Abstract
In April 2016, the Food and Drug Administration approved the first biosimilar monoclonal antibody (mAb) – Inflectra/Remsima (Celltrion) based off the original product Remicade (infliximab, Janssen). Biosimilars promise significant cost savings for patients, but the unavoidable differences between innovator and copycat biologics raise questions regarding product interchangeability. In this study, Remicade and Remsima were examined by native mass spectrometry, ion mobility and quantitative peptide mapping. The levels of oxidation, deamidation and mutation of individual amino acids were remarkably similar. We found different levels of C-terminal truncation, soluble protein aggregates and glycation that all likely have a limited clinical impact. Importantly, we identified over 25 glycoforms for each product and observed glycoform population differences, with afucosylated glycans accounting for 19.7% of Remicade and 13,2% of Remsima glycoforms, which translated into a 2-fold reduction in FcγRIIIa binding for Remsima.
While this difference was acknowledged in Remsima regulatory filings, our glycoform analysis and receptor binding results appear to be somewhat different from the published values, likely due to methodological differences between laboratories and improved glycoform identification by our laboratory using a peptide map-based method. Our mass spectrometry based analysis provides rapid and robust analytical information vital for biosimilar development. We have demonstrated the utility of our multiple attribute monitoring workflow using the model mAbs Remicade and Remsima, and have provided a template for analysis of future mAb biosimilars.
Introduction
In 2014, six of the ten top-selling drugs were monoclonal antibodies (mAbs) with over $50 billion in sales.1 The patents protecting many top-selling mAbs are expiring within the next three years.2 Many pharmaceutical companies have generic or biosimilar versions of these mAbs in development. Biosimilar approvals are expected to provide a 15% to 30% savings for patients that need these life changing treatments.3 Availability of affordable biosimilars will likely change the standard of care by making mAbs accessible to a broader group of patients, including those in earlier disease stages. In order to achieve biosimilarity, products must demonstrate rigorous analytical and clinical comparability in terms of safety and efficacy. All mAbs are produced by recombinant manufacturing processes that inherently yield heterogeneous products; therefore, biosimilars cannot be identical to their template innovator product. Both innovator mAbs and biosimilar are heterogeneous populations of variants characterized by differences in glycosylation, oxidation, deamidation, glycation and aggregation state.
Their heterogeneity could potentially affect target protein binding through the Fab domain, receptor binding through the FC domain, and protein aggregation induced immunogenicity.4,5 Hence, analytical characterization of such heterogeneity in the biosimilar product, as well as comparisons to the heterogeneity of the innovator product, is critical to assure a similar level of safety and efficacy of the two products. The concept “totality of evidence” used to assess biosimilarity was emphasized by the FDA for the review process.6,7 The FDA integrates various types of information from state-of-the-art multiple attribute monitoring methods (MAM) which allow for evaluating combinations of analytical attributes from a single method as well as using multiple complementary methods to assess biosimilarity. First, structural assessments are combined with functional studies such as bioactivity and receptor binding assays to evaluate whether the biosimilar is highly similar to the reference product.
The critical quality attributes (CQAs) of mAb e.g., oxidation, glycosylation profiles, glycation, and the amount of aggregation along with the results of protein bioactivity assays are quantified in order to assess the biosimilarity of two products. Based on this analytical assessment the extent of required toxicological and clinical evaluations are determined.7 Thus, unequivocally showing complete analytical comparability of biosimilar to the reference product could result in lessening the scope of clinical studies.7,8. Hence, biosimilar developers are incentivized to perform complete analytical comparisons of CQA for their own and innovator products using state-of-the art methodologies.
Infliximab, or Remicade (RC), was developed by Janssen and approved in 1998. Remicade was one of the first therapies to target TNF-α, and its annual sales exceeded $9 billion worldwide in 2014.9 However, patent exclusivity for infliximab has since expired and the first infliximab biosimilar, Remsima (RS), was approved in Europe in 2013 and more recently in the USA in 2016. Remsima, also known as Inflectra or CTP-13, was developed and manufactured by Celltrion and licensed to Hospira/Pfizer.10 RS is the first ever biosimilar mAb approved both in Europe and the US. Currently, several other infliximab biosimilars are either undergoing clinical testing or have already been submitted for FDA/EMA approval, including Flixabi (Samsung), Infimab (Epirus/Ranbaxy) and STI-002 (MabTech).
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In order to demonstrate the biosimilarity between RS and RC, Celltrion generated an extensive data package that included a battery of analytical methods, bioassays, and two clinical studies showing equivalency in efficacy, pharmacokinetics, adverse events, and immunogenicity in ankylosing spondylitis (AS) and rheumatoid arthritis (RA).11,12,14 Given its approval status, the package effectively proved the two antibodies were similar, yet not identical. Among differences, the levels of charged variant species attributed to C-terminal truncation and dimer levels were reported and ultimately deemed non-consequential.10,11,13 Like other IgG1 therapeutics, infliximab is a glycoprotein containing an N-glycosylation site at Asn300 in the Fc region of the protein. Both RC and RS contain a heterogeneous mixture of N-glycans that, when analyzed by orthogonal methods, were assessed overall to be similar, as evidenced in a recent publication by the manufacturer.15 However, released regulatory documentation acknowledged apparent differences in RS glycosylation, specifically, in the levels of fucosylated glycans, and how this difference affected FcγIIIa receptor (FcγRIIIa) binding and antibody-dependent cell-mediated cytotoxicity (ADCC).10,11,13 Mediating ADCC is not thought to be a key component of infliximab’s efficacy in the treatment rheumatologic diseases (e.g. RA or AS), but it is presumed to be partially responsible for its activity in inflammatory bowel disease (IBD) through the modulation of local immune cell populations.11,14 Due to glycosylation and ADCC differences, the Canadian regulatory agency did not approve the use of RS for treatment of IBD, and requested additional clinical data in the IBD patients.14 Following RS’s approval, various rheumatology and gastroenterology societies, as well as patient advocate groups, issued statements against the biosimilar products.17-19 However, more recent post-approval data suggests RS is just as safe and efficacious as RC, and the biosimilar is rapidly gaining acceptance in Europe.20
As more biosimilar mAbs gain regulatory approval, it is important that a clear framework for a rapid characterization of innovator and biosimilar products exists that could identify clinically relevant differences. Our analysis aims to bring a level of transparency and simplicity for biosimilar characterization. We applied a comprehensive mass spectrometry (MS) based strategy using bottom-up, middle down, and intact strategies.
These data were then integrated with ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) analyses, as well as data from select biophysical techniques and receptor binding assays to comprehensively evaluate biosimilarity. Additionally, we sought to fill in the gaps found in the current literature regarding quantifiable comparisons between RC and RS, specifically quantification of the various glycoform levels and FcγRIIIa binding. Our ultimate goal was to develop a template, which can be applied towards future analytical comparisons of biosimilar mAbs.
