Pharmaceutical industries are increasingly using biological or biotechnological products for drug development. The biologics or biopharmaceuticals, as they are commonly called, are generally composed of large, complex proteins which may or may not be glycosylated and are of human, plant or animal origin. These products have the potential to induce anti-drug-antibody (ADA) or cell-mediated immune response in patients. The immune response can range from a transient appearance of antibodies without any clinical significance to severe life threatening conditions such as severe hypersensitivity-type reactions, decrease in efficacy and induction of autoimmunity, including antibodies to the endogenous form of the protein. Anti-Drug Antibody (ADA) responses can lead to allergic reactions, reduction or neutralization of drug activity and potential cross reactivity. Moreover, in preclinical studies, ADA can affect drug exposure, affecting the interpretation of the toxicity, pharmacokinetic and pharmaco-dynamic data.
Factors causing immunogenicity :
The factors that cause immunogenicity of these products could be
patient-related, disease-related and product-related. The
patient-related factors could be: genetic factors, age of the patient, disease related factors including other treatments and exposure to similar immunogens owing to concomitant treatment, duration, route of administration and treatment modalities, and previous exposure to similar products. The
patient’s underlying disease can also be responsible for inducing immunogenicity. Some patients with chronic infections may be more prone to an immune response against a therapeutic protein. Patients with autoimmune disease, with an altered immunological tolerance to self-proteins, may react differently. In some conditions such as malnutrition, advanced metastatic disease, organ failure, an immune response against a therapeutic protein might be less likely to occur due to an impaired immune system. For some products, it has been reported that the susceptibility to an antibody response can be different for different indications.
Product-related factors that might influence an immune response are: the origin and nature of the active substance (structural homology, post translational modifications), modification of the native protein (e.g. pegylation), product and process related impurities such as break down products, aggregates and host cell proteins, source of the protein, manufacturing process (impurity profile, contaminants), formulation and stability characteristics (degradation products, aggregates) of the protein, its dose, dosing interval and duration of treatment.
Thus to ensure clinical safety and efficacy of the numerous biologics used in drug development, immunogenicity testing has become very vital. Food and Drug Administration (FDA) in the USA has a policy to include immunogenicity as part of the review of clinical safety assessments for biologic license applications. Similar requirements exist for other regulatory agencies such as European Medicines Agency (EMEA) in Europe. However as of now, no comprehensive guidelines exist on the approaches that are required for immunogenicity testing during product development. Similarly, there is no single source of the requirements for non-clinical or clinical studies and no specific guidance for performing or interpreting assays.
The various analytical approaches that have been used to monitor or detect the presence of antidrug antibodies in the last couple of decades are:
1) Immunological based technologies (e. g. Western blots, immunoassay)
2) Chromatographic formats (e.g. size exclusion, capillary electrophoresis)
3) Surface plasma resonance biosensors and
4) Bioassays (e. g. immortalized cell lines).
Antibodies are needed for western blot, immune precipitation, and protein purification among other methods. Manufacturing a protein made recombinant involves cumbersome steps - cloning the cDNA, expressing the protein and purifying the protein. These processes can be bypassed by outsourcing the whole process of manufacturing the peptides or the antibodies to a vendor who can synthesize the specific peptides of choice as well as manufactures antibodies.
Synthetic peptides could be used for the following purposes:
- To verify the structure of naturally occurring peptides
- To study the relationship between structure and activity of biologically active proteins and peptides and establish their molecular mechanisms and
- To develop new peptide-based immunogens, antibodies, hormones, vaccines, etc.
Synthetic peptides may range from 2 to 120 amino acids. Small synthetic peptides (in general less than 10 amino acids in length) have the advantage of rarely inducing antibody formation and can be tailored to move across blood brain barrier. Peptides for antibody production are generally of 15 to 25 amino acids. Such peptides can be conjugated to a protein carrier such as KLH, BSA etc. which can induce antibody production and used for both in vitro and in vivo assays.