Process Related Impurities and their Impact on Product Quality-An FDA Perspective and Recommendations (With Emphasis on Host Cell Protein (HCP) Impurities)

Barbara Rellahan, M.S., Ph.D. Division of Monoclonal Antibodies Office of Biotechnology Products OPS, CDER

Disclaimer

The contents of this presentation may not necessarily reflect FDA official policy Presentation Outline

1. Definitions/Regulations/Guidance

2. Regulatory Concerns/Considerations

3. Host Cell Protein Assays

4. Examples of HCP review issues Types of Impurities (ICH Q6B)  Product-related = ‘…molecular variants arising during manufacture and/or storage, which do not have properties comparable to those of the desired product …’(e.g. aggregates, fragments, oxidation, deamidation).

 Process-related = ‘…those that are derived from the manufacturing process, cell culture or downstream processing’ (e.g. HCP, DNA, insulin, methotrexate, anti-foam).

 Contaminants = ‘…all adventitiously introduced materials not intended to be part of the manufacturing process’ (e.g. adventitious viral and microbial agents, endotoxin, mycoplasma etc) and probably also leachables. Regulation and Guidance

 21 CFR 610.13 Purity ‘Products shall be free of extraneous material except that which is unavoidable…”

 ICH Q3C Impurities: Guideline for Residual Solvents, Class 1, 2 and 3.

 ICH Q6B Specifications for Biotechnology Products

 ICH Q11 Development and Manufacture of Drug Substances

Regulatory Concerns/Considerations  Safety/Immunogenicity • Some process impurities represent a direct clinical safety risk for the patient (e.g. infection, hypersensitivity, endotoxin shock, immediate toxicity). • Process related impurities may not represent a direct risk for the patient but may influence the overall safety profile of a product and so they must be identified and controlled. • Immunogenicity: Impurities may be direct targets of an immune response, or may impart adjuvant activity and induce or augment anti-drug antibody responses).

 Efficacy • Impurities may be biologically active or induce an immune response to an endogenous protein.

Regulatory Concerns/Considerations for HCPs  Safety/Efficacy

Data indicated a HCP impurity might be responsible for cytokine release.

Wang, X. et al., 2009. Biotech. Bioeng. 103:446. Regulatory Concerns/Considerations for HCPs

 Safety/Immunogenicity

. Clinical trials of a therapeutic protein were recently placed on hold after it was noted that patients treated with the drug were more likely to test positive for antibodies to HCPs. Potential safety issues would be cross-reactivity of anti-HCP response with endogenous proteins, an increase in infusion related reactions or augmentation of anti-product immunogenicity.

Regulatory Concerns/Considerations for HCPs  Efficacy A co-eluting HCP impurity with enzymatic activity was identified. The clinical team had concerns because there was information in the literature that linked the HCP with induction of inflammatory mediators which could exacerbate symptoms in the intended patient population. The HCP impurity sequence was also conserved between species and anti-HCP responses in the patient population could cross-react with the endogenous protein, this could play a role in product efficacy. Process-Related Impurity Control

 Identifying and characterizing all process-related impurities.

 Developing and/or implementing appropriate control strategy. This could consist of but not be limited to: • Raw material testing • Control of introduction into the process (e.g. agents added to the bioreactor on an ‘as needed’ basis) • Leachable/extractable studies • Small scale removal studies (may include spiking) • Process characterization • Validation of removal at commercial scale • In-process and/or release testing including qualification/validation of assays. • Control of product stability

Process-Related Impurity Control Issues

Assay qualification/validation If the assay to detect the impurity does not sensitively and accurately detect the impurity, data based on this assay supporting removal or control may not be meaningful.

Host Cell Protein Assays Host cell protein (HCP) impurities represent a unique challenge because of their complexity and diversity.

In addition, HCP assays almost always rely on the ability of an anti-HCP antiserum to detect HCP impurities, so the performance of the assay is tightly linked to the quality of this reagent.

If the anti-HCP antiserum does not recognize a majority of the potential HCP impurities, the assay is not going to provide data/information that accurately reflects the level of HCP impurities present. Host Cell Protein Assays

1. ELISA (or Western blot) assay- a) Process-specific assay: The anti-HCP antiserum is raised against a host cell protein pool that is derived from the cell line used for production. Generally a vector transfected parental cell line is used.

b) Commercial assay- The anti-HCP antiserum was raised against host cell proteins derived from a ‘generic’ cell line (CHO, NSO, E. coli).

2. HCP specific assay- For proteins that are known to represent a significant HCP impurity (e.g., co-purifying protein, other proteins expressed by expression construct such as GFP).

3.Characterization assays: 2D-Difference Gel Electrophoresis (DIGE), mass spectrometry Host Cell Protein Assay Qualification The anti-HCP antiserum needs to be qualified to determine the approximate percent coverage of potential HCP impurities. One informative method is 2D gel/western blot comparison. Multiple assessment types might be informative.

2D-SDS analysis of Western blot analysis of 2D- representative HCP pool SDS gel using anti-HCP stained with sensitive protein antiserum. stain.

OBP Recommended Practice (OBP-RP-004): Qualification Requirements for the Host Cell Protein (HCP) Release Assay

The following comment should be communicated to sponsors as a non-hold comment. It is recommended that the comment be send as early in the product development cycle as possible to provide sponsors guidance on validation requirements for the HCP assay. The information/data requested should be provided in BLAs/NDAs to support use of the HCP assay.

Provide a summary description of the assay and the source (in-house or commercial) of the antiserum used for detection of host cell protein impurities (HCPs). The anti-HCP antiserum needs to be qualified for its ability to detect potential HCP impurities. This assessment should include 2D SDS-PAGE gels of the range of HCPs detected by a sensitive protein stain, such as silver stain, compared to the range detected by western blot analysis using the antiserum employed in the assay. It is possible to use a similarly sensitive and discriminating assay in lieu of the 2D SDS-PAGE assay. If an alternative pathway is pursued, consultation with the Agency is recommended. These data should be used to determine the approximate percent of potential HCP impurities that are recognized by the HCP antiserum. It is the Agency’s experience that analysis of HCP coverage by a 1-dimensional SDS-PAGE gel method is not sufficiently informative for this purpose.

Generation of an Anti-Host Cell Protein Antiserum

The goal should be to generate an antiserum with as broad HCP coverage as possible, but one that still has good reactivity to HCP impurities that are likely to be present in the final product.

What is the best way to prepare the HCP immunogen? I don’t know but here are some scientific (non-regulatory) considerations… Use of Column Eluates/Fractions Columns 1 2

Harvest/lysate from mock transfected cells.

• Eluates from mock purification run (s) (under worst case conditions??) used as immunogen for anti-HCP antiserum.

• Any step/process that removes proteins from the preparation has the potential to decrease the ability of the resulting antiserum to detect contaminating HCP impurities.

Use of Column Eluates/Fractions Columns 1 2

Harvest/lysate from mock transfected cells.

ThisEluates scenario from may mock increase purification the immunoreactivity run (s) (under worst to a casepool conditions??)of predominant used HCPas immunogen impurities butfor resultsanti-HCP in anantiserum. antiserum that is tightly linked to a given process and one that may not account well for process variability. Antiserum coverage of and make up of HCPs in multiple runs would need to be assessed. New antiserum may need to be made after process changes. This approach, by itself, is not generally recommended. Cell Lysates or Culture Supernatants Potential Pros and Cons Immunogen Pros Cons

Cell Lysates •Contains broad range of •May contain immunodominant proteins that are not potential HCPs impurities present in (or present as a small percentage of) purified product. •Cells can be washed and •Reactivity to very broad range of HCPs may so made free of extraneous minimize immune response to more relevant HCP cell culture media impurities and limit ability to accurately quantitate impurities. actual HCP impurities. Culture •HCPs present could •May not contain broad range of potential HCP supernatant potentially represent a impurities, assay wouldn’t be sensitive to process more relevant pool of variability. potential HCP impurities •Preparation may be contaminated with compared to a total cell immunogenic extraneous cell culture impurities. lysate. Immuno-reactivity to non-HCP process-related impurities needs to be addressed. Generation of an Anti-Host Cell Protein Antiserum

Scientific Points to Consider

 Immunize multiple animals (more than one species?) with HCP immunogen and pool antisera that demonstrate good coverage of potential HCP impurities.

Consider using different immunogens and pooling antiserum to generate a broadly reactive reagent (e.g., mock transfected cell lysate, mock eluate from early purification step).

 Prime animals with one immunogen (e.g., cell lysate) and boost with another (e.g., supernatant, eluate fraction).

Generation of an Anti-Host Cell Protein Antiserum Scientific Points to Consider  TheIt goal is likely should that be an to anti generate-HCP antiseruman antiserum will with need as to broad be HCP coveragereplaced as possible, at some but point. one Bethat sure does that have the good immunogen reactivity to HCP impuritiesand immunization that are mostprocedure likely used to be to present generate in the final product.original antiserum can be replicated if needed to

Immunizegenerate multiple a new reagentanimals with with equal HCP orimmunogen better and pool antiseraperformance that demonstrate compared good to coverage the original of potential antiserum. HCP impurities. New procedures can be used of course as long as the Considernew antiserum using different is qualified immunogens appropriately (and pooling and shown antiserum to to generatebe comparablea broadly reactive to or better reagent than (e.g., the antiserummock transfected being cell lysate,replaced. mock eluate from early purification step).

Examples of Issues DMA has had With HCP Assays

Example 1: General underestimation of HCP impurity levels due to poor coverage of anti-HCP antiserum. Process Specific versus Commercial HCP Assays – Example 1A

Silver stain of HCP assay standards

Western blots

Commercial Assay Process Specific Assay In House versus Specific HCP Assays– Example 1A

Process Specific Assay

Commercial Assay

Release specification Requested a 2-fold increase in specification when using the in- house assay compared to the commercial assay. Example 1B: At the time of licensure, HCP assay was found to be deficient and the sponsor committed to develop a more sensitive HCP assay that recognized a broader range of potential HCP impurities.

New, improved assay with better HCP impurity coverage was not implemented until after a significant DS manufacturing change was made.

Results using the new assay identified a higher level of HCP impurities were present in the new process compared to the old process that had not been identified during process development using the original HCP assay. Some lots would fail the commercial HCP release specification.

Sponsor had to alter new process to include a step that provided additional clearance of HCPs. This process was re-validated prior to submission of an supplement requesting approval for the new process. Examples of Issues DMA has had With HCP Assays

Example 2: Replacement anti-HCP antiserum does not provide as good coverage of HCP impurities as the original antiserum. HCP assay was not a component of lot release, just a New HCP antiserum Original antiserum characterization assay. New drug substance process implemented for licensed product. Replaced the old HCP assay with new assay prior to validation of the new process. No antiserum from original assay available to compare HCP levels of Process 1 to Process 2 validation lots. New HCP antiserum did not detect as broad a range of HCP as the Antiserum dilution original antiserum. Process 1 DS results Data from Process 1 DS Lot HCP ng/mL HCP ng/mL comparing the two HCP Old method New Method assays also suggested the 1 47.1 13.1 new assay was detecting a reduced level of HCP 2 37 7.10 impurities compared to the 3 54.2 22.3 original method. 4 35.5 5.00

A CR letter was issued for 5 26.2 25.3 introduction of a drug 6 39.6 13.5 substance process due to 7 25.9 17.5 issues surrounding DP stability and whether new HCP impurity was impacting DP stability.

Examples of Issues DMA has had With HCP Assays

Example 3: HCP protein co-purifies with product and is present at levels that exceed the capacity of the anti-HCP antiserum to quantify it accurately resulting in under-estimation of HCP content of product. Also do not recognize that there is a co- purifying protein.

Antigen Excess

Sponsor uses a platform HCP Product with assay. During qualification of co-purifying protein. the assay for a new product, they noted the assay demonstrated dilutional dependence, particularly with more purified process pools. The more they diluted the product pools, the higher the Anti-HCP antiserum HCP result. Active HCP Impurity Present at Relatively High Levels Multiple assays used to identify and/or characterize the HCP impurity in the purified DS failed. The HCP impurity needed to be separated from the product before it could be identified. HCP impurity was found to be biologically active.

Regulatory concern: Potential for the impurity to impact safety or efficacy of the product. The impurity could have biologic activity on its own, or an immune response to the protein could impact efficacy. HCP impurity could be considered to be a component of the drug product. It was recommended that the sponsor not initiate pivotal trials until they had developed a process that better controlled the HCP impurity. Lessons Learned/Conclusions

 HCP assay qualification needs to include a sensitive determination of the degree of HCP coverage provided by the anti-HCP detection antibody. Data must demonstrate that the HCP assay detects the majority of potential HCP impurities.

 This aspect of assay qualification needs to be performed early in development so that assessments of process capabilities in regard to HCP removal are valid and process changes can be made if there is sub-optimal HCP clearance.

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