Growing pursuit of the biosimilar market has resulted in a need for new approaches for precise, sensitive and accurate bioanalysis. We shall describe how a combination of the biopharma market, regulatory guidelines and the molecular characteristics of biologics has inspired the application of surface plasmon resonance and flow cytometry-based ligand-binding assays to streamline evaluation of biosimilars.

The pressure to contain healthcare costs and the number of first-to-market biologic drugs coming off patent — 30 licensed biological drugs by 2015 — are driving the development of biosimilars.  Rituximab (Rituxan – Biogen Idec/Genentech) will lose European patent protection in November 2013.  This drug, with $6.8 billion in annual sales, represents the first patent of a monoclonal antibody (mAb) biotherapeutic.  This expiration is only the first in a number of looming patent expirations for biotherapeutics, including, but not limited to, trastuzumab (Herceptin – Roche), cetuximab (Erbitux – Lilly/Merck KgA) and natalizumab (Tysabri – Biogen Idec).¹

The global market size of the biosimilars industry was estimated at $2.5 billion in 2011, and global demand for biosimilars, and mAb biosimilars in particular, is estimated to grow at 8% and 17%, respectively, between 2012 and 2016.² Importantly, the advent of biosimilars will bring more affordable drugs to market; estimates indicate that the cost of biosimilars will be between 50% and 75% that of innovator biologics.  

The European Medicines Agency (EMA), having approved its first biosimilar in 2006, is now reviewing its first application for approval of a biosimilar mAb — a version of infliximab (Remicade – Janssen/J&J). The U.S. Food and Drug Administra-tion (FDA) has received nine biosimilar investigational new drug applications, and some copy biologics have already been approved in China and India.1 Specifically, with several diabetes drug patents expiring in 2015, some insulin copy biologics and insulin analogues are already available in India.¹

Investments in the development of biosimilars development can be quite lucrative — in Europe, revenue from the sale of biosimilars reached $172 million in 2010, and may be as high as $4 billion by 2020.³

Regulatory Requirements
Unlike small molecule drugs, biologics are extremely complex and no single analytical test or preclinical or clinical study can demonstrate that the biosimilar is sufficiently similar to the reference or the innovator biologic.  A biosimilar product cannot be considered an identical copy of the innovator biologic because even very small differences in the cell line or manufacturing process can have a large impact on potential side effects observed during treatment; two similar biologics can potentially trigger very different immunogenic responses.  Therefore, substitution of a biologic with a biosimilar may have significant clinical consequences, which creates safety concerns from a regulatory perspective. 

Although regulatory agencies are considering biosimilars on a case-by-case basis, they have issued some guidelines on what types of in vitro studies should be performed in the evaluation of biosimilarity. Of note is the EMA’s draft guidance, titled Guideline on Similar Biological Medicinal Products Containing Monoclonal Antibodies, published in November 2010.⁴ The EMA’s guidance recommends measuring, among other parameters, binding to the target antigen and binding to all FcΥreceptors, FcRn and complement. Binding to Fc receptors and complement mediates antibody-dependent cell-mediated cytotoxicity and is an important mechanism of action for mAbs.  Thus, assessing differences between binding of innovator and biosimilar drugs to Fc receptors and complement is critical for demonstrating similarity in potency.

The recent FDA draft guidance, Scientific Considerations in Demonstrating Biosimilarity to a Reference Product, is less specific in its recommendations, but indicates a strategy for evaluating biosimilarity based on “totality of the evidence.”⁵ In other words, comparing a biosimilar with an innovator using multiple, orthogonal assays is like matching fingerprints — the more multivariate the fingerprint, the more likely that a match is predictive of clinical biosimilarity.

Most recently, the Indian regulatory agencies, the Department of Biotechnology and the Central Drugs Standards Control Organization, issued their own guideline on the development of similar biologics, in part to attract investment from global biopharma companies.⁶

One limitation of these regulatory guidance documents is that they do not actually specify how similar a biosimilar must be to the innovator product.  Demonstration of biosimilarity is left up to the company developing the biosimilar therapeutic. 

Confirming Similarity
Developing a biosimilar therapeutic can be expensive, with costs potentially reaching 80% of the cost of developing an innovator biologic drug and about 20 times as high as for developing a small molecule generic.³

Each new biosimilar faces the challenge of proving that any differences in potency and safety from the innovator drug are not clinically significant. Especially in the case of mAbs, which are large and complex, chemical differences between biosimilars and innovators may be numerous. Such cases require rigorous demonstration of biosimilarity as a proxy for therapeutic “interchangeability,” the ultimate (though probably unprovable) standard.

Therefore, there is a critical need for increasingly accurate and precise nonclinical, in vitro assays for measuring drug potency, as these are the cornerstone of quality control of manufactured therapeutics. A recent survey showed that 32% of drugmakers declared that innovations in assay technology were required to meet the demands of proving biosimilarity.⁷  Such assays can better determine lot-to-lot variability in the manufactured product, assess the impact of process changes on drug quality, assess drug stability, and more. Therefore, increasing the precision of an assay improves the assay’s statistical power, facilitating the comparison between biosimilars and innovators.

Innovations in in vitro assay development are being welcomed by regulatory agencies, which are championing the “risk-based” or “step-wise” approach to evaluating biosimilarity, suggesting that the results of very sensitive, highly predictive nonclinical assays can help shape the direction of further testing [FDA guidance]. For example, appropriate pharmacodynamic (PD) markers can be a very sensitive indication of potential clinical differences between two drugs. Regulatory agencies have identified immunogenicity testing as an area enhanced by in vitro ligand-binding analysis. Regulatory guidance (ICH Q6B, 1999) states: “When an antibody is the desired product, its immunological properties should be fully characterized. Binding assays of the antibody to purified antigens and defined regions of antigens should be performed, as feasible, to determine affinity, avidity and immunoreactivity (including cross-reactivity).”

We’ve developed two bioanalytical methods that measure the binding of any therapeutic antibody to any Fc receptor: surface plasma resonance (SPR) measures binding of antibody to recombinant soluble Fc receptor; and flow cytometry measures antibody binding to cells that express the receptor. Both methods have been formatted as parallel line assays and demonstrate high levels of accuracy, precision and linearity, making them valuable for comparability, potency and stability assays. These assays also show greater precision and reproducibility than traditional cell-based assays such as antibody-dependent cell-mediated cytotoxicty (ADCC). Additionally, both are readily able to detect structural differences between two mAbs such as glycosylation that can affect function.

Given the high cost of advancing from in vitro assays to preclinical studies, there is high interest in establishing assays that provide valuable preclinical data about the properties of drugs and their physiological interactions without the need for animal experiments. SPR and flow cytometry are ideal techniques to use for this type of study.  Below, we describe use of SPR and flow cytometry to quantify the binding between therapeutic mAbs, alemtuzumab (and its variants) and infliximab, to molecules mediating cytotoxicity.

Flow Cytometry
Flow cytometry is an essential tool for in-depth cell analysis. In a traditional flow cytometer, cells in a liquid stream pass through a laser beam, which excites any fluorescent molecules on the cell. Emitted fluorescence is then measured by detectors tuned to specific wavelengths. With the capacity to simultaneously measure multiple parameters on hundreds of individual cells per second, flow cytometry is a powerful technology with a wide variety of applications in pharmaceutical development.  As shown in Figure 1, flow cytometry can be a sensitive, information-rich method for measuring mAb binding to Fc receptors on the cell surface.

When developing a flow cytometry assay for PD assessment of a therapeutic, factors to consider include:

• Appropriate fluorochromes, dyes and conjugates to get the clearest data from samples
• Incubation temperatures and periods most suited to the matrix and/or analytes
• Appropriate quality control checks on instrumentation to ensure cytometers are performing reproducibly

Surface Plasmon Resonance
Surface plasmon resonance (SPR) is a powerful technique for measuring the binding of any pair of interacting molecules, including drugs and targets, and antibodies and antigens. Interactions are measured in real time, enabling the determination of kinetic parameters. The SPR signal is proportional to the mass of analyte and does not require any type of label. Figure 2 shows the principle by which the SPR signal is generated.

SPR is a very versatile platform with many different applications throughout pharma development. The measurement of the kinetics of critical molecular interactions between the drug and its target and other key receptors (e.g., Fc receptors in the case of antibodies) is a precise and accurate means of comparing biosimilars and innovators.

Effect of Differential Glycosylation of Alemtuzumab on Binding to FcΥRIII
During the production of mAbs, differences can arise in post-translational modifications of the protein, leading to various glycoforms.  Glycosylation variants can be seen if different cell lines are used in the production of the therapeutic, which may occur in the case of a biosimilar.  Importantly, differences in glycosylation can have profound effects on FcR binding and thus mAb therapeutic effect.  The production of alemetumab highlights this concern. Although alemtuzumab can be produced in suspension CHO cells with much greater yield than in traditional rat cells, alemtuzumab from CHO cells is improperly glycosylated compared to that from rat and human cells. Glycoengineered alemtuzumab, made in CHO cells transfected with glycosyltransferases to change glycosylation to being more comparable to that of production in rat cells, showed higher antibody-dependent cell-mediated cytotoxicity compared to the wild type drug.

In order to determine the mechanism of cytotoxicity, we used SPR and flow cytometry binding assays to measure the binding of alemtuzumab and its glycosylated forms to several Fc receptors.

To demonstrate that the assays could detect differences in the measured binding, the SPR assay was first qualified by adjusting the concentration of alemtuzumab to 50%, 70%, 80%, 100%, 120%, 130% and 150% of the reference alemtuzumab concentration⁸. Determination of the relative potency was performed using a 5-parameter logistic parallel line model using Statlia software. The curves of the different starting concentration samples were all determined to be parallel to the reference curve with p>0.05. The assay had good linearity with a correlation coefficient of 0.993 and measured potencies in the range of 91.4 to 100.6%⁸.

The curves showing binding between the various forms of alemtuzumab and FcΥRIII (CD16a) are shown in Figure 3. A negative control antibody, engineered to have no CD16a binding, showed no response in this assay. The glycoengineered antibodies Glyco1 and Glyco2 showed enhanced CD16a binding compared to the wild type alemtuzumab with relative potencies of 300% and 411% respectively. The glycoengineered forms of alemtuzumab did exhibit small deviations from strict parallelism [p=0.017 and p=0.038 respectively at p>0.05]. These data correlated with the increased ADCC activity previously observed8. Precision between replicates at each concentration was generally <5%.

Similar analyses were performed for CD32a and CD64 although the magnitude of the signal obtained was markedly different, reflecting the strength of binding to the different forms of the receptors. Unlike CD16a, CD32a and CD64 bound to wild type alemtuzumab with higher affinity than to its glycosylated forms (data not shown).

The assay was also tested for other therapeutic monoclonals — namely, bevacizumab (Avastin – Roche/Genentech), rituximab (Rituxan) and eculizumab (Soliris – Alexion) (Figure 4). Eculizumab, a monoclonal antibody against C5 which has a hybrid Fc domain of IgG2 and IgG4 and hence no Fc binding, was used as a negative control in the assay. Although no absolute measurements of potency were determined for these therapeutic monoclonals, their relative binding to CD16a was as follows: alemtuzumab bound CD16a with the strongest affinity, followed by rituximab and then bevacizumab. Eculizumab, as expected, exhibited no measurable binding.

As with the SPR assays, a flow cytometry-based assay was qualified for alemtuzumab using the same nominal concentrations to mimic different potencies of 50 to 150% of the reference. The assay performance for CD16a was very similar to that observed using the SPR assay. The correlation coefficient was 0.99 with measured potencies in the range of 97.9 to 108.0%.

As shown in Figure 5, the glycosylated variants of alemtuzumab exhibited higher binding to CD16a than the wild type alemtuzumab. Relative potency values could not be obtained due to the lack of parallelism due to both Glyco1 and Glyco2 reaching a higher plateau of binding than the wild type reference.

Binding of Infliximab to Neonatal Fc Receptor FcRn and Complement Component C1q
The EMA draft guidance recommends testing biosimilars for their binding to the Fc receptor FcRn. The neonatal, intracellular FcRn receptor is responsible for transport of IgG across the placenta. FcRn binds to IgG and albumin at low pH but not at high pH. This receptor is responsible for “salvage” of internalized IgG or albumin and therefore endows these proteins with a long half life.
Structurally, the molecule is similar to Class I MHC and consists of a specific heavy chain combined with b-2 microglobulin.

Although FcRn is not commercially available, we obtained a supply of recombinant dimeric receptor suitable for SPR assays. We coupled the receptor directly to the chip surface and qualified the assay using the same protocol as for the alemtuzumab assays. Although the magnitude of the signal obtained was very low, the data were consistent and reproducible (Figure 6). In the qualification of the assay, the r2 value was 0.96 and the measured potencies were from 81.0 to 110.9%.

The final assay that was developed to satisfy the requirements of the EMA biosimilar monoclonal antibody guidance was to measure C1q binding. C1q is the first component of the complement cascade and binds to IgM or IgG which is complexed with antigen. A large hexameric molecule, its binding affinity in solution is extremely low. In fact, it has proved impossible to detect binding of IgG to C1q, which was immobilized on an SPR chip. Although it is possible to measure binding of C1q to immobilized IgG, this assay will be difficult to convert for potency or comparability studies.

We developed an ELISA to measure C1q binding. The therapeutic mAbs were serially diluted and coated to microtiter plate surface, C1q was added and, following washing, the bound material detected using anti-C1q-HRP conjugate. The assay qualification data showed a correlation coefficient of 0.98 with measured accuracies of between 100.4 and 114.8% (Figure 7).

Our studies of Fc receptor binding by mAbs indicate that binding to soluble or cell-surface Fc receptors can be accurately measured by surface plasmon resonance or by flow cytometry. We observed excellent precision for replicates, typically <5% CV.
The data were amenable to potency determination by the parallel line method, and we could easily distinguish variant glycoforms and different antibodies. Results showed high correlation with results of antibody-dependent cell-mediated cytotoxicity (ADCC) experiments, without the variability due to biological complexity and statistical noise. ADCC experiments testing the effect of Fc receptor binding to alemtuzumab yielded more variable results, with CVs of individual triplicates in the range of 0.2 to 18.6% (mean CV=5.4%).⁸

Flow cytometry and SPR methods are accurate, robust, reproducible and currently in use within a GMP setting for comparison of biosimilar drug lots with innovator. The assays are sensitive to changes in potency related to glycosylation, aggregation, concentration or protein modification. However, many changes can occur in biologics without affecting a potency readout, so careful physico-chemical characterization is still essential. 

References

1. Mullard A. Can next-generation antibodies offset biosimilar competition? Nat Rev Drug Discov. 2012 Jun 1;11(6):426-8.
2. BCC Research. Biosimilars: Global Markets. Available at: http://www.bccresearch.com/report/biosimilars-global-market-bio090a.html Accessed September 4, 2012.
3. Nair, A. Pharmaceutical Technology. 2011 Feb; 35(2):18.
4. European Medicines Agency. Guideline on Similar Biological Medicinal Products Containing Monoclonal Antibodies. EMA/CHMP/BMWP/403543/2010. Nov 2010.
5. United States FDA. Scientific Considerations in Demonstrating Biosimilarity to a Reference Product. Feb 2012.
6. India launches ‘similar biologics’ guidelines at BIO2012 http://www.in-pharmatechnologist.com/Regulatory-Safety/India-launches-similar-biologics-guidelines-at-BIO2012
7. 9th Annual Report and Survey of Biopharmaceutical Manufacturing, BioPlan Associates, Inc, April 2012, www.bioplanassociates.com
8. Harrison A et al. Methods to measure the binding of therapeutic monoclonal antibodies to the human Fc receptor FcΥRIII (CD16) using real time kinetic analysis and flow cytometry. J Pharm Biomed Anal. 2012 Apr 7;63:23-8.

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James Hulse, Ph.D. is managing scientific director, EMD Millipore, Discovery and Development Solutions, North America.
Chris Cox, Ph.D. is a freelance bioanalytical lab consultant. For more information, contact Dr. Hulse at james.hulse@emdmillipore.com.