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Biosimilar drug development and the importance of analytical characterization Posted 25/09/2015

Author: Fiona M Greer, BSc (Hons), MSc, PhD, Global Director, BioPharma Services Development, SGS Life Science Services

Abstract

The development of a biosimilar requires comprehensive physicochemical characterization at many stages of the development pathway. This article reviews the steps that one must follow during the development process.  

Keywords: biosimilarity, biosimilars, head-to-head comparability, physicochemical characterization

Comparability studies require side-by-side data to demonstrate Biosimilarity

Numerous biosimilar drug products have reached the market in Europe since the first, Sandoz’s version of the human growth hormone somatropin, gained EU approval back in 2006. Since then, the first two monoclonal antibodies have received the go-ahead in 2013 – Celltrion’s Remsima and Hospira’s Inflectra, which are both versions of Janssen’s Remicade (infliximab), a tumour necrosis factor alpha blocker indicated for a range of autoimmune conditions. Despite this progress, no biosimilars are yet approved in the US, but numerous products are in development. In the US, in July 2014, Sandoz was the first to apply for biosimilar approval in the US under the Food and Drug Administration’s (FDA) new biosimilars pathway, for the filgrastim biosimilar Zarzio.  This application was granted approval in early 2015.

The EU took the lead with its first guidance documents for similar biological medicinal products, which were published in 2005, with discussions having commenced a couple of years earlier. Other countries soon followed suit, some adopting the European Medicines Agency (EMA) guidelines, some a modified version of them, and others writing their own. But it was not until 2009 that the Biologics Price

Competition and Innovation Act (BPCI Act) was published in the US, introducing the 351(k) new pathway to market into the Public Health Services Act. This is the new route being taken by Sandoz with Zarzio.

Meanwhile, in Europe, EMA has now produced extensive guidelines, some of which have already been revised. First, there is the overarching biosimilar guideline that contains the general principles, and there is also a set of general guidelines covering quality. These guidelines include the quality comparability exercise, clinical and non-clinical guidance, and immunogenicity requirements. In addition, there are product-specific guidelines.

The new 351(k) pathway in the US requires a comparison to be made between a potential biosimilar, and a single reference product that has been approved under the normal 351(a) route for biologicals. The application must include analytical studies that demonstrate the biological is highly similar to its reference, minor differences in clinically inactive components notwithstanding. It may also include animal studies, including assessments of toxicity, and clinical studies. The BPCI Act provides for the approval of two types of biosimilars – a biosimilar that is highly similar to the original and a so-called interchangeable biosimilar, which requires clinical switching studies to be carried out.

Risk-based approaches

FDA uses a risk-based approach in evaluating biosimilarity. The agency will consider the totality of data submitted, including structural and functional characterization and non-clinical evaluations, human pharmacokinetic and pharmacodynamic studies, clinical immunogenicity, and clinical safety testing.  FDA suggests a meaningful fingerprint-like analysis algorithm should be used, covering a large number of product attributes.

A recent FDA guideline [1], issued in August 2014, introduces the concept of four categories of assessment outcome following the initial analytical characterization. The ‘holy grail’ is ‘highly similar with fingerprint-like similarity,’ where a product is deemed nearly identical to its reference product, and only minimal studies required to demonstrate biosimilarity. Next is ‘highly similar,’ which also meets the standard for biosimilarity but more extensive studies will be required.  ‘Similar’ applies where the analysis is inconclusive, and further data or studies will be necessary following consultation with FDA. Finally, there is ‘not similar,’ if a product does not measure up to the reference product, so the 351(k) pathway is not appropriate.

Analytical characterization

So when is analytical characterization required? The development pathway of a biosimilar is somewhat different from a novel biotherapeutic, certainly in the early stages, with a greatly increased requirement for physicochemical analytics compared to a novel biological molecule.

First, the target (reference) molecule must be extensively characterized to determine the variability of quality attributes. Multiple batches of the originator are studied to determine the exact amino-acid sequence and its post-translational modifications. Determining the amino-acid sequence entails tandem mass spectrometry (MS/MS)de novosequencing approaches. These data form the quality target protein profile (QTPP) for the biosimilar.

For the production of the biosimilar, characterization surveys may help in the selection of an appropriate cell line, allowing biosimilarity to be designed into the molecule from the outset. Once the biosimilar has been expressed, various regulatory guidelines require comparative data for the manufacture of biosimilars side-by-side with the originator molecule. This will require extensive data on both the primary and higher-order structure, which can be determined using a variety of orthogonal analytical methods.

The recently revised EMA quality guideline provides some additional clarification about analytical strategies. State-of-the-art analytical methods must be used to assess composition, physical properties, primary and higher order structure, purity, product-related substances and impurities to be compared between the biosimilar and the originator. The biological activity must also be examined. Quantitative ranges must be established for these quality attributes. It is also important to use material from the final process in the clinical trials if further comparability exercises are to be avoided. While the formulation does not need to be the same as the original product, its suitability does need to be demonstrated.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q6B [2], although slightly dated now, can be used as anaide memoireto ensure all the molecule’s physical attributes are covered. Another rich source of information on appropriate techniques is the updated 2009 EMA monoclonal antibody guideline [3].  Experiments will include MS on the intact protein plus the released light and heavy chains, going through the N- and C-terminal sequence, peptide mapping, monosaccharide and sialic acid analysis, the secondary structure and folding, and studying aggregation using appropriate techniques. The complexity and size of the molecule, together with the potential structural variations, present quite a challenge. The potential variations in quality attributes such as deamidation, glycosylation, C-terminal clipping and so on can be extensive, rendering the number of variations to be deduced rather extensive.

The starting point for an antibody, guided by ICH Q6B, is the intact molecular mass measurement, which can be carried out on the whole molecule, or on reduced and released light and heavy chains. Using modern mass spectrometers, well-resolved and accurate data at 150k Da can be obtained, allowing the glycoforms to be assessed. This intact mass is a useful starting point as a comparison tool, allowing various batches of antibodies to be studied.

Peptide mass mapping is a particularly powerful structural confirmational tool. The protein is digested, following reduction and alkylation if necessary, using specific proteases to produce a mixture of peptides that can then be analysed by mass spectrometry to provide a mass fingerprint. Any change in the molecule would result in changes to the mass map, making it an effective identity test. Mapping large molecules, such as an antibody, requires several proteolytic digestions to be performed in parallel and the results combined. The resulting peptides can also be separated by online liquid chromatography–mass spectrometry (LC/MS), and with high energy MS/MS sequencing, the amino acids in the sequence can be confirmed.

A complementary strategy involves the separation of the digested peptides by reverse-phase high-performance liquid chromatography (HPLC) and collection using classical Edman degradation-based sequencing. Once the peptides are identified by mass using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), they are sequenced, allowing isomeric amino acids, such as leucine and isoleucine, to be differentiated.

A similar mass mapping strategy can be applied to one of the most challenging problems – the characterization of disulfide bridges. Specific enzymic digestion is used under non-reducing conditions to produce a mixture of peptides, which are identified by mass using MS. Under normal non-reducing conditions, the disulfide bridge will remain intact, giving a signal in the mass map. If the mixture is then reduced and studied by MS once more, the broken disulfide bridge will produce two new peptides, lower in the spectrum, corresponding to the individual masses of the free thiol-containing peptides.

Post-translational modifications

Glycosylation is, arguably, the most important post-translational modification, but it produces a significant challenge to the analytical chemist. The population of sugar units attached to an individual glycosylation site on any protein depends on the host cell type used, and it will also be a mixture of different glycoforms on the same polypeptide. The carbohydrate profile of a biosimilar may not necessarily be the same as that of the originator protein, so further studies will have to be carried out to prove that they have no impact on safety and efficacy.

Guided by ICH Q6B, the carbohydrate content, the structure of the carbohydrate chains and the glycosylation site need to be considered. Mass mapping strategies can be used to give information on monosaccharide composition, glycan populations and antennary profiles, antennae linkages and Glycosylation sites. These MS studies can be carried out on both underivatised and derivatised samples to determine glycosylation sites for both N-linked and O-linked structures.

The intact glycoprotein can be studied for example with MALDI and/or electrospray MS, and the monosaccharide composition with LC/MS. Many chromatographic techniques, such as ion exchange can also be used for glycan profiling. The glycoprotein can be digested; N-linked glycans can be removed enzymatically; O-linked glycans can be removed via reductive beta-elimination. The fragments can be then analysed using MS and LC methods. Linkage analysis is important; particularly the stereospecificity of oligosaccharide antennae linkages, as certain glycotopes such as Galalpha-1,3-Gal can promote antigenic stimulation in humans.

While MS is a powerful and important tool, a host of other non-MS techniques is required for this comparability exercise, looking at the differences in size, shape, and charge of the molecules. Another routine tool, capillary isoelectric focusing, for example, is useful for studying isoform distribution in a comparative manner.

When determining higher-order structure, a battery of orthogonal tests also needs to be employed. Appropriate biophysical techniques include circular dichroism in the far-UV, which enables the number of beta-sheets, alpha-helices, and other turns to be studied, for example. Fourier transform infrared spectroscopy (FT-IR) and fluorescence spectroscopy enable the study of local tertiary structure. And techniques, such as analytical ultracentrifugation, dynamic light scattering, and fluorescence resonance energy-transfer methods allow examination of any aggregates that may be formed.

In summary, the development of a biosimilar requires comprehensive physicochemical characterization at many stages of the development pathway. First, batches of the originator must be examined to determine its exact protein sequence, post-translational modifications, and the variability of quality attributes. These data form the quality target product profile. Advances in MS instrumentation and proteomic/glycomics strategies enable rapid identification of these structural data. The extensive comparability studies that must then be carried out require side-by-side data to demonstrate biosimilarity, and there is an increasing importance being placed on higher-order structure to link with the biological activity. Clearly, if the regulators are to be convinced that the potential biosimilar and the originator are sufficiently similar for approval to be granted, these comparability studies must be carried out comprehensively and effectively.

References

1. U.S. Food and Drug Administration. Guidance for industry. Clinical pharmacology data to support a demonstration of biosimilarity to a reference product. May 2014 [homepage on the Internet]. 2014 May 9 [cited 2015 Sep 14]. Available from: http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm397017.pdf
2. European Medicines Agency. ICH Harmonised tripartite guideline.Specifications: test procedures and acceptance criteria for biotechnological/biological products Q6B. Step 4 version (1999) [homepage on the Internet]. 2006 Feb 28 [cited 2015 Sep 14]. Available from: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q6B/Step4/Q6B_Guideline.pdf
3. European Medicines Agency. Guideline on development, production, characterisation and specifications for monoclonal antibodies and related products. EMEA/CHMP/BWP/157653/2007. 18 December 2008 [homepage on the Internet]. 2009 Jan 13 [cited 2015 Sep 14]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003074.pdf

Note: A version of this article previously appeared in the December 2014 edition of Pharmaceutical Technology Europe.

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