# US 2019/0169675 A1 · Proteomic analysis of host cell proteins

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- Number: US 2019/0169675 A1
- Document ID: US20190169675A1
- Status: Published application
- Kind: A1
- Application number: 16/324,960
- Filed: 2017-08-11
- Published: 2019-06-06
- Earliest claimed: 2016-08-12
- Assignees: Lonza Ltd.
- Applicants: Lonza Ltd
- Inventors: James Graham
- Web page: https://patentgrep.com/patent/US20190169675A1

## CPC

- C12Q 1/37: involving peptidase or proteinase
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G16B 40/10: Signal processing, e.g. from mass spectrometry [MS] or from PCR
- G16B 45/00: ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
- G16B 50/30: Data warehousing; Computing architectures
- G16C 20/90: Programming languages; Computing architectures; Database systems; Data warehousing
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16C: COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE

## Abstract

Disclosed herein are methods and compositions useful for detecting and/or quantifying host cell proteins during the production of a product, e.g., a recombinant protein, e.g., an antibody.

## Claims

### Claim 1

A simple method of rapidly analyzing a sample, e.g., to provide an assessment of the risk of a protein (e.g., the risk the protein presents if present as a contaminant in a preparation, e.g., a preparation to be administered to a subject, e.g., a pharmaceutical preparation), the method comprising:
a) providing, e.g., forming and/or maintaining, a sample mixture comprising:
i) the sample, which comprises the protein and optionally a product (e.g., a recombinant polypeptide, e.g., an antibody, enzyme, or cytokine), produced via a process; and
ii) a denaturant, e.g., deoxycholate and urea,
under conditions, e.g., concentrations of denaturant, that denatures the protein in the sample 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 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 analyzing the sample and providing an assessment of the risk of a protein.

### Claim 2

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.

### Claim 3 (depends on 1, 2)

The method of either of claim 1 or 2, further comprising evaluating a plurality of different samples, each made by a different process, e.g., evaluating at least 2, 4, 8, 10, 50, 96, 100, 192, 200, 500 or 1,000, different samples.

### Claim 4 (depends on 3)

The method of claim 3, further comprising comparing the assessment of risk for a first and a second different sample.

### Claim 5 (depends on 4)

The method of claim 4, further comprising, responsive to the comparison, selecting, a process for producing the product.

### Claim 6 (depends on 4)

The method of claim 4, further comprising, responsive to the comparison, selecting, classifying, or further processing one of the samples.

### Claim 7

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.

### Claim 8

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.

### Claim 9 (depends on 7, 8)

The method of either of claim 7 or 8, further comprising evaluating a plurality of different processes of making a product, e.g., evaluating at least 2, 4, 8, 10, 50, 96, 100, 192, 200, 500 or 1,000, different processes.

### Claim 10 (depends on 9)

The method of claim 9, further comprising comparing the evaluation for a first and a second different process.

### Claim 11 (depends on 10)

The method of claim 10, further comprising, responsive to the comparison, selecting a process of making the product.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method of any of claims 1-11, wherein the protein is a contaminant or other undesirable component (e.g., a fragment, denatured, or mis-folded version of a product being produced by a set of conditions, or a host cell protein (HCP) or fragment thereof).

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The method of any of claims 1-12, wherein the denaturant, first denaturant, or second denaturant comprises, consists of, or consists essentially of deoxycholate and urea, guanidine hydrochloride, or urea and guanidine hydrochloride.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The method of any of claims 1-13, wherein the concentration of denaturant in the sample mixture is higher than the concentration of denaturant in the sample/enzyme mixture.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The method of any of claims 1-14, wherein:
the concentration of denaturant in sample mixture is sufficiently high to denature the protein, e.g., wherein at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the protein is denatured; and
the concentration of denaturant in sample/enzyme mixture is sufficiently low to not denature the enzyme, e.g., wherein less than 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1% of the enzyme is denatured.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any of claims 1-15, wherein the concentration of the denaturant in the sample mixture is:
i) at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.5, or 8;
ii) 1-10 M, 2-9 M, 3-8 M, 4-7 M, 5-7 M, 6-6.6 M, 6 M, 6.6 M, or 8 M;
iii) 0-10 M, 2-9 M, 3-8 M, 4-7 M, 5-7 M, 0.5-5 M, 0.5-2 M, 0.5 M, 1 M, or 2 M; or
iv) 0.01%-50%, 1%-40%, 1%-20%, 0.5%-10%, 0.01%-5%, or 0.1%-2% (m/v).

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method of any of claims 1-16, wherein the sample mixture comprises a first denaturant (i.e. the denaturant of (a)(ii)), and the sample/enzyme mixture comprises the first denaturant and a second denaturant.

### Claim 18 (depends on 17)

The method of claim 17, wherein the first denaturant is guanidine hydrochloride and the second denaturant is urea.

### Claim 19 (depends on 17, 18)

The method of either of claim 17 or 18, wherein the concentration of the first denaturant in the sample mixture is 1-10 M, 2-9 M, 3-8 M, 4-7 M, 5-7 M, 6-6.6 M, 6 M, 6.6 M, or 8 M.

### Claim 20 (depends on 17, 18, 19)

The method of any of claims 17-19, wherein the concentration of the first denaturant in the sample/enzyme mixture is:
i) 0-10 M, 2-9 M, 3-8 M, 4-7 M, 5-7 M, 0.5-5 M, 0.5-2 M, 0.5 M, 1 M, or 2 M;
ii) less than or equal to 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, or 0.1 M; or
iii) ii) less than or equal to 0.5 or 0.1 M, e.g., essentially 0 M.

### Claim 21 (depends on 17, 18, 19, 20)

The method of any of claims 17-20, wherein the concentration of the second denaturant in the sample/enzyme mixture is:
i) less than or equal to 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, or 0.1 M;
ii) less than or equal to 0.5 or 0.1 M, e.g., essentially 0 M; or
iii) less than or equal to 2 M or 0.5 M.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)

The method of any of claims 1-21, wherein:
the pH of the sample mixture is sufficiently low that deamidation reactions are substantially inhibited e.g., wherein at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the asparagine and glutamine side chains of the protein are unaltered, and
the pH of the sample/enzyme mixture is sufficiently high that the enzyme is active, e.g., wherein the enzyme is at least 50, 60, 70, 80, 90, or 100% active, e.g., operating at 50, 60, 70, 80, 90, or 100% efficiency compared to maximum efficiency.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The method of any of claims 1-22, wherein the pH of the sample mixture is 5.5±1, 0.75, 0.5, or 0.25 (e.g., 5.5±0.5) and the pH of the sample/enzyme mixture is 7.3±1, 0.75, 0.5, or 0.25 (e.g., 7.3±0.5).

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The method of any of claims 1-23, wherein the pH of the sample mixture is 5.5 and the pH of the sample/enzyme mixture is 7.3.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)

The method of any of claims 1-24, wherein the method does not comprise alkylation of cysteine residues of the protein or protein digestion products.

### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)

The method of any of claims 1-25, wherein the sample mixture and/or sample/enzyme mixture comprise a reducing agent, e.g., Tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or beta-mercaptoethanol.

### Claim 27 (depends on 26)

The method of claim 26, wherein the concentration of reducing agent, e.g., Tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or beta-mercaptoethanol, in the sample mixture is higher than in sample/enzyme mixture.

### Claim 28 (depends on 26, 27)

The method of either of claim 26 or 27, wherein:
the concentration of reducing agent, e.g., Tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or beta-mercaptoethanol, in the sample mixture is sufficiently high to substantially reduce the cysteines of the protein, e.g., wherein at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the cysteine residues of the protein are reduced; and
the concentration of reducing agent, e.g., Tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or beta-mercaptoethanol, in the sample/enzyme mixture is sufficiently low to not interfere with other steps of the method or method of manufacturing, e.g., wherein the reducing agent does not significantly accumulate in equipment (e.g., mass spectrometer or analytical column) or produce additional signal in data (e.g., mass spectrometry data).

### Claim 29 (depends on 26, 27, 28)

The method of any of claims 26-28, wherein the concentration of the reducing agent in the sample mixture is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM.

### Claim 30 (depends on 26, 27, 28, 29)

The method of any of claims 26-29, wherein the concentration of the reducing agent in the sample/enzyme mixture is less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1 mM, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1 mM.

### Claim 31 (depends on 26, 27, 28, 29, 30)

The method of any of claims 26-30, wherein the reducing agent is Tris(2-carboxyethyl)phosphine (TCEP).

### Claim 32 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)

The method of any of claims 1-31, wherein the protein digestion products are separated on the basis of one or more (e.g., one, two, three or more) of size, charge, or affinity.

### Claim 33 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The method of any of claims 1-32, wherein separating the protein digestion products comprises using chromatography, e.g., 1-dimensional chromatography, e.g., affinity chromatography, gel filtration chromatography, ion exchange chromatography, reversed phase chromatography, hydrophobic interaction chromatography, high performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis, ion mobility, or any chromatographic method described herein.

### Claim 34 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)

The method of any of claims 1-33, wherein (c) further comprises providing the identity of a protein digestion product, e.g., by mass spectroscopy, e.g., LC/MS, tandem mass spectrometry, or RP-LCMS2.

### Claim 35 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)

The method of any of claims 1-34, wherein (c) comprises separating the protein digestion products using chromatography, e.g., 1-dimensional chromatography, and providing the identity of the protein digestion products, e.g., by mass spectrometry, e.g., LC/MS, tandem mass spectrometry, or 1D RP-LCMS2.

### Claim 36 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)

The method of any of claims 1-35, wherein a plurality, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of protein digestion products in at least 2, 10, 20, 96, 100, 192, 1,000, or 10,000 samples are classified or assigned an identity, structure or composition.

### Claim 37 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)

The method of any of claims 1-36, wherein the protein risk score is a function of one or more of:
an unwanted, e.g., off-target, property in a subject to receiving a preparation comprising the protein and, optionally, a product, e.g., immunogenicity;
an unwanted effect of the protein in a preparation of the product, e.g., a preparation of a drug, e.g., the propensity to cause denaturation, precipitation, or color; and
a value for the abundance of the protein present in the sample.

### Claim 38 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The method of any of claims 1-37, wherein step (d) is repeated to provide a protein risk score for one or more (e.g., at least 2, 10, 50, 100, 200, 500, 1000, or all) proteins identified in the sample.

### Claim 39 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)

The method of any of claims 1-38, wherein step (d) comprises providing a protein risk score, e.g., an immunogenicity risk score, e.g., as generated by the Epibase® platform.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The method of any of claims 1-39, wherein step (d) comprises providing a immunogenicity risk score as generated by the Epibase® platform.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The method of any of claims 1-40, further comprising providing a process risk score to the sample.

### Claim 42 (depends on 41)

The method of claim 41, wherein the process risk score is a function of the one or more protein risk scores of the sample's proteins.

### Claim 43 (depends on 41, 42)

The method of either of claim 41 or 42, wherein the process risk score is calculated based upon the formula:
Process Risk Score=Σ([Protein Abundance]×[Immunogenicity Risk Score]).

### Claim 44 (depends on 41, 42, 43)

The method of any of claims 41-43, wherein the method is repeated to analyze a plurality of samples, e.g., at least 2, 10, 50, 96, 10, 192, or 1000, and
wherein two or more (e.g., all) of the samples are provided using a different process or method of manufacturing, and wherein a process risk score is provided for a plurality of samples (e.g., all samples), or
wherein two or more (e.g., all) of the samples are provided at different time points during a process or method of manufacturing, and wherein a process risk score is provided for a plurality of samples (e.g., all samples).

### Claim 45 (depends on 41, 42, 43, 44)

The method of any of claims 41-44, comprising comparing the process risk score of a process or method of manufacturing, e.g., the process or method of manufacturing used to provide the sample, with a reference.

### Claim 46 (depends on 41, 42, 43, 44, 45)

The method of any of claims 41-45, comprising comparing the process risk score of a first process or method of manufacturing with a process risk score for a second process or method of manufacturing.

### Claim 47 (depends on 41, 42, 43, 44, 45)

The method of any of claims 41-45, comprising comparing the process risk score of a process at a first time point with the process risk score of a process at a second time point.

### Claim 48 (depends on 46)

The method of claim 46, comprising, responsive to the comparison, selecting one of the processes or methods of manufacturing, e.g., for further analysis or for further use, e.g., to make the product, e.g., recombinant polypeptide.

### Claim 49

A database (e.g., memorialized or recorded on a computer readable medium) comprising a library of identifying characteristics for HCPs or protein digestion products and protein risk scores derived from cell culture supernatant of a cell culture (e.g., a CHO, eg., a GS-CHO, cell culture).