Comparative studies of biosimilar medicinal products

In view of the impending phase-out period for the IP protection of many drugs, and the declining number of new small-molecule drugs discovered and developed each year,  the share of biosimilar drugs in the pharmaceutical and biotech market, is becoming more and more apparent. 

According to the EMEA „Guideline on similar biological medicinal products containing biotechnology derived proteins as active substance: non-clinical and clinical issues” (EMEA/CHMP/BMWP/ 42832/2005), biosimilar products are defined as „similar biological medicinal products”. Biosimilars are much more challenging to regulate than chemically synthesized small-molecule drugs due to their complexity in structure, and the fact that they are produced in living cells. 

Fulfilling the increasing biotech and pharmaceutical industry needs, BioCentrum offers various analytical services, as well as molecular biology and biochemistry assays, which can be successfully applied in biosimilar drug development.

Electrophoretic pattern 
Electrophoretic patterns and data on the identity, homogeneity and purity can be obtained by SDS-PAGE electrophoresis. Additionally to the SDS-PAGE 2D electrophoresis, IEF-SDS-PAGE and Western-Blot are also available. 

Liquid chromatography services 
Protein and peptide analysis based on low-, medium- and high-pressure liquid chromatography techniques. UV/VIS and mass spectrometry (MS) detection is applied, while using analytic and semi analytic columns allow to perform an ion-exchange, reverse-phase, hydrophobic and size-exclusion chromatography.

Peptide mapping 
Both identification and comparison studies are performed by enzyme digestion technique. The digest is then analyzed by HPLC coupled with MS detection and MS/MS identification. One can subsequently compare not only the HPLC profiles of both the original and the biosimilar product, but also study the fragmentation pattern of the resultant peptides. Obtained results can be analyzed 
by Mascot bioinformatics database.

Amino acid analysis and sequencing of proteins and peptides 
Amino acid analysis of both proteins and peptides plus determination of protein and peptide sequences using Edman chemistry on an Applied Biosystems model 491 automatic sequencer.

Circular dichroism 
Determination of the percentage of secondary structures of proteins (alpha-helices, beta-structures and unstructured regions) by circular dichroism method. Analysis is carried out on Jasco's spectrophotometer at wavelengths ranging from 190 to 240 nm.

Set of necessary experimentation i.e. crystallization, measurements and calculations, providing high-resolution tertiary structures of proteins in the free state and in complexes with low molecular weight ligands. 

Non-clinical in vitro pharmacodynamic studies 

Selvita offers broad spectrum of pharmacodynamic studies used to analyze biosimilar product’s activity in comparison to adequate reference compounds i.e. medicinal products that have already been granted a marketing authorization. Scope of analysis depends on the type of the similar medicinal product and is designed based on clear understanding of its characteristics and activity.
Selvita offers assays that are able to detect even minor changes in biological response to pharmacologically active substances:

  • Analysis of key protein phosphorylation during the signal transduction
  • Analysis of receptor autophosphorylation (activation) upon biosimilar stimulation
  • Cell-based assays (production of cAMP, an omnipresent cellular second messenger, which plays a key role in signal transduction after ligand binding to the GPCRs)
  • Protein biomarkers expression analysis (with the use of Western Blot or ELISA assays)
  • Characterization of protein-protein and drug-protein interactions (ELISA, fluorescence anisotropy)
  • Radioligand binding assays 
  • Cytotoxicity and proliferation assays (MTS, MTT, BrdU, LDH)