Analytical Methods for the Measurement of Host Cell Proteins and

Chapter 9

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Analytical Methods for the Measurement of Host Cell Proteins and Other Process-Related Impurities Kesh Prakash*,1 and Weibin Chen2 1Emergent

BioSolutions, 300 Professional Drive, Gaithersburg, Maryland 20879, United States 2Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States *E-mail: [email protected]

Host cell proteins (HCPs) and other process-related impurities such as Protein A, insulin, and residual host cell DNA are routinely encountered during the manufacture of biopharmaceutical products. HCP impurities are of significant interest to the biopharmaceutical industry because therapeutic proteins (monoclonal antibodies [mAbs] and recombinant proteins) need to be highly purified from the production host cell line that is utilized to manufacture them. It is imperative that HCPs are removed during the purification (downstream) process and brought down to as low a level as possible in the final drug substance. This is done to ensure product purity and safety. Similarly, clearance of other process-related impurities (host cell DNA, protein A, and insulin) must be demonstrated during the manufacture of biopharmaceutical products. This chapter will discuss analytical methods that cover the detection and clearance of these impurities. In addition, the application of these analytical methods to the quantitation of various process-related impurities in the submitted NISTmAb is described. The results obtained show that the test sample is pure and would meet specification requirements.

© 2015 American Chemical Society

Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The NISTmAb C ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


General Introduction to Host Cell Proteins (HCPs) and Other Process-Related Impurities The production of a biopharmaceutical such as a monoclonal antibody (mAb) involves a variety of steps, typically divided into upstream and downstream processes (1). From any stage of the process, process-related impurities, which can include raw materials, host cell components, media components, leachables from the type of purification process used, and chemical additives, may be derived (2). For example, low levels of process-related impurities such as HCPs and residual DNA (from the host cell) may persist during various stages of the biopharmaceutical manufacturing process. Downstream processing steps intended for removal of such impurities (e.g., Protein A that may leach from the Protein A resin) may also be introduced. Based on the manufacturing process used, other cell culture media-derived impurities (e.g., insulin, bovine serum albumin [BSA], transferrin, β-glucans) also may be present and will need quantification (2). Although there are a number of such potential impurities to consider, this chapter will focus mainly on HCPs, Protein A, insulin, and residual DNA. Cell expression system-derived impurities will vary, depending on the host cell used in the production process. Biopharmaceutical products are generally produced utilizing recombinant technology in host cells ranging from bacteria (e.g., Escherichia coli [E. coli]), yeast (e.g., Pichia pastoris), or cell lines of various origins such as mammalian (e.g., Chinese hamster ovary [CHO] cells), insect (e.g., Sf9 cells), or even plant (e.g., tobacco). Host cell-derived proteins or HCPs arise as a result of cell lysis or secretion during cell culture and harvesting, and they are released into the medium in addition to the product. HCPs are a complex set of proteins that need to be monitored during the development and commercialization of biopharmaceutical products. It is essential that HCPs be removed during the purification process and brought down to as low a level as possible in the final drug product to ensure product purity and patient safety (2, 3). HCPs should be few in number and low in quantity. One of the important properties of HCPs is that they are specific and unique to the host cells and the particular culture process used for manufacture (4, 5). HCPs can vary in isoelectric point (pI) (~3–11) and hydrophobicity, and display a broad range of molecular weights (5 kD to > 250 kD), depending on the host cell and manufacturing process utilized. The number of HCPs in conditioned media (upstream) samples can vary from several hundred (e.g., E. coli) to more than a thousand proteins (e.g., CHO cells), depending on the host cell system used (4, 5). Most of the experience gathered to date has been obtained utilizing E. coli and mammalian cells like CHO and NS0 (2, 5). Additional impurities must also be considered. Purification-derived impurities include host cell DNA. Low levels of host cell DNA originating from cellular DNA also may remain following purification (3, 6). Cell culture and harvest-derived impurities usually consist of raw materials like insulin and others added as growth media supplements for stimulating cell growth. The purification process must be designed appropriately to bring these impurity levels down. In addition, downstream purification process-derived 388


impurities such as Protein A may be present as a result of leaching from Protein A resin (2) used as the capture column during purification of mAbs. The goal across the biopharmaceutical industry is to develop a production process that results in low levels of all process-related impurities—HCPs, host cell DNA, Protein A, insulin, and so forth. All of this is done to ensure product purity and safety prior to administration of these products to patients.


Regulatory Guidance There is no clear regulatory guidance on HCP, Protein A, and insulin levels. However, most biopharmaceutical products reviewed by the U.S. Food and Drug Administration (FDA) contain enzyme-linked immunosorbent assay (ELISA)-based HCP levels of 1 to 100 ng/mg of product (3, 7–10). The ability to set a single numerical limit for an appropriate level of HCP for all products is difficult because the HCPs present can vary from process to process and product to product, and the sensitivity of detection and quantitation is closely linked to the quality of the immunoreagents from which the assay is constructed. According to both U.S. (FDA) and European Union (European Medicines Agency [EMEA]) regulations, HCPs are labelled as process-derived impurities, and guidance to lower their presence by the use of appropriate well-controlled manufacturing processes is given. In particular, 21 CFR 610.13 (6) states that biological products be “… free of extraneous material except that which is unavoidable…,” while ICH Q6B guidance (10) specifically states that biologic product manufacturers should evaluate impurities which may be present and develop meaningful acceptance criteria for the impurities based on their preclinical and clinical experience. However, this guidance also recognizes the challenges that exist with HCP assay-specific data and product- and process-specific reagents. Per ICH Q6B (10), “The absolute purity of biotechnological and biological products is difficult to determine and the results are method-dependent.” Along the same line, the EMEA (11) has stated that “… standardization of the analytical methods would be problematic since the reagents used are product and production system-related.” Although FDA guidance describes the need to test for HCPs, it also allows, through validation, removal of testing requirements once clearance has been shown to be uniform and consistent. An appropriately conducted clearance study carried out as part of process validation can be an acceptable alternative for production lot-to-lot testing (12) even in cases where low quantities of HCPs are present in the drug substance. Additionally, the FDA (10) states that “[i]f impurities are qualitatively and quantitatively (i.e., relative amounts and/or concentrations) the same as in the drug substance, testing of the drug product is not necessary.” As far as the process-related impurities Protein A and insulin are concerned, there is no defined guidance by regulatory agencies (e.g., FDA, EMEA). The general rule of thumb that is followed across the biopharmaceutical industry is to develop and optimize processes to bring all process-related impurities (HCPs, Protein A, and insulin) to as low a level as possible (10, 12). 389


With regard to host cell DNA, FDA requirements in the past stipulated an upper limit of 100 pg per therapeutic patient dose (13). This has now been replaced by World Health Organization (WHO) guidelines that have an upper limit of 10 ng/ patient dose (14). This guideline is followed for products produced in mammalian and non-mammalian systems.

Immunogenicity Pertaining to HCPs; Quality Issues Related to HCPs and Other Impurities Downloaded by CENTRAL MICHIGAN UNIV on January 4, 2016 | http://pubs.acs.org Publication Date (Web): October 15, 2015 | doi: 10.1021/bk-2015-1201.ch009

HCPs mAb products are large molecules that can bind to specific targets. mAbs could be considered to have a potential for immunotoxicity, which is reflected in the clinical experience accumulated on mAbs-induced adverse effects related to immunosuppression, immunostimulation and hypersensitivity (immunogenicity). Moreover, it is well known that anti-drug antibodies can be induced when immune systems recognize a material (e.g., mAb) introduced as foreign, especially for parenterally administered products. Biopharmaceuticals can elicit product-specific anti-drug antibodies (ADA), or antibodies against HCPs can develop in either nonclinical or clinical studies. Anti-HCP antibodies could cause adverse events by inducing immunemediated clinical sequelae such as injection site reactions, flu-like symptoms, and at worst case, anaphylaxis (15). Immunogenicity also may be related to the state of a patient receiving such therapy, as pre-existing antibodies to HCPs have been identified in individuals with no known exposure to biopharmaceutical products (16). In addition, it has been observed that HCPs may act as adjuvants and enhance an ADA immune response to the product itself (3, 12). Although the risk of immunogenicity related directly or indirectly to HCPs is not well published, there are some cases, and the general perception is that it is a risk to be considered. Proteases are one of the types of HCPs that may indirectly influence immunogenicity to the product. Proteolytic HCPs have the potential to digest the desired protein product over time, thus altering biological potency, bioavailability, or—through the creation of immunogenic sequences—inducing an ADA response (17). Similar reports of HCP-related ADAs in biosimilars have appeared recently (18). Insulin In terms of human safety, high doses of insulin (100–200 µIU/kg) is associated with hypoglycemia, coma, convulsions, and even death in very severe cases. There are at least three recombinant human insulin products that have been used or are in use in humans, including Velosulin BR®, Humulin® R, and Exubera® (19). There are no clear reproductive toxicity or carcinogenicity warnings in the labels of these approved products. As no harmful activity has been attributed to insulin so far across products commercialized by the biopharmaceutical industry, it is not expected to have biological or safety effects at the trace levels that may be present 390


in non-insulin drug products due to residual raw material insulin process-related impurity. Protein A


Some published information is available on the toxicity risk attributable to Protein A impurity (20, 21). As Protein A is obtained from the cell wall of the bacterium Staphylococcus aureus, a test for bacterial endotoxin is carried out on every biopharmaceutical product before it is released. DNA For host cell DNA, it has been generally shown that DNA with unmethylated cytosine-guanosine (CpG) dinucleotide motifs (i.e., unmethylated dinucleotides of cytosine and guanine) may be immunostimulatory. As mammalian DNA (e.g., NS0 cells) is known to be methylated at 60 to 90% (22), DNA methylated at this level has a very low risk for immunogenicity. In contrast, bacterial DNA (e.g., E. coli) possesses immunostimulatory potential as DNA-containing fractions mediate immune modulation. It has been shown that this is due to the relative abundance of unmethylated CpGs in bacterial DNA (23). Additionally, DNA fragments observed in host cell DNA are usually smaller than 200 base pairs (bp) and therefore provide substantial safety margins pertaining to oncogenicity and infectivity for biopharmaceutical products that meet the 10 ng DNA/patient dose limit (24, 25). Reduction of DNA fragment size (< 200 bp) reduces the probability that intact oncogenes or viral infectious sequences would be present.

Considerations for Manufacturing Consistency and HCP Clearance A biopharmaceutical product is generally secreted from mammalian cells into the surrounding cell culture medium in a manner similar to secreted native host proteins. When cells die during cell expansion in bioreactors (fermentation), soluble proteins from within the cells may be released into the cell culture media. During cell harvest, cells lyse due to shear stress and may result in increased levels of HCPs. As clarified cell culture media containing HCPs is taken through the downstream purification process, each purification step (consisting of various types of resins, nanoparticle/virus filters, etc.) affects the clearance of HCPs in different ways. The intrinsic features of the product, the isolation procedure, and the downstream purification process are all factors that influence HCP levels. The particular properties of the HCP may result in its co-purification with the product, in rare cases through direct association (26). Therefore, a well-designed and properly scaled up purification process is imperative to help reduce the HCPs to the lowest levels possible. Once the process is optimized, process characterization and validation studies are necessary to demonstrate removal of HCP by each process purification step. Moreover, it is essential to evaluate 391



the robustness of each of these steps to show reliable and consistent removal of HCPs during manufacturing. Well-developed and optimized HCP assays play a major role in ensuring consistency of manufacturing runs. In the end, reliable, robust, and reproducible HCP assays are used to quantitate the level of residual HCPs remaining in the final drug product that is to be administered to patients. A downward trend of HCP clearance, beginning with the cell culture medium all the way through the drug substance/drug product using these assays must be established. HCP levels should be well documented for pre-clinical lots used in toxicology studies, in lots used for clinical trials, and in process validation of the final commercial process.

Methods for Quantitation of HCPs and Other Process-Related Impurities The methods currently used for the measurement of HCPs and host cell DNA are based on a sandwich ELISA (2, 27) format and real-time quantitative polymerase chain reaction (qPCR), respectively. PicoGreen®, a fluorescent nucleic acid stain, is used for host cell DNA quantitation, but this technique has the disadvantage of including the contribution of single-stranded nucleic acids (RNA) and free nucleotides to the measurement signal. In addition, the Picogreen® method is less sensitive than the ThresholdTM or qPCR methods. Therefore, the PicoGreen® method is not often used for quantitation unless it is being used for quick, initial screening purposes. There are both immunological (ELISA) and non-immunological methods (liquid chromatography-tandem mass spectrometry [LC-MS/MS], LC-MS HCP chapter/Volume 3, Chapter 13) to detect and quantitate HCPs (28, 29). Immunological methods are the main methods of choice for measurement of the impurities such as HCP, Protein A, and insulin as they are relatively easy to perform, are quantitative due to their measurement being based on direct antigen (e.g., HCP)-antibody interaction, and provide good sensitivity. The workhorse for HCP quantitation has been the sandwich immunoassay (plate ELISA format, Figure 1), based on polyclonal antibodies raised against a wide range of HCPs. This assay offers a combination of accuracy, precision, reproducibility, sensitivity, relatively high throughput, potential for automation, quantitative value, quick turn-around if properly planned, and a fairly low cost per assay. The polyclonal anti-HCP antibodies are used for both capture and detection, with the detector antibodies being biotin-labelled. Then, a streptavidin-peroxidase conjugate is used followed by the addition of a specific substrate. Absorbance is read in a microplate reader at a specific wavelength. HCP quantities are regressed from the standard curve generated by the software used and corrected for dilution factors. Sample values are reported as the mean concentration of the dilutions yielding spike recovery values (usually 75–125%) in samples containing defined amounts of HCP antigen spiked in them. The ± 25% recovery is generally followed throughout the industry.

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Figure 1. Sandwich enzyme-linked immunosorbent assay (ELISA) format. HCP = host cell protein, HRP = horseradish peroxidase, TMB = 3,3′,5,5′-tetramethylbenzidine. Other immunoassay formats, such as the direct and competition immunoassay methods, are not suitable because they lack the higher sensitivity afforded by the sandwich ELISA. It is pertinent to point out here that the polyclonal anti-HCP reagents used in the sandwich ELISA (plate method) cannot recognize all of the possible HCP species because some HCP species can either be weakly immunogenic or have no immunogenic potential at all. The percent recognition of all possible HCP species present is defined as percent coverage. The methods for coverage determination are challenging and results can vary widely from analysis to analysis, limiting the quantitative power of this technique. It is generally accepted that “good” polyclonal antisera usually react with more than 50% of the HCPs present. Orthogonal methods for assessing product purity may need to be developed. These may involve the use anti-HCP antibodies (in 2-dimensional [2-D] gel western blots) or may be non-antibody based (LC-MS/MS methods, LC-MS HCP chapter/Volume 3, Chapter 13). Non-immunological LC-MS methods (28, 29) can be used to provide increased assurance of product purity and safety. For example, it could be used for identification and/or quantitation of the HCP impurity when present in high abundance, or to identify HCPs for which antibodies are not easily raised for use in the HCP ELISA. Knowledge of the identity of the HCP impurity is gathered and a quantitative method for that particular protein impurity could then be developed if it is deemed immunogenic or risky. Orthogonal LC-MS methods are an emerging technology that shows great promise to provide complementary information to ELISA, and are expected to play an increasing role in biopharmaceutical development as described in the LC-MS HCP chapter/Volume 3, Chapter 13. Protein A and insulin also are commonly measured using ELISA. With regard to the sandwich ELISA formats for quantitation of Protein A and insulin, the interest is focused on a specific protein rather than the entire proteome of a given cell line. Therefore, a majority of the industry uses commercially available antibodies such as polyclonal anti-Protein A antibody and anti-human 393


insulin antibody, respectively, for capture and appropriate biotinylated antibodies for detection. These assays are mostly developed in-house using plate ELISA formats.

Characterization and Development of Reagents with Reference to HCP Critical Reagents


Critical Reagents HCPs are complex proteins numbering anywhere from several hundred (cell lysate from bacteria) to more than a thousand (mammalian cell culture supernatant) species of proteins (4, 5), varying in molecular weight, pI, hydrophobicity, and potential for being immunogenic. The HCP antigen generated will be used as the reference standard in HCP assays, and most importantly, this HCP antigen will be used as the immunogen to raise anti-HCP polyclonal antibodies in various animals (rabbits, sheep, or goats). The antigen is therefore paramount to producing an antiHCP panel having good reactivity to as many HCP antigen species as possible. The HCP antigen and the anti-HCP polyclonal antibodies are the most critical reagents for the sandwich ELISA used for quantitating HCPs. Preparation of the HCP Antigen The HCP antigen preparation is typically made from null cells that do not contain the gene for the target or the product of interest (30). Two methods may be used. Method 1 consists of utilizing the non-transfected parental cell line that was expanded to create the production cell line (4). A non-transfected cell line does not express selection markers or other genes coded for by the expression plasmid. In method 2, mock-transfected cells can be used as null cells to produce HCP antigen (5). These cells are created by transfecting the parental cell line with an empty or blank plasmid that was used to create the production cell line but missing the gene for the actual product. Once the null cells have been identified, the HCP antigen is prepared by a mock production run. The prevalent approach is to use a platform cell culture process that is used for several products, such as monoclonal antibodies, from the same cell line. When a platform cell culture process is used, it is very minimally processed in order to ensure that the material contains a broad range of HCPs. For example, the conditioned media from the bioreactor fermentation run containing all the secreted or released HCPs is used as the HCP antigen source as this contains all the HCPs possibly originating from that particular cell culture process. Therefore, this allows for the use of the antigen for HCP monitoring of multiple products generated from the same platform upstream process using mammalian cell lines (e.g., CHO cells). Preparation of Anti-HCP Polyclonal Antibodies A wide range of animal species are used to generate anti-HCP antisera. The HCP antigen (immunogen), derived from either bacterial or mammalian cells, is combined with an adjuvant (complete or incomplete Freund’s adjuvant) before 394



immunization of the animals (most commonly rabbits, goats, or sheep) (2). During the immunization, each animal receives a priming immunization followed by several (4 to 6) booster shots to produce high titer, high affinity/avidity polyclonal antibodies. Antiserum is collected from each animal prior to the first priming immunization (control bleed) and at certain intervals (~2 weeks) subsequent to the booster shots (sample bleeds). Normally, four to six bleeds are collected from each animal. The test samples obtained from the animal antisera are assessed by a titer immunoassay and western blot to evaluate the antiserum with regard to antibody affinity, specificity, degree of immunoreactivity, and titer. The titer is usually identified as the dilution of antibody yielding a signal at least three- to four-fold higher than the non-specific binding signal seen in the titer immunoassay. High titer responses are typically in the 1:1000 or 1:2000 dilution ranges, whereas low titers are below the 1:500 dilution range. It is critical to assess the level of immunoreactivity of each test bleed. Test bleeds are combined to ensure that the antisera included in the final pool display immunoreactivity to most of the HCP proteins present during the product manufacturing process and do not contain antibodies that react with the product. It is important to perform titer and western blot analysis of the each test bleed prior to combining high titer antisera, and this allows one to also remove sera samples with low titer. Once fully characterized, appropriate sera samples are combined and the total antibody fraction purified. Antibody purification is usually carried out by Protein A or Protein G and/or HCP column affinity chromatography. The choice of use of Protein A or Protein G affinity ligand depends on the animal species from which the antibodies were generated. Anti-HCP antibodies produced in rabbits can be purified on either Protein A or Protein G affinity columns. In general, goat or sheep antibodies are purified using a Protein G resin column. Some use HCP affinity columns to specifically purify the anti-HCP antibody fraction. Both purification methods (Protein A/G, HCP affinity) are in common usage, sometimes in combination. Characterization of Anti-HCP Polyclonal Antibodies The affinity-purified antibodies are used as both capture and detector antibodies in the HCP sandwich ELISA. If prepared and stored properly as a large batch (frozen at −70°C) and used efficiently, these antibodies should last for many years (5–10 years). Initially, the protein concentration of the batch is measured by A280 or bicinchoninic acid assay (BCA) or with any other suitable method. The correct antibody pair (coating or capture antibody and biotin-labelled detector antibody) for the HCP sandwich ELISA is finalized by testing HCP-containing samples obtained from the unit operations of the appropriate purification process. The sensitivity and specificity are assessed by ensuring non-reactivity with the product of interest. One of the methods for characterization of anti-HCP polyclonal antisera is to carry out 1-dimensional (1-D) gel western blot analysis initially to get a gross picture of HCP binding efficiency, followed by a confirmatory 2-D gel western blot analysis using the HCP immunogen used to raise the anti-HCP polyclonal antibodies (Figure 2). In the first dimension, the HCP immunogen is separated 395



into various protein species based on its pI, whereas in the second dimension, it is lined up based on the molecular weight of each protein component. More than 1200 HCP species are present, as shown by silver staining, which is a comprehensive method that labels all protein species present (Figure 2A). Western blot analysis is then done using the anti-HCP polyclonal antibodies raised (Figure 2B). Comparison of the two gels allows one to estimate the coverage (percent coverage) of the raised antibodies. In this particular case, the percent coverage estimated was ~75%. The dark spots (black) in Figure 2B show the HCP species that are recognized and bound by the anti-HCP polyclonal antibodies. Some HCP species of higher molecular weight (100–150 kD) and higher pI values (8–10) seen in Figure 2A are not recognized by the anti-HCP antisera and are therefore not bound (Figure 2B). The percent coverage of HCPs helps to ascertain the quality of the antibodies for the HCP immunogen used. As these coverage analyses are quite complex and require a high level of skill to execute, it is imperative to do this on large-format gels at least a few times and derive an average value. The large-format gels (18 cm or longer) have higher resolution and are therefore more meaningful than small format gels (< 10 cm). Although there is no upper limit to this percent coverage value, the higher the number, the better the coverage of HCPs by that particular batch of antibodies generated. The percent coverage analysis is an important measure of the quality of the anti-HCP polyclonal antibodies. The 2-D gel procedure is prone to artifacts (e.g., HCPs are denatured and may not reflect those bound in solution in ELISA) and highly variable. For this reason, they see their main application as a qualitative comparison of antibody batches as part of an overall evaluation. It is important to emphasize that the coverage estimated by 2-D gel western blots is only approximate because the exact value is very technique-dependent (i.e., having low reproducibility) and is influenced by the amount of protein loaded on the gel and the exact way the western blotting is carried out.

Figure 2. Two-dimensional gel western blot analysis. (A) Host cell proteins (HCPs) seen after silver staining. (B) HCPs recognized and bound by anti-HCP polyclonal antibodies. 396



Good Manufacturing Practice (GMP) Testing As previously stated in this chapter, all of the HCPs present in the HCP immunogen used to raise polyclonal antibodies are not necessarily immunogenic because some could be weakly immunogenic and some not immunogenic at all. Therefore, during the assessment of product purity, the sandwich HCP ELISA may not detect and accurately quantify all of the potential HCPs present at the end of the product purification process. The ELISA is therefore used as only one part of a system of evaluation of product purity. There are other orthogonal methods, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, reversed phase-high-pressure liquid chromatography (RP-HPLC), and LC-MS/MS, which could be used to identify the hard-to-quantitate HCPs. Efforts should be made to identify those unique HCPs that have a predilection to be co-purified with product. As a result, HCP ELISAs used for product purity assessment are usually part of the GMP control system. It is generally carried out as an in-process test, but some companies across the biopharmaceutical industry also perform a Certificate of Analysis (C of A) test. The HCP ELISA needs to have appropriate alert and reject limits in place. Orthogonal methods are most useful as characterization tools when demonstrated to be scientifically sound.

Management of HCP Assay Critical Reagents through the Life Cycle of the Product The most critical reagents for the HCP assay are the HCP antigen and anti-HCP polyclonal antibodies. When stored frozen and properly validated for bench stability, HCP reagents are generally stable for many years. However, as stocks are depleted, it is essential to have a process and plan for their replacement. The general rule-of-thumb is to prepare a high volume of anti-HCP antibodies soon after the HCP immunogen/calibration standard is prepared. This is the standard that is prepared initially from the null or mock transfected cells. When the supply is exhausted, a new pair of reagents (HCP antigen and anti-HCP antibodies) should be generated following the same procedure that was used for the previous set of reagents in order to maintain consistent performance of the new and previous HCP assays. Often across the industry, the manufacturing process is modified to improve the yield of the biopharmaceutical product. In such a situation, the original HCP assay reagents might have been produced according to the previous or old process that is not similar to the newly developed process. The old process reagents may not yield HCP values similar to those obtained using the new pair of reagents (HCP antigen and anti-HCP antibodies). If the upstream cell culture process has changed significantly, a new reagent may be required. The new reagent performance should be evaluated for accuracy again in the HCP assay using spike recovery, precision/reproducibility, sensitivity, range of detection, lack of interference from various sample matrices, and linearity in all in-process samples and the final drug substance. Some laboratories in the industry also use a technique called 2D-difference gel electrophoresis (DIGE), as it allows for a direct comparison of HCP populations between new and old processes (31, 32). In 2D-DIGE, different samples (up to 5) can be simultaneously analyzed and 397



compared on the same gel by labeling with CyDyes. This helps in evaluating if the changes between the two processes are large enough to merit going through the development of a new HCP assay. It is important to demonstrate that the new HCP assay is appropriate by showing that HCP clearance (fold clearance from step-to-step), beginning with the conditioned media (step 1, harvest) all the way down to the drug substance (final step), is similar or better than that observed with the old HCP assay. In order to achieve this goal, the new HCP antigen should be prepared using a method similar to that used for making the old or previous version of the HCP antigen. After measuring the protein concentration of the new HCP antigen, a comparison of the new and old HCP standards should be carried out by SDS-PAGE gel characterization to demonstrate similarity between the two. With regard to the anti-HCP polyclonal antibody preparation, it is better to use the same animal species that had been used for raising the old antibody reagent. The new anti-HCP polyclonal antibodies need to be characterized by 2-D western blot coverage analysis, as described above. In particular, a side-by-side comparison of the old and new anti-HCP polyclonal antibodies using the same sandwich HCP ELISA format is essential to show the suitability of the new reagents in the new HCP assay. Assay qualification using appropriate parameters (accuracy; precision, including repeatability and reproducibility; range; linearity; and sensitivity) should be performed. Bridging studies also should be performed using several samples from step 1 (harvest), all in-process steps, and the final drug substance. Identical samples should be tested in the old and new HCP sandwich ELISAs, and data obtained should be compared. The anti-HCP polyclonal antibodies generally are very stable when stored frozen (−80°C), and maintaining storage integrity throughout the life cycle of the product it is intended for is critical.

NIST Sample: Assay Protocols and Summary of Results The NISTmAb described throughout the current series is intended as an analytical reference material that mimics a typical biopharmaceutical drug substance. Therefore, to serve as a representative material, it should be free of process-related impurities to a level expected for a typical drug substance. For this reason, a subset of process-related impurity methods were applied to the NISTmAb sample to evaluate HCPs, Protein A, insulin, and residual DNA levels.

Sample The NISTmAb sample, received from NIST, was described to be a purified, humanized IgG1κ mAb produced in murine myeloma suspension cells. The antibody was at a concentration of 100 mg/mL in 12.5 mM L-His, 12.5 mM L-His HCl (pH 6.0).

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Assay Protocols The protocols used for quantitation of HCP and the other process-related impurities (Protein A, insulin, and host cell DNA) in the sample (IgG1κ mAb, # 203972) sent by NIST are shown in brief below:


HCP Assay HCP antigen standard was derived from the HCP antigen (immunogen) from an in-house murine cell line in order to mimic as closely as possible the profile one may expect from the NISTmAb. HCP antigen (ranging from 125 ng/mL to 5 ng/mL), the NISTmAb sample (in serial dilutions, 1:10, 1:50, 1:100, etc.) and controls (samples with no NISTmAb and positive controls) were added to an ELISA plate coated with anti-HCP polyclonal antibodies (capture antibody). Following incubation, biotinylated anti-HCP antibody (detector antibody) was added and incubated. Next, a streptavidin-peroxidase conjugate was added and incubated. Finally, the substrate ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was added and incubated before stopping the reaction with the addition of ABTS stop solution. The absorbance was measured at 405 nm using a microplate reader. HCP level present in the NIST sample was regressed from the standard curve generated by the software and corrected for the dilution factor. The sample HCP concentration was reported as the mean concentration of the dilutions yielding spike recovery values of 75 to 125%. This has been discussed under “Methods for quantitation of HCPs and other process-related impurities” earlier in this chapter. The HCP level in the sample, after correction based on a mAb product concentration of 100 mg/mL, was calculated to be 1.8 ng/mg (Table 1). System suitability requirements of the standard operating procedure (SOP) used for this assay were successfully met. In general, system suitability requirements pertain to obtaining a correlation coefficient (R) of ≥ 0.990 with the HCP antigen standard curve, negative control showing a value below the lower limit of quantitation (LLOQ), and the positive control (1000 ng/mL) to fall within 750 to 1250 ng/mL in the HCP assay.

Protein A Assay Standards (ranging from 12.5 to 0.1 ng/mL to generate a standard curve), the NISTmAb sample, and controls were added to an ELISA plate coated with commercially available polyclonal anti-Protein A antibody (capture antibody). Following incubation, biotin-labelled anti-Protein A antibody (detector antibody) was added and incubated. Next a streptavidin-peroxidase conjugate was added and incubated. Finally, the colorimetric substrate ABTS was added and incubated before stopping the reaction with the addition of ABTS stop solution. The absorbance was measured at 405 nm using a microplate reader. The Protein A level in the NIST sample was regressed from the standard curve generated by the 399



software and corrected for the dilution factor. Sample Protein A concentration was reported as the mean concentration of the dilutions yielding spike recovery values of 75 to 125%. The Protein A level in the sample, after correction based on a mAb product concentration of 100 mg/mL, was calculated to be < 0.01 ng/mg (Table 1). System suitability requirements of the SOP used for this assay were successfully met. In general, system suitability requirements pertain to obtaining an R value of ≥ 0.995 with the Protein A standard curve, negative control showing a < LLOQ value, and the positive control (2.5 ng/mL) to fall within 1.875 to 3.125 ng/mL in the Protein A assay.

Table 1. NIST Sample Results in Process-Related Impurity Assays Process-Related Impurity Method

Assay System Suitability

LLOQa

Result Obtainedb

Host Cell Protein (HCP) ELISAc

Met requirements

5 ng/mL

1.8 ng/mg

Protein A ELISA

Met requirements

0.1 ng/mL

< 0.01 ng/mg (No signal observed)

Insulin ELISA

Met requirements

4 μIU/mL

< 0.4 μIU/mg (No signal observed)

Host Cell DNA qPCRd

Met requirements

0.3 pg/mL

< 2.00 × 10−5 ng/mg (No signal observed)

a

LLOQ is the lower limit of quantitation of each impurity assay. the assays generally calculated using the following formula:

c ELISA = enzyme-linked immunosorbent assay. chain reaction.

d

b

Results obtained in all

qPCR = quantitative polymerase

Insulin Assay Standards (ranging from 250 µIU/mL to 1.95 µIU/mL to generate a standard curve), the NISTmAb sample, and controls were added to an ELISA plate coated with commercially available anti-human insulin antibody (capture antibody). Following incubation, biotinylated anti-human insulin antibody (detector antibody) was added and incubated. Next, HRP-labeled streptavidin conjugate was added and incubated. The colorimetric substrate TMB (3,3′,5,5′-tetramethylbenzidine) was then added and incubated before stopping the reaction by adding sulfuric acid. The absorbance was measured at 450 nm using a microplate reader. The insulin level in the NIST sample was regressed from the standard curve generated by the software and corrected for the dilution factor. The sample insulin concentration was reported as the mean concentration 400


of the dilutions yielding spike recovery values of 75 to 125%. The insulin level in the sample, after correction based on a mAb product concentration of 100 mg/mL, was calculated to be < 0.4 µIU /mg (Table 1). System suitability requirements of the SOP used for this assay were successfully met. In general, system suitability requirements pertain to obtaining an R value of ≥ 0.990 with the insulin standard curve, negative control showing a < LLOQ value, and the positive control to fall within 80 to 120 μIU/mL in the insulin assay.


Residual Host Cell DNA Assay The assay was performed in a 96-well format using the Applied Biosystems 7500 Real-Time PCR System and Sequence Detection System (SDS) software using Power SYBR Green chemistry, with forward and reverse primers specific for short interspersed repetitive elements (SINE) found within the mouse genome. The NISTmAb sample, NS0 genomic DNA calibrators, and controls were added to a reagent master mix containing primers and SYBR Green. Quantitation of DNA in the NISTmAb sample was regressed from an assay-specific standard curve generated by the software. System suitability requirements of the SOP used for this assay were successfully met. In general, system suitability requirements pertain to obtaining an R value of ≥ 0.990 with the NS0 DNA standard curve, and no template control showing a Ct > Mean Ct of Standard Calibrator 0.001 pg/10 μL or undetermined quantity in the host cell DNA assay. After running all of the process-related impurity assays described above, the results we obtained on the NIST sample mAb at MedImmune are shown below in Table 1.

Conclusions The current chapter has described the various process-related impurities one would encounter in a biopharmaceutical product (mAb). It is pertinent to mention here that the HCP values are critically evaluated from the point of view of the type of disease these products are clinically used for (acute or chronic) as well as the route of administration and the total dose used in patients. As described in the section on regulatory guidance, the numbers shown for process-related impurities in Table 1 for the NIST sample are within the levels generally seen in FDA-reviewed products for HCP (3, 7) (ELISA-based HCP levels of 1–100 ng/mg) and the host cell DNA levels that need to be met per WHO guidelines (14) (upper limit of 10 ng/patient dose for monoclonal antibodies). There are no clear guidelines on the limits (upper levels) allowed for other processrelated impurities such as Protein A and insulin in biopharmaceutical products, although the aim is to have them as low as possible. The NISTmAb also showed very low levels of these potential process impurities. In summary, the results obtained on the NISTmAb sample for all four process-related impurities (HCP, Protein A, insulin, and host cell DNA), as shown in Table 1, are (a) extremely low for HCP and (b) below the LLOQ for Protein A, insulin, and host cell DNA. 401


With regard to these process impurities, the NISTmAb material has at or near comparable levels expected for a regulatory submission. Therefore, although not intended for clinical use, the material can serve as a representative material typical of a purified drug substance.

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