Proteomic analysis of host cell proteins

A simple method of rapidly analyzing a sample to provide an assessment of the risk of a protein, the method comprising:

• a) providing, e.g., forming and/or maintaining, a sample mixture comprising:
• i) the sample, which comprises the protein (e.g., a HCP) and optionally a therapeutic product (e.g., a recombinant polypeptide), produced via a process;
• and
• ii) a first denaturant, e.g., guanidine hydrochloride,
• under conditions, e.g., concentrations of a first denaturant, that denatures the protein in the sample at temperature of between 30 and 60° C., e.g., 45-55° C., e.g., 50±3° C., 50±2° C., 50±1° C., or 50° C.;
• b) providing, e.g., forming and or maintaining, a sample/enzyme mixture comprising:
• i) sample mixture;
• ii) a second denaturant, e.g., urea;
• and
• iii) an enzyme preparation comprising an enzyme for which the protein is a substrate, e.g., a proteolytic enzyme, e.g., an enzyme which cleaves proteins at a preselected or defined target site, e.g., trypsin, lysC, GluC, or AspN, with the sample mixture,
• under conditions in which the enzyme maintains substantial activity and reacts with, e.g., cleaves, the protein to provide protein digestion products;
• c) separating the protein digestion products using chromatography, e.g., 1-dimensional chromatography, providing the identity of the protein digestion products, e.g., by mass spectrometry, e.g., LC/MS, and using one or more protein digestion products to provide the identity of a protein associated with the protein digestion products;
• d) assigning a protein risk score to a protein identified in the sample, thereby analyzing the sample and providing an assessment of the risk of a protein.

A method of evaluating a process of making a product, e.g., an evaluation that incorporates assessment of the risk presented by a protein other than the product, e.g., a contaminant, produced by the process, comprising:

• a) providing, e.g., forming and/or maintaining, a sample mixture comprising:
• i) the protein and optionally a product (e.g., a recombinant polypeptide, e.g., an antibody, enzyme, or cytokine), produced by the process;
• and
• ii) a denaturant, e.g., deoxycholate and urea
• under conditions, e.g., concentrations of denaturant, that denatures the protein in the sample, e.g., at temperature of between 10 and 30° C., e.g., 18-26° C., e.g., 20±3° C., 20±2° C., 20±1° C., or 20° C.;
• b) providing, e.g., forming and or maintaining, a sample/enzyme mixture comprising:
• i) sample mixture (e.g., an aliquot of sample mixture from a));
• and
• ii) an enzyme preparation comprising an enzyme for which the protein is a substrate, e.g., a proteolytic enzyme, e.g., an enzyme which cleaves proteins at a preselected or defined target site, e.g., trypsin, lysC, GluC, or AspN, with the sample mixture,
• under conditions in which the enzyme maintains substantial activity and reacts with, e.g., cleaves, the protein to provide protein digestion products;
• c) separating the protein digestion products, e.g., by using chromatography, e.g., 1-dimensional chromatography, providing the identity of the protein digestion products, e.g., by mass spectrometry, e.g., LC/MS, and using one or more protein digestion products to provide the identity of a protein associated with the protein digestion products;
• and
• d) assigning a protein risk score to a protein identified in the sample,
• thereby evaluating a process of making a product, e.g., an evaluation that incorporates assessment of the risk presented by a protein other than the product, e.g., a contaminant, produced by the process.

A method of evaluating a process of making a product, e.g., an evaluation that incorporates assessment of the risk presented by a protein other than the product, e.g., a contaminant, produced by the process, comprising:

• a) providing, e.g., forming and/or maintaining, a sample mixture comprising:
• i) the protein and optionally a product (e.g., a recombinant polypeptide, e.g., an antibody, enzyme, or cytokine), produced by the process;
• and
• ii) a first denaturant, e.g., guanidine hydrochloride,
• under conditions, e.g., concentrations of denaturant, that denatures the protein in the sample, e.g., at temperature of between 30 and 60° C., e.g., 45-55° C., e.g., 50±3° C., 50±2° C., 50±1° C., or 50° C.;
• b) providing, e.g., forming and or maintaining, a sample/enzyme mixture comprising:
• i) sample mixture (e.g., an aliquot of sample mixture from a));
• ii) a second denaturant, e.g., urea;
• and
• iii) an enzyme preparation comprising an enzyme for which the protein is a substrate, e.g., a proteolytic enzyme, e.g., an enzyme which cleaves proteins at a preselected or defined target site, e.g., trypsin, lysC, GluC, or AspN, with the sample mixture,
• under conditions in which the enzyme maintains substantial activity and reacts with, e.g., cleaves, the protein to provide protein digestion products;
• c) separating the protein digestion products, e.g., by using chromatography, e.g., 1-dimensional chromatography, providing the identity of the protein digestion products, e.g., by mass spectrometry, e.g., LC/MS, and using one or more protein digestion products to provide the identity of a protein associated with the protein digestion products;
• and
• d) assigning a protein risk score to a protein identified in the sample,
• thereby evaluating a process of making a product, e.g., an evaluation that incorporates assessment of the risk presented by a protein other than the product, e.g., a contaminant, produced by the process.