), ii) between lppm and lOOOppm, iii) at least lppm, lOpppm, 50ppm, lOOppm, 500ppm, or lOOOppm, or iv) no greater than lppm, 5ppm, lOpppm, 20ppm, 30ppm, 40ppm, 50ppm, lOOppm, 500ppm, or lOOOppm.

In some embodiments, the level of GS-CHO HCP below the reference level is a specification for commercial release of the drug product, e.g., under Section 351(a) of the Public Health Service (PHS) Act, and, optionally wherein the level of GS-CHO HCP is acquired for one, two, or more samples or batches.

In some embodiments, i) the recombinant polypeptide is a homopolymeric or

heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin, or ii) the recombinant polypeptide is one disclosed in Tables 1 or 2.

In some embodiments, the recombinant polypeptide is an antibody, e.g., a monoclonal antibody, e.g., a therapeutic antibody.

In some embodiments, i) the sample is derived from a downstream processing step in a production process of the recombinant polypeptide, ii) the sample is derived from an upstream processing step in a production process of the recombinant polypeptide, iii) the sample is derived from a final product of the recombinant polypeptide, or iv) the method comprises two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the recombinant polypeptide.

In some embodiments, the detectable antibody is: i) directly detectable, ii) amplified by a fluorimetric reagent, or iii) biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, disclosed herein are methods of manufacturing a recombinant polypeptide drug product, the method comprising: a) providing or obtaining a sample of a recombinant polypeptide preparation from culture of a GS-CHO cell; b) acquiring a value for the GS-CHO host cell protein (HCP) in the sample, and, c) optionally evaluating the acquired value, e.g., by comparing the acquired value with a reference value; and d) optionally responsive to the evaluation, classifying, selecting, accepting, releasing, processing into a drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into commerce, selling or offering for sale the preparation, wherein the value is a function of, is proportional to, or was obtained by, the binding of a polyclonal GS-CHO antibody to the sample, thereby manufacturing a recombinant polypeptide drug product.

In some embodiments, the value was determined by any method described herein.

In one aspect, disclosed herein are kits comprising: a) a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support; b) optionally a detectable antibody; c) optionally a wash buffer; and d) optionally a detecting reagent, e.g., a detecting reagent that is the same as the capture reagent or the same as the capture reagent only modified to be amplified by a fluorimetric reagent, and optionally wherein the detecting antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

In one aspect, disclosed herein are purified preparations of polyclonal anti-GS-CHO antibody, optionally wherein the anti-GS-CHO antibody is a sheep, goat, horse, rabbit, bovine, murine, hamster, or human antibody.

In some embodiments, the preparation is substantially free from non-HCP specific IgGs, e.g., the preparation contains no more than 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% non-HCP specific IgGs or the preparation contains less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% non-HCP specific IgGs.

In one aspect, disclosed herein are methods of determining whether a batch or lot of a recombinant polypeptide drug product meets or satisfies a release specification, wherein the release specification is a preselected reference level of GS-CHO HCP the method comprising: a) providing or obtaining a sample of a recombinant polypeptide preparation; b) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex; d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP; e) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and f) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP meets, satisfies, or is below the release specification, and optionally wherein processing comprises classifying, selecting, accepting, releasing, processing into a drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into

commerce, selling or offering for sale the preparation, thereby determining whether a batch or lot of a recombinant polypeptide drug product meets or satisfies a release specification.

In one aspect, disclosed herein are methods of making a polyclonal anti-HCP antibody preparation, the method comprising: a) acquiring a sample comprising antibodies, e.g., antisera, from an animal (e.g., a sheep, rabbit, mouse, rat, hamster, goat, etc.) immunized with HCP of a host cell (e.g., a cell line, e.g., a mammalian, rodent, or insect cell or cell line, e.g., a CHO, SP2, NSO host cell); and b) separating anti-HCP antibody from the sample, e.g., by contacting the sample with an HCP-affinity reagent, e.g., protein A, and optionally wherein b) comprises: b.l) separating antibodies, e.g., IgG antibodies, from the sample to provide an antibody preparation; and b.2) separating anti-HCP antibody from the antibody preparation, e.g., by contacting the antibody preparation with an HCP-affinity reagent, thereby producing a polyclonal anti-HCP antibody preparation.

In some embodiments, the purification removes: i) at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs, e.g., at least 99% non-HCP specific IgGs, or ii) more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs, e.g., more than 99% non-HCP specific IgGs.

In some embodiments, the polyclonal antibody preparation is substantially free from non-HCP specific IgGs, e.g., contains no more than 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% non-HCP specific IgGs or contains less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% non-HCP specific IgGs

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts an exemplary purification process for sheep GS-CHO HCP antibody.

DETAILED DESCRIPTION

For recombinant biopharmaceutical proteins to be acceptable for administration to human patients, it is important that residual contaminants resulting from the manufacture and purification process are removed from the final biological product. These process contaminants include culture medium proteins, immunoglobulin affinity ligands, viruses, endotoxin, DNA, and host cell proteins (HCPs). HCPs may generate a range of undesirable effects that may impact on

the safety profile of a product, including immune response, adjuvant activity, direct biological activity or product interaction/degradation. These host cell contaminants include process-specific HCPs, which are process-related impurities/contaminants in the biologies derived from recombinant DNA technology.

U.S. and foreign regulations often require removal of such contaminants. For example, the U.S. Food and Drug Administration (FDA) requires that biopharmaceuticals intended for in vivo human use should be as free as possible of extraneous immunoglobulin and non-immunoglobulin contaminants, and requires tests for detection and quantitation of potential contaminants, such as HCPs. The International Conference on Harmonization (ICH) provides guidelines on test procedures and acceptance criteria for biotechno logical/biological products. The guidelines suggest that for HCPs, a sensitive immunoassay capable of detecting a wide range of protein impurities be utilized. Although there are commercial assays and reagents available to detect immunoglobulins, DNA, endotoxins, viruses, etc., there are currently no commercial reagents or analytical methods available for the detection and quantification of cell line specific GS-CHO HCPs.

The present disclosure provides, inter alia, a robust, sensitive HCP ELISA platform assay and methods of use, to support multiple expression systems, including e.g., mammalian expression systems, e.g., the CHOK1SV expression system. The quality of an HCP ELISA method is defined mainly by 2 parameters (1) limit of quantitation (LOQ), and (2) percentage coverage of the HCPs that could be present as contaminants. Currently available HCP ELISAs provide 200ng/ml - lOOOng/mg for LOQ and 40% - 60% for coverage by spot counting. The ELISA described herein provide an LOQ of 2ng/mg and 71% coverage by spots matching (which is a better approach than spot counting), satisfying both of these parameters. The platform assay can support HCP detection in different products from a stable cell line and process (e.g., GS-CHO).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of and/or for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a cell" can mean one cell or more than one cell.

As used herein, the term "host cell protein" or "HCP" refers to any protein produced or encoded by the organism used to produce a recombinant polypeptide product and unrelated to the intended recombinant product. HCPs are undesirable in the final drug substance.

As used herein, the term "GS-CHO" refers to a Chinese hamster ovary (CHO) cell that expresses a recombinant glutathione synthetase (GS). For example, a CHO-K1SV GS (Lonza Biologies, Inc.).

As used herein, the term "GS-CHO HCP" refers to a host cell protein derived from a GS-CHO cell.

As used herein, the term "endogenous" refers to any material from or naturally produced inside an organism, cell, tissue or system.

As used herein, the term "exogenous" refers to any material introduced to or produced outside of an organism, cell, tissue or system. Accordingly, "exogenous nucleic acid" refers to a nucleic acid that is introduced to or produced outside of an organism, cell, tissue or system. In an embodiment, sequences of the exogenous nucleic acid are not naturally produced, or cannot be naturally found, inside the organism, cell, tissue, or system that the exogenous nucleic acid is introduced into. In one embodiment, the sequences of the exogenous nucleic acids are non-naturally occurring sequences, or encode non-naturally occurring products.

As used herein, the term "heterologous" refers to any material from one species, when introduced to an organism, cell, tissue or system from a different species.

As used herein, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" are used interchangeably and refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA thereof, and polymers thereof in either single- or double-stranded form. The term "nucleic acid" includes, but is not limited to, a gene, cDNA, or an

mR A. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless specifically limited, the term encompasses molecules containing analogues or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally or non-naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91 -98 (1994)).

As used herein, the terms "peptide," "polypeptide," and "protein" are used

interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds, or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. In one embodiment, a protein may comprise of more than one, e.g., two, three, four, five, or more, polypeptides, in which each polypeptide is associated to another by either covalent or non-covalent bonds/interactions. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.

As used herein, "product" refers to a molecule, nucleic acid, polypeptide, or any hybrid thereof, that is produced, e.g., expressed, by a cell which has been modified or engineered to produce the product. In one embodiment, the product is a naturally occurring product or a non-naturally occurring product, e.g., a synthetic product. In one embodiment, a portion of the product is naturally occurring, while another portion of the product is non-naturally occurring.

In one embodiment, the product is a polypeptide, e.g., a recombinant polypeptide. In one embodiment, the product is suitable for diagnostic or pre-clinical use. In another embodiment, the product is suitable for therapeutic use, e.g., for treatment of a disease. In one embodiment, the product is selected from Table 1 or Table 2. In one embodiment, the modified or engineered cells comprise an exogenous nucleic acid that controls expression or encodes the product. In other embodiments, the modified or engineered cells comprise other molecules, e.g., that are not nucleic acids, that controls the expression or construction of the product in the cell.

In one embodiment, the modification of the cell comprises the introduction of an exogenous nucleic acid comprising a nucleic acid sequence that controls or alters, e.g., increases, the expression of an endogenous nucleic acid sequence, e.g., endogenous gene. In such embodiments, the modified cell produces an endogenous polypeptide product that is naturally or endogenously expressed by the cell, but the modification increases the production of the product and/or the quality of the product as compared to an unmodified cell, e.g., as compared to endogenous production or quality of the polypeptide.

In another embodiment, the modification of the cell comprises the introduction of an exogenous nucleic acid encoding a recombinant polypeptide as described herein. In such embodiments, the modified cell produces a recombinant polypeptide product that can be naturally occurring or non-naturally occurring. In such embodiments, the modified cell produces a recombinant polypeptide product that can also be endogenously expressed by the cell or not. In embodiments where the recombinant polypeptide product is also endogenously expressed by the cell, the modification increases the production of the product and/or the quality of the product as compared to an unmodified cell, e.g., as compared to endogenous production or quality of the polypeptide.

As used herein, "recombinant polypeptide" or "recombinant protein" refers to a polypeptide that can be produced by a cell described herein. A recombinant polypeptide is one for which at least one nucleotide of the sequence encoding the polypeptide, or at least one nucleotide of a sequence which controls the expression of the polypeptide, was formed by genetic engineering (of the cell or of a precursor cell). E.g., at least one nucleotide was altered, e.g., it was introduced into the cell or it is the product of a genetically engineered rearrangement. In an embodiment, the sequence of a recombinant polypeptide does not differ from a naturally occurring isoform of the polypeptide or protein. In an embodiment, the amino acid sequence of

the recombinant polypeptide differs from the sequence of a naturally occurring isoform of the polypeptide or protein. In an embodiment, the recombinant polypeptide and the cell are from the same species. In an embodiment, the recombinant polypeptide is endogenous to the cell, in other words, the cell is from a first species and the recombinant polypeptide is native to that first species. In an embodiment, the amino acid sequence of the recombinant polypeptide is the same as or is substantially the same as, or differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% from, a polypeptide encoded by the endogenous genome of the cell. In an embodiment, the recombinant polypeptide and the cell are from different species, e.g., the recombinant polypeptide is a human polypeptide and the cell is a non-human, e.g., a rodent, e.g., a CHO, or an insect cell. In an embodiment, the recombinant polypeptide is exogenous to the cell, in other words, the cell is from a first species and the recombinant polypeptide is from a second species. In one embodiment, the polypeptide is a synthetic polypeptide. In one embodiment, the polypeptide is derived from a non-naturally occurring source. In an embodiment, the

recombinant polypeptide is a human polypeptide or protein which does not differ in amino acid sequence from a naturally occurring isoform of the human polypeptide or protein. In an embodiment, the recombinant polypeptide differs from a naturally occurring isoform of the human polypeptide or protein at no more than 1 , 2, 3, 4, 5, 10, 15 or 20 amino acid residues. In an embodiment, the recombinant polypeptide differs from a naturally occurring isoform of the human polypeptide by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15% of its amino acid residues.

"Acquire" or "acquiring" as the terms are used herein, refer to obtaining possession of a physical entity, or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent.

"Acquiring a sample" as the term is used herein, refers to obtaining possession of a sample, e.g., a tissue sample or nucleic acid sample, by "directly acquiring" or "indirectly acquiring" the sample. "Directly acquiring a sample" means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample. "Indirectly acquiring a sample" refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample). Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient. Exemplary changes include making a physical entity from a starting material, dissecting or scraping a tissue; separating or purifying a substance (e.g., a sample tissue or a nucleic acid sample); combining two or more separate entities into a mixture; performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a sample includes performing a process that includes a physical change in a sample or another substance, e.g., as described above.

"Downstream processing step" as the term is used herein, refers to any step in the production process of the recombinant polypeptide that follows after/post a chosen separation step, e.g., after/post separation of the cells from the media containing the recombinant protein product.

"Upstream processing step" as the term is used herein, refers to any step in the production process of the recombinant polypeptide that precedes or is prior to a chosen

separation step, e.g., prior to separation of the recombinant protein product from cell containing media.

"Final product" as the term is used herein, refers to a recombinant polypeptide substantially purified from other cellular components. In some embodiments, a final product is a recombinant polypeptide that is formulated for storage, shipping, and/or use as a drug.

"Production process" as the term is used herein, refers to a method or series of steps that produces a recombinant polypeptide. In some embodiments, a production process is a manufacturing process designed to produce a recombinant polypeptide. In some embodiments, a production process is a method for purifying a recombinant polypeptide. In some embodiments, a production process further comprises evaluation steps, analysis steps, and/or determination steps based on said evaluation and analysis. In some embodiments, the final product of a production process is a recombinant polypeptide formulated for use as a drug.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

GENERAL DESCRIPTION OF THE PROCESS

The methods described herein may be characterized as an enzyme linked immunosorbant assay (ELIS A). The general method of an ELISA and ELISA variations are known to those skilled in the art. The following is a general description of the methods described herein solely to illustrate the timeline of steps involved in the methods. The disclosure should not be limited to or restricted to this description in anyway. The components are merely described for purposes of illustration.

A coating agent (e.g., anti-GS-CHO HCP antibody) is immobilized onto a solid support (e.g., microtiter plate). Once the coating agent has been immobilized on the solid support the remaining binding sites on the solid support are blocked using a blocking buffer (e.g., 0.2% casein in IX DPBS). The blocking buffer contains a component capable of non-specifically binding to the solid support to saturate the open binding sites, therefore preventing binding of free ligand to any excess sites on the solid support. The specific conditions of the coating and blocking incubation periods are selected to maximize coating of the solid support; and variations are known to those skilled in the art. After coating and blocking of the solid support, the standards and/or samples (e.g., samples being tested for HCPs) to be analyzed are appropriately diluted in a suitable dilution buffer (e.g., 0.2% casein in IX DPBS) and added to the immobilized support. The specific conditions of the standard/sample incubation period are selected to maximize sensitivity of the assay and minimize dissociation; variations are known to those skilled in the art.

Any non-immobilized standard/sample (e.g., HCP) is removed by washing the solid support with a suitable wash buffer (e.g., 0.05% Tween® 20 in IX DPBS), a suitable number of times (e.g., 3X with 300μ1). The specific wash buffer and number washes at any wash step are selected to minimize background and maximize sensitivity; and variations are known to those skilled in the art. Any immobilized standard/sample can then be detected either indirectly or directly. For indirect detection, an antibody (primary antibody) against the antigen of interest in the standard/sample (e.g., anti-GS-CHO HCP antibody) is added to the solid support; and the incubation conditions selected to maximize signal amplification. Any non-immobilized antibody is then removed by washing the solid support with a suitable washing buffer, a suitable number of times. An antibody conjugated to a moiety that is detectable by some means (detecting antibody) and capable of binding to the immobilized standard or HCP in sample is then added to the solid support. Any unbound detecting antibody is then removed by washing the solid support with a suitable washing buffer, a suitable number of times. The level of the antigen of interest in the standard/sample (e.g., HCP) bound to the coating agent can be determined using a detection system compatible with the detection antibody employed. A suitable detection means will be known to one of skill in the art.

In the instance direct detection of the antigen of interest in the standard/sample is employed, the primary antibody is conjugated to a moiety that is detectable by some means and is thus also the detecting antibody, i.e., the primary antibody and detecting antibody are the same. Any unbound detecting antibody is then removed by washing the solid support with a suitable washing buffer, a suitable number of times. The level of the antigen of interest in the standard/sample (e.g., HCP) bound to the coating agent can be determined using a detection

system compatible with the primary/detection antibody employed. A suitable detection system will be known to one of skill in the art.

The results demonstrate, inter alia, a robust, sensitive HCP ELISA platform assay to support multiple host cell expression systems, including e.g., CHOK1 SV expression system and GS-CHO expression system. The improved sensitivity of the ELISA assay is at least about-40-fold, better than a HCP ELISA method not using the polyclonal HCP antibody purified as described herein. During the HCP antibody purification, over 99% of total IgGs showed no immunoresponse to the HCP and were therefore eliminated from the final HCP antibody. As there is no exactly matched standards for quantitation cross the industry and regulation, the sensitivity of HCP assay is assay specific for cell line and process. The methods described herein can be used to generate a robust, sensitive HCP ELISA platform assay for any recombinant host cell, including but not limited to, for example mammalian host cells (e.g., CHO, e.g., GS-CHO), eukaryotic host cell, prokaryotic host cells, insect cell. Additionally, the methods of the invention can be used in conjunction with regulatory requirements for the production of recombinant proteins.

In some embodiments, the HCP ELISA assay of the present invention demonstrates acceptable accuracy, precision, and linearity for robust and reliable HCP detection at concentration ranges comprising 0.1 -100, 0.5-100, 1-100, 1.5-100, 2-100, 2.5-100, 3-100, 0.1-90, 0.5-90, 1-90, 1.5-90, 2-90, 2.5-90, 3-90, 0.1 -80, 0.5-80, 1-80, 1.5-80, 2-80, 2.5-80, 3-80, 0.1-70, 0.5-70, 1-70, 1.5-70, 2-70, 2.5-70, 3-70, 0.1-60, 0.5-60, 1-60, 1.5-60, 2-60, 2.5-60, or 3-60 ng/ml. In some embodiments, the HCP ELISA assay demonstrates acceptable accuracy, precision, and linearity for robust and reliable HCP detection at a concentration range comprising 2 through 60 ng/ml. In some embodiments, the HCP ELISA assay demonstrates acceptable accuracy, precision, and linearity for robust and reliable HCP detection at a concentration range comprising 3 through 80 ng/ml. In some embodiments, the limit of detection of the HCP ELISA assay of the present invention is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/ml. In some embodiments, the limit of detection of the HCP ELISA assay of the present invention is 0.5 ng/ml. In some embodiments, the limit of detection of the HCP ELISA assay of the present invention is 2 ng/ml. In some embodiments, the limit of quantitation of the HCP ELISA assay of the present invention is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/ml. In some embodiments, the limit of detection of the HCP ELISA assay of the present invention is 3 ng/ml.

ANTI-GS-CHO HCP ANTIBODIES

The methods described herein can provide an anti-GS-CHO HCP antibody for the detection of HCP. Anti- GS-CHO HCP antibodies can be made by standard molecular biology techniques. The antibodies can be derived from any species. The antibodies can be monoclonal or polyclonal or any variation of an "antibody" described herein. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a polyclonal antibody made in sheep. The antibodies can be directly detectable through the attachment of a detectable label, e.g., a chemical modification, enzyme conjugation, fluorescent dye labeling, luminescence labeling, etc. The antibodies can also not be directly detectable, and require a secondary means for detection. Antibodies with these various properties can be purchased or made by standard molecular biology techniques.

SAMPLES

Samples can include, but are not limited to samples taken from a cell culture at any period during recombinant protein production, e.g., any point during upstream or downstream processing. For example, samples may be taken from a cell culture (e.g., prior to separation of the recombinant protein product). Samples may be taken during the downstream processing process, e.g., post separation of the cells from the media containing the recombinant protein product. A sample may be taken at any and at multiple points during a purification process. A sample can include a sample of the final product. Accordingly, samples may include both in process products (prior to final formulation) and final products (e.g., final formulated products post all purification steps).

SOLID SUPPORT

Solid supports can include any surface to which a coating agent, e.g., anti-GS-CHO HCP antibody, or equivalent thereof can be immobilized on. Solid supports used for immobilization can be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, e.g., surfaces, particles, porous matrices, etc.

Examples of commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, and assays plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like including 96-wel microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water insoluble matrices such as cyanogens bromide-activated carbohydrates are suitably employed for coating reagent immobilization. The immobilized coating agent can be coated on a microtiter plate, for instance a multi-well microtiter plate that can be used to analyze several samples at one time. Solid support surfaces can also include but are not limited to, a membrane, e.g., a nitrocellulose membrane, a polytetraluorethylene membrane, cellulose acetate membrane, cellulose nitrate membrane, a solid surface coated with molecules containing hydrophobic groups, a solid surface coated with molecules containing hydrophilic groups. Solid support surfaces can be in the form of a microtiter plate, e.g., a polystyrene microtiter plate, cell culture plate, or any variation thereof.

CAPTURE REAGENT

The term "capture reagent" refers to an antibody which binds directly to the antigen of interest (e.g., GS-CHO HCP). The capture antibody can also be a detecting antibody with protein modification, e.g., biotinylation. The antibodies can be derived from different species, including but not limited to, human, rabbit, mouse, rat, sheep, goat, chicken, human, horse, dog, cat, hamster, monkey, chimpanzee, ovine, equine, porcine, bovine, primate, etc. In some

embodiments, the primary antibody is an anti-GS-CHO HCP antibody produced in sheep and affinity purified.

DETECTING REAGENT

The term "detecting reagent" or "detection reagent" refers to a labeled antibody used to detect an "antigen" or "antibody". The detecting antibody can also be a primary antibody. The antibodies can be derived from different species, including but not limited to, human, rabbit, mouse, rat, sheep, goat, chicken, human, horse, dog, cat, hamster, monkey, chimpanzee, ovine, equine, porcine, bovine, primate, etc. The label used on the detecting antibody can be any detectable functionality that does not interfere with the binding of HCP to the antibody.

Examples of suitable labels are those numerous labels known for use immunoassays, including moieties that may be detected directly, such as fiuorochrome, chemiluminscent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Suitable labeling methods that can be used in the present invention include, but are not limited to, isotope labeling, chemical modification, enzyme conjugation, fluorescent dye labeling, luminescence labeling, and other labeling methods commonly known to those skilled in the art.

Commercially available antibodies to a wide variety of antigens are known in the art. Those skilled in the art will be aware of a variety of labeling methods for an antibody or other detection agent. Labeling methods include but are not limited to, an enzyme such as horse radish peroxide (HRP), alkaline phosphatase (AP), beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, glactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP,

lactoperoxidase, or microperoxidase, biotin/avidin, biotin/streptavidin, biotin/Streptavidin-β-galactosidase with MUG, spin labels, bacteriophage labels, stable free radicals, or other enzymes and the like. A detection agent can also be labeled with radioactive isotopes, e.g., 1251, 32P, 14C, 3H, and 131I, or other isotope. Fluorescent labels can include but are not limited to fluorophores such as rare earth cheats or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.

4,737,456), luciferin, fluorescin isothiocynate (FITC), rhodamine, Texas Red, Alexa488, Cy5, Cy3, Alexa610, 7-AAD, propidium iodide, Cy7, phycoerythrin, etc. A detection agent can be labeled by a fiuorochrome (a fluorescent dye) that can be detected by fluorescent plate reader, a fluorescent microscope, a fluorometer, a camera, or scanner. A detection agent can also be labeled by a lumichrome which can be detected by luminescence methods. Alternatively a detection agent can be labeled biotin, which can bind to avidin or streptavidin. Avidin or streptavidin can be used as detection agents which can bind to biotin, biotinylated antibodies, or biotinylated polypeptides.

Conventional methods are available to bind these labels covalently to proteins or polypeptides. For instance, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat.

Nos. 3,940,475 (fiuorimetry) and 3,645,090 (enzymes); Hunter et al. Nature 144:945 (1962); David et al. Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods 40:219-230 (1981); and Nygren J. Histochem. and Cytochem. 30:407-412 (1982). Preferred labels herein are fluorescent to increase amplification and sensitivity to 8 pg/ml, more preferably biotin with streptavidin- -galactosidase and MUG for amplifying the signal.

The conjugation of such label, including the enzymes, to the antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques. See, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

DETECTION SYSTEM

The term "detection system" refers to a means which can be used to give a readout comprising information related to the quantity or relative amount of a protein or agent in a sample. The choice of a detection system depends on the choice of the detection antibody used. For example, if a detecting antibody is labeled with an enzyme, in which a chemical reaction can result in color or a chemiluminescence signal; the detection system may include a suitable substrate and any necessary reagents associated with the chemical reaction and a means of detecting the chemical reaction, for example, visual inspection, a device capable of detecting the signal, e.g., an absorbance plate reader, a chemiluminescence plate reader, CCD camera, etc; alternatively, if the detection antibody is fluorescently labeled, a fluorescence microscope, a fluorescence plate reader, a fluorescence cell sorter, a fluorescence scanner, camera, etc. may be used; alternatively, if the detecting antibody is isotope labeled, X ray film or other isotope sensitive material may be used.

What is claimed is:

A method of detecting, monitoring, identifying, or quantifying a GS-CHO host cell protein (HCP) in a sample comprising a recombinant polypeptide, e.g., therapeutic polypeptide, the method comprising:

a) providing or obtaining a sample comprising a recombinant polypeptide;

b) incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a support, e.g., an insoluble or solid support, to form an immobilized antibody-GS-CHO HCP complex;

c) separating the sample from the immobilized antibody-GS-CHO HCP complex;

d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCPs;

e) detecting the presence of GS-CHO HCP bound to the antibody using a detection means for the detectable antibody; and

f) optionally quantifying the level of GS-CHO HCP detected;

g) optionally identifying one of more of the GS-CHO HCP detected; and

h) optionally quantifying one or more of the GS-CHO HCP identified.

2. The method of claim 1, wherein:

i) the recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin, or

ii) the recombinant polypeptide is one disclosed in Tables 1 or 2.

3. The method of claim 1 or 2, wherein the recombinant polypeptide is an antibody, e.g., a monoclonal antibody, e.g., a therapeutic antibody.

4. The method of any one of claims 1-3, wherein:

i) the sample is derived from a downstream processing step in a production process of the recombinant polypeptide,

ii) the sample is derived from an upstream processing step in a production process of the recombinant polypeptide,

iii) the sample is derived from a final product of the recombinant polypeptide, or iv) the method comprises two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the recombinant polypeptide.

5. The method of claim 1 , wherein the detectable antibody is:

i) directly detectable,

ii) amplified by a fluorimetric reagent, or

iii) biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3 ',5,5'-tetramethyl benzidine.

6. A method of manufacturing a recombinant polypeptide, e.g., therapeutic polypeptide, drug product, the method comprising:

a) providing or obtaining a sample of a recombinant polypeptide preparation;

b) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex;

c) separating the sample from the immobilized antibody-GS-CHO HCP complex;

d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP;

e) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and

f) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP is below a preselected reference level, optionally wherein processing comprises one or more of: formulating the polypeptide preparation; processing the polypeptide preparation into a drug product; combining the polypeptide preparation with a second component, e.g., an excipient or buffer; changing the concentration of the polypeptide in the preparation; lyophilizing the polypeptide preparation; combining a first and second aliquot of the polypeptide to provide a third, larger, aliquot; dividing the polypeptide preparation into smaller aliquots; disposing the polypeptide preparation into a container, e.g., a gas or liquid tight container; packaging the polypeptide preparation; associating a container comprising the polypeptide preparation with a label (e.g., labeling); shipping or moving the polypeptide preparation to a different location, thereby manufacturing a recombinant polypeptide drug product.

7. A method of manufacturing a recombinant polypeptide, e.g., therapeutic polypeptide, drug product, the method comprising:

a) culturing a GS-CHO cell cultured under conditions suitable for production, e.g., expression and secretion, of a recombinant polypeptide,

b) separating the secreted recombinant polypeptide from the host cell;

c) subjected the secreted recombinant polypeptide to one or more purification steps to produce a recombinant polypeptide preparation;

d) taking a sample of a recombinant polypeptide preparation;

e) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex;

c) separating the sample from the immobilized antibody-GS-CHO HCP complex;

f) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP;

g) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and

h) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP is below a preselected reference level, optionally wherein the processing comprises one or more of: formulating the polypeptide preparation; processing the polypeptide preparation into a drug product; combining the polypeptide preparation with a second component, e.g., an excipient or buffer; changing the concentration of the polypeptide in the preparation; lyophilizing the polypeptide preparation; combining a first and second aliquot of the polypeptide to provide a third, larger, aliquot; dividing the polypeptide preparation into smaller aliquots; disposing the polypeptide preparation into a container, e.g., a gas or liquid tight container; packaging the polypeptide preparation; associating a container comprising the polypeptide preparation with a label (e.g., labeling); shipping or moving the polypeptide preparation to a different location, thereby manufacturing a recombinant polypeptide drug product.

8. The method of claim 6 or 7, wherein the reference level is:

i) about lOOppm (e.g., less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1 ppm), ii) between lppm and lOOOppm,

iii) at least lppm, lOpppm, 50ppm, lOOppm, 500ppm, or lOOOppm, or

iv) no greater than lppm, 5ppm, lOpppm, 20ppm, 30ppm, 40ppm, 50ppm, lOOppm,

500ppm, or lOOOppm.

9. The method of claim 6 or 7, wherein the level of GS-CHO HCP below the reference level is a specification for commercial release of the drug product, e.g., under Section 351(a) of the Public Health Service (PHS) Act, and, optionally wherein the level of GS-CHO HCP is acquired for one, two, or more samples or batches.

10. The method of claim 6 or 7, wherein

i) the recombinant polypeptide is a homopolymeric or heteropolymeric polypeptide, e.g., a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or an antibody fragment, e.g., a human antibody or a humanized antibody or fragment thereof, e.g., a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep, or cow antibody, typically of rabbit origin, or

ii) the recombinant polypeptide is one disclosed in Tables 1 or 2.

11. The method of claim 6 or 7, wherein the recombinant polypeptide is an antibody, e.g., a monoclonal antibody, e.g., a therapeutic antibody.

12. The method of any one of claims 6-11 , wherein:

i) the sample is derived from a downstream processing step in a production process of the recombinant polypeptide,

ii) the sample is derived from an upstream processing step in a production process of the recombinant polypeptide,

iii) the sample is derived from a final product of the recombinant polypeptide, or

iv) the method comprises two, three, four, five, six, seven, eight, nine, ten, or more than ten samples derived from one or more steps in a production process of the recombinant polypeptide.

13. The method of claim 6 or 7, wherein the detectable antibody is:

i) directly detectable,

ii) amplified by a fluorimetric reagent, or

iii) biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3 ',5,5'-tetramethyl benzidine.

14. A method of manufacturing a recombinant polypeptide drug product, the method comprising:

a) providing or obtaining a sample of a recombinant polypeptide preparation from culture of a GS-CHO cell;

b) acquiring a value for the GS-CHO host cell protein (HCP) in the sample, and, c) optionally evaluating the acquired value, e.g., by comparing the acquired value with a reference value; and

d) optionally responsive to the evaluation, classifying, selecting, accepting, releasing, processing into a drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into commerce, selling or offering for sale the preparation,

wherein the value is a function of, is proportional to, or was obtained by, the binding of a polyclonal GS-CHO antibody to the sample,

thereby manufacturing a recombinant polypeptide drug product.

15. The method of claim 14, wherein the value was determined by the method of any of claims 1 -12.

16. A method of evaluating a process of manufacturing a recombinant polypeptide in a GS-CHO host cell, the method comprising:

a) culturing a GS-CHO cell cultured under conditions suitable for production, e.g., expression and secretion, of a recombinant polypeptide,

b) separating the secreted recombinant polypeptide from the host cell to provide a first polypeptide preparation;

c) optionally taking a sample of the first polypeptide preparation;

d) subjecting the first polypeptide preparation to a purification step to produce a second polypeptide preparation;

e) taking a sample of the second polypeptide preparation;

f) optionally subjecting the second polypeptide preparation to a second, third, fourth, fifth, or sixth purification step to provide subsequent polypeptide preparations;

g) optionally taking a sample of any subsequent polypeptide preparations provided in step f);

h) contacting and incubating each of the samples taken with a polyclonal anti-GS-CHO

HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex;

i) separating each of the samples from the immobilized antibody-GS-CHO HCP complex;

j) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP;

k) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and

1) based on said quantification, making a determination about the process,

thereby evaluating a process of manufacturing a recombinant polypeptide.

17. A method of evaluating a process of purifying a recombinant polypeptide, the method comprising:

a) subjecting the first polypeptide preparation to a purification step to produce a second polypeptide preparation;

b) taking a sample of the second polypeptide preparation;

c) optionally subjecting the second polypeptide preparation to a second, third, fourth, fifth, or sixth purification step to provide subsequent polypeptide preparations;

d) optionally taking a sample of any subsequent polypeptide preparations provided in step c);

e) contacting and incubating each of the samples taken with a polyclonal anti-GS-CHO

HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex;

f) separating each of the samples from the immobilized antibody-GS-CHO HCP complex;

g) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP;

h) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and

i) based on said quantification, making a determination about the process,

thereby evaluating a process of purifying a recombinant therapeutic.

18. The method of claim 16 or 17, comprising comparing the level of GS-CHO HCP to a reference level, e.g., wherein if the level of GS-CHO HCP is below the reference level validating the process for use in the production of the recombinant polypeptide.

19. The method of claim 18, wherein the reference level is:

i) between lppm and lOOOppm,

ii) at least lppm, lOpppm, 50ppm, lOOppm, 500ppm, or lOOOppm, or

iii) no greater than lppm, 5ppm, lOpppm, 20ppm, 30ppm, 40ppm, 50ppm, lOOppm, 500ppm, or lOOOppm.

20. The method of claim 16 or 17, wherein the purification step comprises one or more or any combination of size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), reverse phase chromatography (RPC), immobilized metal chelate chromatography, ammonium sulfate precipitation, thiophilic adsorption, protein A chromatography, protein G chromatography, protein L chromatography, and affinity chromatography.

21. The method of claim 16 or 17, wherein the purification step comprises:

i) Protein A chromatography and one or more affinity chromatography steps,

ii) protein A chromatography and CNBr chromatography, e.g., protein A chromatography and subsequent CNBr chromatography, or

iii) protein A chromatography and NHS chromatography, e.g., protein A chromatography and subsequent NHS chromatography.

22. A kit comprising:

a) a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support;

b) optionally a detectable antibody;

c) optionally a wash buffer; and

d) optionally a detecting reagent, e.g., a detecting reagent that is the same as the capture reagent or the same as the capture reagent only modified to be amplified by a fluorimetric reagent, and

optionally wherein the detecting antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.

23. A purified preparation of polyclonal anti-GS-CHO antibody, optionally wherein the anti-GS-CHO antibody is a sheep, goat, horse, rabbit, bovine, murine, hamster, or human antibody.

24. The purified polyclonal preparation of claim 23, wherein the preparation is substantially free from non-HCP specific IgGs, e.g., the preparation contains no more than 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% non-HCP specific IgGs or the preparation contains less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% non-HCP specific IgGs.

25. The purified polyclonal preparation of claim 23, purified by a method comprising: protein A chromatography, and one or more affinity chromatography steps (e.g., CNBr chromatography, NHS chromatography).

26. A reaction mixture comprising a polyclonal preparation (e.g., a polyclonal preparation described herein) of anti-GS-CHO antibody and a recombinant polypeptide selected from Table 1 or Table 2.

27. The reaction mixture of claim 26, comprising an antibody that binds to the anti-GS-CHO antibody.

28. A method of determining whether a batch or lot of a recombinant polypeptide drug product meets or satisfies a release specification, wherein the release specification is a preselected reference level of GS-CHO HCP the method comprising:

a) providing or obtaining a sample of a recombinant polypeptide preparation;

b) contacting and incubating the sample with a polyclonal anti-GS-CHO HCP antibody immobilized to a solid support to form an immobilized antibody-GS-CHO HCP complex; c) separating the sample from the immobilized antibody-GS-CHO HCP complex;

d) contacting the immobilized antibody-GS-CHO HCP complex with a detectable antibody that binds to GS-CHO HCP;

e) quantifying the level of GS-CHO HCP bound to the capture reagent using a detection means for the detectable antibody; and

f) processing at least a portion of the preparation as drug product if the level of GS-CHO HCP meets, satisfies, or is below the release specification, and

optionally wherein processing comprises classifying, selecting, accepting, releasing, processing into a drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into commerce, selling or offering for sale the preparation,

thereby determining whether a batch or lot of a recombinant polypeptide drug product meets or satisfies a release specification.

29. The method of claim 28, wherein the value was determined by the method of any of claims 1 -21.

30. A method of making a polyclonal anti-HCP antibody preparation, the method comprising:

a) acquiring a sample comprising antibodies, e.g., antisera, from an animal (e.g., a sheep, rabbit, mouse, rat, hamster, goat, etc.) immunized with HCP of a host cell (e.g., a cell line, e.g., a mammalian, rodent, or insect cell or cell line, e.g., a CHO, SP2, NSO host cell); and

b) separating anti-HCP antibody from the sample, e.g., by contacting the sample with an HCP-affinity reagent, e.g., protein A, and

optionally wherein b) comprises:

b. l) separating antibodies, e.g., IgG antibodies, from the sample to provide an antibody preparation; and

b.2) separating anti-HCP antibody from the antibody preparation, e.g., by contacting the antibody preparation with an HCP-affinity reagent,

thereby producing a polyclonal anti-HCP antibody preparation.

31. The method of claim 30, wherein separating antibodies comprises purifying a polyclonal anti-HCP antibody from the antisera through a purification method comprising protein A chromatography.

32. The method of either of claims 30 or 31 , wherein the HCP-affinity reagent comprises HCP coupled to a substrate, e.g., an insoluble or solid substrate, e.g., an agarose bead, e.g., Sepharase, e.g., cyanogen bromide (CNBr) or N-hydroxysuccinimide (NHS) chromatography) derivatized substrate.

33. A method of producing a polyclonal anti-HCP antibody preparation for use in an ELISA for detection of HCPs in a recombinant polypeptide preparation, the method comprising: a) acquiring a sample comprising antibodies, e.g., antisera, from an animal (e.g., a sheep, rabbit, mouse, rat, hamster, goat, etc.) immunized with HCP of a host cell (e.g., a cell line, e.g., a mammalian, rodent, or insect cell or cell line, e.g., a CHO, SP2, NSO host cell); b) separating antibodies from the antisera with by contacting the antisera with a protein A affinity reagent to provide an antibody preparation;

c) separating anti-HCP antibody from the antibody preparation by contacting the antibody preparation with an HCP-affinity reagent,

thereby producing a polyclonal anti-HCP antibody preparation.

34. The method of any one of claims 30-33, wherein the purification removes:

i) at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs, e.g., at least 99% non-HCP specific IgGs, or

ii) more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% non-HCP specific IgGs, e.g., more than 99% non-HCP specific IgGs.

35. The method of any one of claims 30-33, wherein the polyclonal antibody preparation is substantially free from non-HCP specific IgGs, e.g., contains no more than 0.5%, 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% non-HCP specific IgGs or contains less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% non-HCP specific IgGs.

36. The method of any one of claims 1 -21 , wherein the polyclonal antibody is made by method of any one of claims 30-35.

37. A polyclonal anti-HCP antibody preparation produced by the method of any one of claims 30-35.

38. A purified polyclonal anti-HCP antibody preparation produced by the method of any one of claims 30-35.

39. A method of detecting, monitoring, identifying, or quantifying a host cell protein (HCP) (e.g., a CHO HCP) in a sample comprising a recombinant polypeptide, the method comprising:

a) providing or obtaining a sample comprising a recombinant polypeptide;

b) contacting and incubating the sample with a polyclonal anti-HCP antibody (e.g., an anti-CHO HCP antibody) immobilized to a solid support to form an immobilized antibody-HCP complex, wherein the polyclonal antibody has been produced by the method of any one of claims 28-35;

c) separating the sample from the immobilized antibody-HCP complex;

d) contacting the immobilized antibody-HCP complex with a detectable antibody that binds to GS-CHO HCPs;

e) detecting the presence of HCP bound to the antibody using a detection means for the detectable antibody; and

f) optionally quantifying the level of HCP detected;

g) optionally identifying one of more of the HCP detected; and

h) optionally quantifying one or more of the HCP identified.

40. The method of any one of claims 1-21 , wherein the polyclonal anti-GS-CHO HCP antibody is produced by a method of any one of claim 30-35.