Glatiramer acetate is an immunomodulatory used to reduce the frequency of relapses in the treatment of multiple sclerosis. It consists of a heterologous mixture of synthetic peptides comprising L-glutamic acid, L-alanine, L-tyrosine, and L-lysine. The peptide oligomers have an average molecular weight of 5 – 9kDa. The breadth of structural variation in the population is a result of the copolymerization process of these individual amino acids. As a result, this molecule poses unique challenges in structural comparability.

Biosimilar Characterization Considerations

  • Amino acid composition analysis – to provide a comparability assessment of the levels of each of the four amino acids in different batches/products.
  • N-terminal sequence analysis – to provide a comparative, quantitative assessment of the levels of the four amino acids across the first 15 N-terminal residues within the oligomers.
  • C-terminal sequence analysis – to provide a comparative quantitative assessment of the uncapped carboxyl C-termini.
  • Peptide mapping  – to provide comparative peptide mass and fragment ion information using LC-MS (with MSe) in various forms and from various enzymatic digests.
  • Intact molecular weight distribution – to provide a comparative assessment of the molecular weight distribution of the various batches/products
  • NMR analysis – to provide a comparative primary, secondary and tertiary structure analysis and also assess the comparative levels of capped and uncapped C-termini
  • Circular Dichroism (CD) and FT-IR analysis – to provide an orthogonal further assessment of secondary and tertiary structure
  • Size Exclusion Chromatography with UV and Multiangle Light Scattering (SEC-UV and SEC‑MALS)
  • Imaging Capillary IsoElectric Focusing (icIEF)

Product Specific Technical Considerations

The difficulties with analyzing Glatiramer acetate relate to the complexity of the product, which contains random but consistent oligomers containing the four amino acids mentioned above. This creates difficulty in designing methodologies for assessing the C-terminal sequence and in interpreting the peptide mapping data. The fact that a small portion of the C-termini are likely to be capped (diethylamido-) and a small portion of the N-termini are likely to be Pyroglutamate further complicates the issue.

BioPharmaSpec has designed a suite of analyses to fully characterize and compare generic material, while taking these structural features into account.