With the advent of biotechnology, more and more drug products currently available in the market are biotech derived human recombinant protein biotherapeutics which have replaced the animal derived drug products. This has helped in global availability of quality drug products at a competitive price with very little side effects. To utilize the growing demand for the biotechnology derived drug products, many multinational agencies have established the biopharmaceutical units in different parts of the world using Recombinant DNA technology. The technology involves identifying genes, isolating genes, modifying genes, re-expressing genes in other hosts or organisms, identify new genes and the proteins they encode, to manufacture large quantities of specific gene products such as hormones, vaccines and other biological agents of medical interest. The advantages of the recombinant protein biotherapeutics include high purity, high specific activity, steady supply and batch-to-batch consistency. All protein biotherapeutics are potentially immunogenic and hence could elicit immunogenicity response. These responses could be of different types such as binding antibodies or neutralizing antibodies. Immunogenicity of protein biotherapeutics is a major concern especially when the biological function of the drug and the endogenous counterpart are neutralized by antidrug antibodies (ADA). Hence the regulatory agencies insist that the immunogenicity response should be assessed by validated sensitive assay formats during the different stages of drug development and the antibody response be characterized. In this document, an effort has been to provide an overview of importance of safety assessment of the drug product for market launch, regulatory concerns and different methods for immunogenicity assessments. The document also includes a case study on immunogenicity assessment using GYRO LAB system available at Accutest Biologics Private Limited.

The development of recombinant DNA technology has dramatically increased the use of protein biotherapeutics such as antibodies, hormones and enzymes. These are useful in the treatment of a wide range of diseases, including cancerous conditions, infections, diabetes, and rheumatoid arthritis. The relative success of these new drugs has stimulated the development of new candidates that are evaluated in clinical studies for desired and adverse effects in patient groups. While all protein biotherapeutics are potentially immunogenic in nature, recombinant human biotherapeutics, however, are not expected to evoke an immune response in humans given their similarity to endogenous proteins. Recombinant human proteins do display reduced immunogenicity compared with non-human sequences (Wadhwa & Thorpe,2007), yet formation of ADA was noted after patient treatment with such therapeutics (Sauerborn et al.,2010; Schernthaner,1993). Therefore ADAs pose a challenge in clinical medicine due to possibility of neutralization of endogenous protein thus reducing the efficacy of a biotherapeutics.

Protein biotherapeutics
Protein biotherapeutics derived from recombinant DNA technology are of high purity, high specific activity and with batch-to-batch consistency. Unlike the generic small molecule drug products, these are large molecules such as peptides, proteins, hormones and therapeutic monoclonal antibodies with molecular weight ranging from 3000-150000 Da. These protein drug products are the human gene sequences expressed in suitable host and purified for the specific drug product of interest.

The production of recombinant protein biotherapeutics and successful launch involves multiple critical steps such as selecting the target protein, determining the amino acid sequence for expression, optimization of codons and synthesis of DNA. Other critical steps include, building the construct with various expression vectors, screening the best expression route, production and purification of proteins, characterization of protein, locking the API, performing the preclinical studies and clinical studies.

Immunogenicity of therapeutic proteins
Immunogenicity is the ability of a substance to trigger immune response. Immunogenicity triggered by Biopharmaceuticals is mainly linked to production of ADA and unwanted immunogenicity. However, in case of vaccine candidates it is wanted immunogenicity.

A well-documented example of biotherapeutics immunogenicity is the neutralizing antibody responses that developed in patients receiving treatment with human erythropoietin. This was associated with the development of pure red cell aplasia (PRCA) among patients with chronic renal failure (Casadevall et al.,2002). Erythropoietin is a hormone that is required for red blood cell (RBC) development, and PRCA manifests as severe sudden-onset of anemia that is characterized by the absence of red cell precursors in the bone marrow (Boven et al.,2005b). In PRCA, the neutralizing antibodies blocked exogenous and endogenous EPO functions. The patient became severely anaemic and required regular blood transfusion. Several deaths were also reported. When EPO therapy was ceased, antibody levels declined and the condition was reversed. Therefore, risk based immunogenicity assessment of the protein biotherapeutics has been recommended to confirm that the drug product does not elicit immune response which potentiates the neutralization of administered /endogenous protein.

A non-self recognition and interaction of protein therapeutic by immune cells initiates the immunogenicity process. There are essentially two ways in which a protein therapeutic may elicit immune responses in the subjects administered with the drug product under investigation. The first is an adaptive immune response if the therapeutic agent is recognized as Non-Self or ‘foreign’ protein which subsequently leads to production of specific antidrug antibody response. Foreign antigens trigger a ‘classical’ immune reaction that is dependent upon T-cell activation. This mechanism requires interaction of antigen with APC that, in turn, prime naïve T-cells. Primed T-cells may then interact with B-cells displaying the antigen within a major histo-compatability complex (MHC) molecule. Interaction with co-stimulatory molecules further activates T-cells and stimulates cytokine secretion, leading to the proliferation of B-cells and antibody production. Depending on the nature of drug product, its purity (with the contaminant protein, DNA), the antibody response can be as follows:

• Antibodies which bind and neutralize protein
• Antibodies which bind, but do not neutralize protein
• Antibodies against non-product related proteins e.g. host cell derived proteins
• Antibodies against product and non-product related proteins
• No antibodies

Thus the nature of antibody response has to be evaluated using sensitive validated bioanalytical methods to know whether the immune response has an effect on neutralization of the protein drug.

Regulatory considerations and immunogenicity testing
Regulatory agencies have recognized the importance of immunogenicity testing of protein biotherapeutics for safety and efficacy evaluation. In order to demonstrate clinical safety and efficacy, immunogenicity testing is now a key component of biotherapeutics drug development process and is required as per ICH, US-FDA and EMA guidance documents.

The formation of neutralizing antibodies can affect safety and efficacy, but non-neutralizing antibodies can also be a concern due to effects on half-life and biodistribution of the product (Shankar et al.,2006). Hence risk based immunogenicity assessment is recommended. Wherever the endogenous counter parts are available, appearance of neutralizing antibody in subject samples to the treatment regime will be viewed as serious concern and regulatory acceptance of such products becomes questionable (e.g. Insulin). The regulatory guidance recommends use of multi-tiered strategy for immunogenicity testing using validated bioanalytical approaches for various assay steps such as, screening, confirmatory, characterization of the confirmed positives for antibody titer and for assessing neutralizing abilities with class of antibody response.

Factors influencing immunogenicity
There are multiple factors causing immunogenicity of the protein drugs administered to the subjects. Some of the factors triggering the immunogenicity are presented in Fig 1 below.

These factors can be classified under major groups such as, product, patient, assay, process and administration related, which are to be suitably addressed at an early stage when product and processes are finalised. The variation in human gene sequence can lead to the drug to be recognised as a foreign or non-self particle provoking an immune response. Protein aggregation can increase the immunogenicity of biotherapeutics. The level of contaminant proteins and DNA components from the expression systems or from the host cell is considered to be another major concern and these are to be assessed by highly sensitive methods. The route of administration, dose and frequency of administration can also account for immune responses. Subcutaneous route of administration is more likely to induce immunogenicity than the intravenous route due to slow absorption and possibility of recognition by antigen presenting cells. Post translational modifications and glycosylation pattern are likely to induce conformational changes in the structure which may lead to immunogenicity.

The bioanalytical methods developed to evaluate the immunogenicity should be of adequate sensitivity and these are to be validated as per regulatory requirements (USFDA and EMA guidance)to detect low levels and low affinity ADA in presence of high circulating concentration of drugs.

Strategy for immunogenicity testing
The immunogenicity should be assessed in the pre-clinical and clinical subject samples using validated bioanalytical approaches. The method developed should be able to detect the false positive samples and should be performed preferably using the drug specific critical reagents including positive control antibodies and appropriate cut-point criteria. The immunogenicity methods are quasi quantitative as surrogate positive controls are used as assay system suitability. However, the positivity is declared based on the statistically derived screening assay cut point during validation using the experimental values for minimum of 200-300 drug free naïve matrix data points (preferably using disease state matrix, for clinical 50-100 individual matrix). The method should also be robust to detect ADA in presence of high circulating drug. The method should have adequate sensitivity for detecting the ADA in neat matrix (250ng/mL for clinical and up to 500-1000ng/mL for Non-Clinical samples). All the study samples are to be tested first for screening assay, screened positives to be taken for confirmatory assay and the confirmed positives samples for further characterization- antibody titer assessment. The confirmed positives also need to be tested for presence of neutralizing antibodies using functional assay preferably cell based assays.

Method of immunogenicity testing
The methods to detect and characterize ADA response include immunoassays that can identify ADA (capable of binding to antigen) and bioassays that can distinguish between neutralizing and non-neutralizing antibodies Some of the sensitive immunoassay formats used in Accutest Biologics are ELISA, Electro chemiluminescence (Meso Scale Discovery), GYRO Lab, cell based assays (neutralizing Abs); In vitro immunogenicity and LCMS/MS based assays as indicated in Fig 2 below.

Following section discusses each assay platforms:
ELISA (Enzyme Linked Immunoadsobant Assay)
Species independent bridging acid dissociation ELISA is commonly adopted to detect ADA in presence of Drug–Antibody Complex. This can be done by coating the drug on an ELISA plate and detecting the captured ADA with the help of biotin labeled drug using SA-HRP and TMB substrate. The samples with absorbance values above plate specific cut-point are treated as positive for ADA. These are taken for confirmatory assay and the confirmed positives are tested for antibody titer assessment.

Electro chemiluminescence using MSD (ECL- MSD)
The ECL method offers higher sensitivities and higher assay ranges as compared to traditional ELISA. The method is highly suitable for evaluating PK and immunogenicity. Generally, assay duration is short (2-3 hours) due to homogenous assay format. The method is similar to ELISA however all reaction take place on an electrode surface hence background values are very low.

SPR (Surface Plasmon Resonance)
SPR also offers a good platform for label-free interaction of antigen and antibody on a gold coated sensor chip which measures the binding response based on mass deposited on the surface. The SPR offers online acid dissociation benefits and ability to handle multiple samples with 6 concentrations simultaneously. Antibody titration becomes much easier on SPR assay format.

Cell based assays
These are generally employed for detecting the neutralizing antibodies in the pre-clinical or clinical samples. The assays include proliferation assays, MTT assays, ADCC assay, CDC assay, intracellular messenger levels, multiplexing assays, receptor up/ down regulation studies, uptake assay and gene expression assays. These methods are to be developed and validated as per the assay design using the specific cell models.

LCMS/MS
Besides the characterization services LCMS/MS based method is also used for ADA detection and quantification in the biological matrix. This method does not require use of the labeled critical reagents.

In Vitro immunogenicity
This method is mainly used for assessing the T-Cell activities and also to assess the cytokine production on a multiplex assay format using cell based assays. The cells are given a trigger of the test item to evaluate level of proliferation and the cytokine secretion in the cell culture supernatants. The levels of cytokines are measured with the help of MSD or LUMINEX assay format. The T-cell proliferation is measured by analysis of cell surface markers using Flow Cytometer.

GYROS immunoassay format
GYRO LAB Immunoassay system is a highly sensitive assay format which offers advantage of high sensitivity, high throughput, broad range and requirement of ultra low sample and critical reagent volume. The GYROS system uses 200nL of reaction mixture in micro column on a CD which can analyze multiple samples at a time. The partially automated system involves one step of reagent preparation followed by automated sample processing.

The following case study describes detection and quantification of intact human IgG on Gyrolab™ immunoassay platforms. In this immunoassay format, a biotinylated reagent was introduced into a microstructure in the CD to saturate a capture column packed with porous beads coupled with streptavidin. The samples containing intact human IgG were then introduced into the microstructures where intact IgG is captured in the capture column. Finally, a detecting reagent labeled with a suitable fluorophore is added. The integrated signal in the capture column represents the total response from the sample. The positive control samples were loaded at 7 different concentrations in duplicate and the data was evaluated in Gyrolab Evaluator version 3.3. The results of the experimental runs are presented below

Results
The results indicated that all the seven concentrations of positive controls exhibited a high percentage recovery in the matrix with very low %CV (<8%). The background values were found negligible. The experiment demonstrated that the observed concentration is similar to the expected concentration indicating the assay format is precise and accurate.

Summary
The development of recombinant technology has helped in large scale production of protein biotherapeutics useful for many indications and in diseased conditions. These protein biotherapeutics may elicit unwanted immunogenicity and may pose significant clinical, scientific, and manufacturing challenges. The observation of immunogenicity is thus a major concern for industry, regulatory agencies and for the efficacy of the drug product. Besides increasing the quality of drug development processes, it is also important to develop and validate bioanalytical methods to detect very low levels ADA responses in presence of high quantity of circulating drug concentration. Accutest Biologics has made available multiple immunogenicity testing assay formats for assessment of ADA responses in preclinical and clinical study samples for submission to the regulatory agencies. Besides the commonly used assay format like ELISA and MSD, Accutest is also equipped with GYRO Lab. The data from GYROS immuno assay format demonstrated that the assay is sensitive, precise and accurate for detecting the very low levels of ADA response in matrix. u