The biosimilar comparability exercise is a series of studies characterizing quality attributes of a reference and a biosimilar product. Approval of the biosimilar product by regulatory agencies is based on demonstrating high similarity between reference and biosimilar products. Guidelines for the conducting of biosimilar comparability exercises have been published by the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA).
Protein Primary Structure
Amino Acid Sequence Companies that develop a biosimilar protein for marketing as a comparable clinical treatment to replace an originator biopharmaceutical product initially invest a significant amount of time in determining the primary protein sequence of the reference protein. They then reverse-engineer the gene sequences that will produce an identical protein sequence in a comparable mammalian cell, for example the Chinese hamster ovary (CHO) line used by Visser et al. . With monoclonal antibody proteins, comparison of the primary sequences of the biosimilar and reference products is accomplished by determining the following parameters:
- Intact molecular mass determination.
- Determination of molecular masses of reduced heavy and light chains.
- Determination of peptide sequences by peptide mapping of heavy and light chains, followed by mass determination of individual peptides.
Disulfide Bond Analysis: In order to make a comparison of the disulfide bond structures (intra- and inter-chain disulfide bonds) it is again necessary to first have data on the disulfide bonds present in the reference compound. Comparability studies involve side-by-side reference and biosimilar peptide mapping of DTT reduced or non-reduced protein samples and then analysis by LC-Mass Spectroscopy.
Glycosylation: For biopharmaceuticals such as growth factors and monoclonal antibodies, minimizing lot-to-lot variation in glycosylation profiles is critically important to preserving the structural and functional qualities of the finished products. For a detailed description of manufacturing variability due to changes in glycosylations. In comparability exercises, it is important to show the range of glycosylation variants, and to evaluate any possible effects of these variants on the biological function that might potentially affect safety or efficacy. Structures of N- and O-linked glycans are determined by LC-mass spectroscopy applications.
Size Exclusion HPLC: Size exclusion HPLC divides analytes based on molecular size using a physical filter, generally matrices composed of porous beads. This HPLC technique is used both for sample preparation for MS, but also for the detection of protein aggregates in pharmaceutical products, including biologicals.
Other Protein Modifications: Other protein modifications that need to be characterized in the biosimilarity exercise include oxidized methionines, deamidated asparagines, N/C terminal variations, and glycation. Techniques such as cation exchange (CEX) chromatography or capillary electrophoresis are commonly used to initially assess charge heterogeneity. Determination of structures is then by LC/MS of appropriate fragments.
Higher Order Structure
Other techniques used in the comparability exercise that reflect quality attributes related to the secondary and tertiary structures of the reference and biosimilar proteins. These techniques include:
- Secondary Structure: Fourier-Transformed Infra-Red (FTIR) Spectroscopy
- Secondary Structure: Circular Dichroism (CD)
- Thermodynamic Properties: Differential Scanning Calorimetry (DSC)
- Tertiary Structure: Hydrogen Deuterium Exchange Mass Spectroscopy (HDX-MS)
- 3-D Structure: X-Ray Crystallography
For a more detailed description of these methods, see here.
Bioassays & Functional Assays
In order to give an indication of the possible efficacy of a biopharmaceutical prior to conducting animal or human studies, functional in vitro assays need to be conducted based on the mechanism of action that would support the proposed biological action. Biochemical assays for antibodies usually include assays testing binding affinity of the antigen to antibody via a variety of in vitro principles. For antibody biopharmaceuticals, cell-based assays include effector function such as antibody-dependent cellular cytotoxicity (ADCC) assays and complement dependent cytotoxicity (CDC) assays. Other cell-based assays include antigen-targeted apoptosis assays.
Biosimilars are required to demonstrate a high level of safety and efficacy for treatment of a particular medical indication in humans. Regulatory agencies require that physical properties of the finished product, such as protein concentration, pH, osmolarity, and excipient components (buffers, stabilizers, etc.) be as close to the reference product as possible. For more details see the white papers in our download section.
Aggregates & Particulates
Presence of aggregates or particulates can be a problem for a biosimilar if there is a definite increase in the amounts of these larger species in the finished product with respect to the reference product. Aggregates are a multimerization of the active drug product. Aggregates can be detected via analytical ultracentrifugation-sedimentation velocity (AUC-SV) or by size exclusion liquid chromatography with light scattering detection (SE- HPLC-LS). Particulates are larger species ranging in size from sub-micron (< 1 µM) to the subvisible category (> 2 µM to 25 µM). Methods of detection include light obscuration (LO) or micro-flow imaging (MFI) for subvisible particles and dynamic light scattering (DLS) or field flow fractionation with light scattering detection (FFF-LS) for submicron particles. For additional information see our download section.
Impurities of biopharmaceuticals relevant to comparability studies are categorized as product-related impurities or host-related impurities. Product-related impurities can be truncations of the full-length protein, covalent aggregates of the protein, or other modifications of the full-length protein. Host related impurities can be any other non-product protein that carries through in the purification and originates from the host. In order to detect possible product-related impurities, a number of analytical liquid chromatography methods can be used. Other techniques such as capillary electrophoresis, SDS-PAGE , and 2-D electrophoresis can also be used. For additional information see our download section.