All protein biotherapeutics are potentially immunogenic and hence could elicit immunogenicity response. These responses could be of different types such as binding antibodies or neutralising antibodies. Immunogenicity of protein biotherapeutics is a major concern especially when the biological function of the drug and the endogenous counterpart are neutralised by Anti-Drug 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 characterised.
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 Anti-Drug Antibodies (ADA) was noted after patient treatment with such therapeutics (Sauerborn et al.,2010 ; Schernthaner,1993 ). Therefore, ADAs pose a challenge in the biotherapeutics industry and in clinical medicine due to possibility of neutralisation of endogenous protein thus reducing the efficacy of a biotherapeutics. Immunogenicity is therefore a key limitation for the clinical use of biotherapeutics and the drug development process requires a number of analytical methods to support efficient product development with high safety or low risk potential.
Recombinant DNA technology is not only an important tool in scientific research, but is also responsible in enormous progress in the diagnosis, treatment and production of protein derived biotherapeutics of high purity, high specific activity, steady supply and batch-to-batch consistency. These protein biotherapeutics are inherently highly specific for the target with minimum risk for non-mechanism based toxicity and safety issues. Protein biotherapeutics augment normal growth factors, hormones, enzymes and are able to modulate protein/protein interactions. Protein biotherapeutics are aapplicable in multiple therapeutic areas and for a variety of antigen targets
Unlike the generic small molecule drug products, protein biotherapeutics are large molecules (higher molecular weight) 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. Table 1 summaries some of differences in small and large molecule therapeutics.
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, optimisation of codons and synthesis of DNA. Other critical steps include, buildingtheconstruct with various expression vectors, screening the best expression route, production and purification of proteins, characterisation of protein, locking the API, performing the preclinical studies and Clinical studies.
Immunogenicity is the ability of a substance to trigger immune response. All protein biotherapeutics have the ability to induce immunogenicity. Immunogenicity triggered by Biopharmaceuticals is mainly linked to production of anti drug antibodies and unwanted immunogenicity. However, in case of vaccine candidates it is wanted immunogenicity.
A well-documented example of biotherapeutics immunogenicity is the neutralising 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 anaemia that is characterised by the absence of red cell precursors in the bone marrow (Boven et al.,2005b ). In PRCA which is a very rare serious adverse drug reaction seen in the patients treated erythropoietin for renal anaemia, the neutralising 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 in the regulatory guidance to confirm that the drug product does not elicit immune response which potentiates the neutralisation of administered /endogenous protein biotherapeutics compromising the efficacy of the drug product.
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 recognised as Non-Self or ‘foreign’ protein which subsequently leads to production of specific anti-drug 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 neutralise protein
• Antibodies which bind, but do notneutralise protein
• Antibodies against non-product related proteins e.g. host cellderived proteins (expression system related proteins)
• Antibodies against product and non-product related proteins
• No antibodies.
Thus the nature of antibody response has to be evaluated with the help of sensitive validated bioanalytical methods to know whether the immune response has effect on neutralisation of the protein drug in the subjects which could be detrimental.
Regulatory agencies have recognised the importance of screening for protein biotherapeutics arising from concerns over its safety and efficacy. In order to demonstrate clinical safety and efficacy, immunogenicity testing is now a key component of biotherapeutics drug development. Immunogenicity testing is required under ICH, US-FDA and EMA guidance documents to characterise the ADA response.
The formation of neutralising antibodies can affect safety and efficacy, but non-neutralising antibodies can also be a concern due to effects on half-life and bio distribution of the product (Shankar et al., 2006). Neutralising antibodies can deplete both endogenous and exogenous protein. Hence risk based immunogenicity assessment is recommended. Wherever the endogenous counter parts are available, appearance of neutralising 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, characterisation of the confirmed positives for antibody titer and for assessing neutralising abilities with class of antibody response.
There are multiple factors causing immunogenicity of the protein drugs administered to clinical 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 which can be a key factor in causing adverse events associated with immunogenicity in the clinic (Rosenberg,2006). The level of contaminant proteins and DNA components from the expression systems or from the host cell is considered to be another major concern for immunogenicity. The levels of such contaminants are to be assessed by highly sensitive methods to ensure that the levels are at or below the acceptable limits as per regulatory norms. Immunogenicity assessment may become necessary for the host cell related protein in certain drug product depending on its expression system and on the nature of the drug product under investigation. 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 confrormational changes in the structure of the drug product which may lead to immunogenicity. PRCA was observed in patients treated with the epoetin-α formulation that contained polysorbate 80 (Villalobos et al.,2005).
The elicitation of ADA against biotherapeutics can have detrimental effects on drug safety, efficacy, and pharmacokinetics. The immunogenicity of biotherapeutics is, therefore, an important issue. The bioanalytical methods developed to evaluate the pharmacokinetics profile and immunogenicity should be of adequate sensitivity and these are to be validated as per regulatory requirements (USFDA and EMA guidance). The immunogenicity method developed and validated should be able to detect low levels and low affinity anti-drug antibodies in presence of high circulating concentration of drugs.
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 samples to be used). 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 characterisation- antibody titer assessment. The confirmed positives also need to be tested for presence of neutralising antibodies using functional assay preferably cell based assays. The strategy for immunogenicity assessment is summarised in Fig 2.
To study the immunogenicity of protein therapeutics, ADA detection and characterisation methods are required. Such methods should be adequately sensitive to recognise and detect low affinity and low titer ADA in both clinical and non clinical samples. A range of techniques exist which are useful for investigating the presence of antigen-specific antibody. These methods include immunoassays that can identify ADA (capable of binding to antigen) and bioassays that can distinguish between neutralising and non-neutralising antibodies (Wadhwa & Thorpe,2007). Some of the sensitive immunoassay formats used in Accutest Biologics are ELISA (bridging, acid dissociation), Electro chemiluminescence (Meso Scale Discovery), GYRO Lab, cell based assays (neutralizing Abs); In vitro immunogenicity and LCMS/MS based assaysas indicated in Fig 3 below.
Following section discusses each assay platforms:
Species independent bridging acid dissociation ELISAs are 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. Depending on the quality of critical reagents ELISAs can assay sensitivity below 50ng/mL
This 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 observed.
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 chip surface. The SPR offers online acid dissociation benefits and ability to handle multiple samples with 6 concentrations simultaneously. Antibody titration becomes much more easier on SPR assay format.
These are generally employed for detecting the neutralising antibodies in the pre-clinical or clinical samples. The assays generally 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.
Besides the characterisation 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.
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.
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 analyse 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
The results indicated that all the 7 different concentrations of positive controls exhibited a high percentage recovery in the matrix with very low %CV for standard points (<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.
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.
Dixit (graduate from University of Illinois) is a scholastic personality with several patents and publications to his credit and with more than 25 years of rich experience in pharmaceuticals, biopharmaceutical and CRO industries in the area of drug discovery and bio-analytical services. He has extensively worked on method development and validation of various assay platforms such as LC-MS, ELISA, MSD, SPR, RIA/RIPA and Cell based assays for Immunogenicity and pharmacokinetics evaluation of non-clinical and clinical study samples under GLP and GCLP compliant practices for regulatory submission studies.