Introduction

Biopharmaceuticals are foreign to the body and with potential to elicit immune response when they are administered to patient. The immunogenicity of a biopharmaceutical, such as antibody drug and protein drug, can be detected through the analysis of anti-drug antibodies (ADAs). The ADAs arose in body may affect the distribution of antibody drugs and protein drugs which bind with the target or ligand to achieve drug efficacy. The presence of ADAs also may result in increased clearance rate and hypersensitivity to the drug. Although all of biopharmaceuticals, especially antibody drugs, are humanized with more human-like structures, the clinical and nonclinical immunogenicity assessment are still very critical steps in drug development.

Factors involved in development of immune response against drug

Many non-drug factors as genetic factors, age, therapeutic regimen, pre-existing antibodies and etc. may influence immune responses to biopharmaceuticals and lead to inter-patient variability¹. Here we only have a glance at product-related factors.

1. Therapeutic proteins & antibodies

Immune tolerance to low abundance endogenous proteins is weaker. It is easy to induce ADAs against analogue therapeutic proteins like cytokines and growth factors, which are low-level in normal human body. The variations in the amino acid sequence as fused proteins may form new T cell epitopes and neo-epitopes for B cells. Changes in the protein structure caused by post-translational modifications, denaturation, aggregation of the therapeutic protein or chemical modifications can also trigger immune response. Residual impurities as host cell DNA (HCD) and host cell protein (HCP) in products can act as adjuvant and trigger stronger immune response to therapeutic proteins and antibodies.

2. Therapeutic peptide

The active pharmaceutical ingredient (API) of generic peptide drug may not be the primary target of the immunogenicity assessment, because the immune response against API has been well defined over decades of clinical use. However, it can be immunogenic post the modifications to the API sequence. Immunogenicity can also be attributed to process-related impurities as HCP for peptide drug manufactured by recombinant expression².

3. Oligonucleotide therapeutics

The oligonucleotide itself, modifications, carrier components (e.g., PEGylated lipid nanoparticles), and conjugates such as peptides or antibodies all can affect the immunogenicity of the final products³.

How to generate good ADA positive control

ADA positive control can be polyclonal antibody (pAb) or monoclonal antibody (mAb). The pAb can recognize multiple epitopes of the biopharmaceutical, which has high consistency with the ADA level in actual samples. The mAb can only recognize single epitope of the biopharmaceutical, while it is more specific. A neutralizing mAb can be used as positive control to monitor neutralizing ADA. The technological methods for antibody development are diverse and powerful, but there are still several aspects to consider.

1. Immunogen preparation

It would be best to directly use final product with representative of a complete and mature process as immunogen. Antibody fragmentation can increase the one-time success rate of ADA and anti-idiotope (anti-ID) mAb against antibody dug. However, fragmentation is not recommended to the protein drug with fused Fc, antibody drug conjugate (ADC) and bispecific mAb, because of sequence variations and potential structure changes. The molecular size of some therapeutic peptides may be over thousands of Daltons, but it is still very hard to stimulate high level of ADA in animal without conjugation to carrier protein. Similarly, it is a critical step to conjugate the payload and oligonucleotide to the carrier protein before immunization. From experiences, using KLH as a carrier protein has the highest success rate.

2. Purity of ADA

A good ADA pAb generation should has at least 2 steps of purification. First, the serum of animal should be purified with protein A or protein G resin. Then the eluted IgG will be further incubated with the drug-conjugated resin to enrich the specific ADA. The mAb against drug can be produced by the hybridoma cell line or recombinant expression. In vivo antibody production with hybridoma may not be a good choice, especially to the anti-ID mAb, because of the contamination of total IgG from mouse immune system.

3. Specificity of ADA

Due to the high abundance of the total IgG in human serum, the ADA against antibody drug should nearly have no cross-reaction with total human IgG. Therefore, people choose to remove Fc from antibody drug before immunization to obtain antibody specific to idiotopes. Further purification with total human IgG conjugated resin can remove the cross-reacting antibody against CH1 & CL constant region and same FR in variable region. However, cross-absorption with total human IgG does not work well to the antibody drugs with Lamda type light chain or IgG4 subtype heavy chain, because they account for a very small proportion in total human IgGs. It is recommended to choose a similar mAb with Lamda type light chain or IgG4 subtype heavy chain to do the further purification.

4. Titer of ADA

ADA against antibody drugs or protein drugs always pose very good titers, but some common buffer components may cause interference in ELISA and result in very low or unrepeatable ADA titer. For example, the buffer component as polysorbate (tween) is chemically similar to polyethylene glycol (PEG) which may react with anti-PEG ADAs. Moreover, the final purified ADA can be used for a preliminary matrix interference test to check the risk of decreased titer or a very high minimal required dilution. Sometimes, ADAs against antibody drug need a new round of purification with total human IgG conjugated resin because the cross-reaction leads to the lower titer and false negative in spike-in assay with human serum samples.

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References

[1] Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection Guidance for Industry U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) January 2019 Pharmaceutical Quality/CMC.

[2] Anne S De Groot, Brian J Roberts, Aimee Mattei, Sandra Lelias, Christine Boyle, William D Martin, Immunogenicity risk assessment of synthetic peptide drugs and their impurities, Drug Discovery Today,Volume 28, Issue 10, 2023,103714.

[3] Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics Guidance for Industry U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) June 2024 Clinical Pharmacology.