Immunogenicity and Biosimilar Protein Development

Michael J. Anderson, Ph.D. • Manager, Immunogenicity Immunoassay Technologies US, ICON Development Solutions

John Chappell • Director, Immunoassay EU, ICON Development Solutions

Immunogenicity is the ability of a therapeutic drug to elicit an immune response, specifically the production of antibodies, in study subjects. An antibody response specific for a therapeutic protein can range from the production of clinically irrelevant antibodies with no impact on drug distribution, clearance, or efficacy to a severe anti-drug antibody response that manifests in serious safety issues. In addition, the production of antibodies may neutralize the bioactivity of the protein therapeutic, leading to decreased efficacy of the drug and possible cross-neutralization of endogenous proteins. Hypersensitivity reactions (anaphylaxis), immune complex deposition within the kidney, and cross-reactivity with endogenous proteins are all safety issues that have been seen in the clinic. Patient immune responses to therapeutic proteins can result in serious, life threatening safety events, thus, the issue of immunogenicity requires significant attention.

Immunogenicity testing is a critical facet of drug development, with regulatory agencies requiring anti-drug antibody testing for both pre-clinical and clinical trials. Although not predictive of clinical immunogenicity, detection of anti-drug antibodies in pre-clinical studies is essential to properly interpret pharmacokinetic (PK) and safety data. In the clinic, development of anti-drug antibodies can lead to serious consequences for the patient as mentioned above. Because of the potential safety risk, development of sensitive, drug tolerant immunoassays to detect anti-drug antibodies in clinical trials is essential. The development of these assays and interpretation of sample analysis results will become even more important within the realm of biosimilar drug development.

Biosimilar proteins are not generic (identical to the innovator) drugs; rather they are biologic molecules that are “similar” to the innovator product. Traditional small molecule drugs are relatively easy to replicate with defined chemical processes, leading to generic drugs that are identical to the reference product. Due to their small molecular weight, the immune system tends to ignore small molecule therapeutics, and immunogenicity testing is not a required analytical test when developing a true generic small molecule drug. On the contrary, due to their large molecular weight, protein therapeutics can easily be recognized by the immune system. Large molecule drugs have complex, three-dimensional molecular structures and are manufactured within living systems using extremely complex manufacturing processes. These living systems typically use cell-based production methods, utilizing E. Coli or a variety of mammalian cell lines, none of which produce a protein identical to the human body. This leads to a molecule that is inherently difficult to reproduce.

As demonstrated in Figure 1, the size and complexity differences between a common protein therapeutic (monoclonal antibody) and aspirin, a traditionally small molecule drug, are significant. These differences underscore the complexity of attempting to produce a biologic drug that is similar to the innovator molecule. It is known that small manufacturing or storage changes can lead to physical modifications of a molecule, which can alter the immunogenicity profile.^1-3 The same principles will apply to the production of biosimilars. Due to the nature of protein therapeutic production, minor manufacturing differences between the biosimilar product and the innovator product can contribute to differences in glycosylation, aggregation and protein folding, all of which can result in an altered immunogenicity response.

The potential immunogenicity of a therapeutic protein can be influenced not only by the manufacturing processes mentioned above, but also by the type of disease, route of administration and dose.^2-4 Biosimilar proteins will most likely not be identical to the innovator drug, and because of this, immunogenicity becomes a major concern when developing biosimilar proteins. The potential exists for serious clinical consequences due to anti-drug immune responses with all protein therapeutics. A safe immunogenicity profile of the innovator product does not indicate that the biosimilar protein will be safe. As exemplified by the PRCA cases in the late 1990s, one small modification in the manufacturing and packaging process can lead to severe consequences in the clinic. In this case, seemingly minor changes in formulation and packaging (which some attribute to the use of pre-filled syringes with uncoated rubber stoppers) led to patient production of anti-drug antibodies which cross-reacted with and neutralized endogenous erythropoietin.^5-6 This case underscores the need for a comprehensive plan to develop and validate the necessary analytical assays to address immunogenicity concerns.

Immunogenicity of protein therapeutics is a complicated field in itself, and the complexity multiplies when moving into the realm of biosimilars. The ability to properly detect anti-drug antibodies is an imperfect science, with all available approaches having certain limitations. Traditionally, Enzyme Linked Immunosorbent Assays (ELISAs) or variants thereof, have been the work horse of immunogenicity testing. However, ELISA based assays have lacked in both drug tolerance and sensitivity, being unable to detect low-titer, low-affinity antibodies in the presence of the therapeutic drug. This, coupled with the lack of standardization of assay validations, sample analysis, and data analysis within the industry, has lead to immunogenicity results that can be difficult to interpret. In recent years, the use of electrochemiluminescent assay platforms, combined with development of acid dissociation techniques, procedures to remove excess therapeutic drug from patient samples, and homogeneous sample incubations to optimize for drug tolerance have led to more sensitive and drug tolerant anti-drug antibody assays. These improved analytical techniques will prove valuable when assessing the immunogenicity of biosimilar protein therapeutics in comparison to the innovator product.

The immunogenic potential of a biosimilar protein can only be evaluated within proper clinical trials using sensitive analytical methods designed to detect low affinity anti-drug antibodies. For higher risk molecules (replacement therapies, proteins with endogenous counterparts), the neutralizing potential of the anti-drug antibody will have to be evaluated. When developing monoclonal antibodies, neutralizing antibody assays may or may not have to be performed as the presence of neutralizing antibodies becomes an issue of interpreting efficacy data rather than a safety concern. Because of the limitations that most likely existed with the anti-drug antibody assays when the innovator product was being developed, comparison of recently generated immunogenicity data with the data generated as part of the innovator drugs approval will not suffice. A full comparison between the innovator product and biosimilar therapeutic may have to be performed using current, more sensitive assays.

Proper immunogenicity assessment of biosimilars requires an in-depth understanding of the design of assays to detect anti-drug antibodies, implementation of proper validation procedures, and experience in analysis and interpretation of sample results. Use of an inappropriate anti-drug antibody assay format, improper validation testing or analysis of sample results may lead to misinterpretation of safety data. Anti-drug antibody assays must be properly designed and developed if they are to perform as intended. Many platforms and assay formats are available, and, depending on the protein therapeutic and its target, not all are appropriate for use. There are many variables that must be considered when developing an assay to detect anti-drug antibodies. The design of the clinical trial (single vs. multiple dose study), anticipated therapeutic drug levels in the immunogenicity samples, therapeutic target of the drug, mechanism of action of drug, and patient disease state all need to be considered when developing immunogenicity assays. Once the appropriate assay has been developed, the assay must be properly validated. Validation must include statistical determinations of both screening and confirmatory cut points. The use of arbitrarily set cut points to determine sample reactivity is improper and scientifically invalid. In order to ensure that the assay performs as expected when analyzing study samples, validation testing must also include assessment of signal precision, from which the in-study acceptance criteria can be generated. During sample analysis, data must be properly analyzed to ensure that the assay is performing as intended and that samples are not being reported as false negatives.

Development of biosimilar proteins will present drug developers with a variety of challenges, both in manufacturing and in the clinic. Immunogenicity will prove to be one of the biggest challenges. Proper design and validation of an assay to detect anti-drug antibodies and accurate interpretation of sample analysis results will prove integral to developing a biosimilar protein.

References:

¹ Chirino, A.J., and Mire-Sluis, A. Characterizing biological products and
assessing comparability following manufacturing changes. Nat Biotechnol 2004. 22, 1383-1391.

² Crommelin, D.J.A., Storm, G., Verrijk, R., de Leede, L., Jiskoot, W., and Hennink,
W.E. Shifting paradigms: biopharmaceuticals versus low molecular weight
drugs. Int J Pharm 2003. 266, 3-16.

³ Schellekens, H. Biosimilar epoetins: how similar are they? Eu J Hosp Pharm 2004.
3, 8-12.

⁴ Sharma, B. Immunogenicity of therapeutic proteins. Part 1: impact of
product handling. Biotechnol Adv 2007. 25, 310-317.

⁵ Boven K, Knight J, Bader F et al. Epoetin-associated pure red cell aplasia in patients with chronic kidney disease: solving the mystery. Nephrol Dial Transplant 2005; 20 [Suppl 3]: iii33–iii40

⁶ Casadevall N, Nataf J, Viron B et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 2002; 346: 469–475