European Biosimilar Company – Structural And Physicochemical Characterization And Comparability

PROJECT OVERVIEW

Our client is developing a biosimilar to Simponi and required the below studies to be performed:

1. Determination of the primary sequence of an EU RMP Golimumab sample. The primary amino acid sequence of a biosimilar must be shown to be identical to the innovator at the protein level. In order to determine the primary amino acid sequence of a monoclonal antibody, the following analyses should be performed:
   • On-line LC/ES-MS/MS and/or MALDI-TOF/TOF analysis, as appropriate, with MS/MS and/or MSe analysis of the peptides obtained from multiple digests of the product. The experiments should be carried out to provide determination/confirmation of the light and heavy chain protein sequences.
   • Where the MS-based sequencing does not distinguish the isobaric amino acids Isoleucine and Leucine, peptides containing these amino acids can be purified and further subjected to Automated Edman Degradation (N-terminal sequencing) in order to identify the Isoleucine/Leucine positions unambiguously.
2. A structural and physicochemical comparability assessment of three EU RMP Golimumab samples

The methods to be performed in such a comparability study are outlined in the ICH Q6B guideline and include:

• Amino acid composition analysis
• N-terminal Sequencing
• C-terminal Intactness
• Peptide mapping
• Disulfide bridge analysis
• Glycosylation analysis
  • Monosaccharide composition analysis
  • Oligosaccharide population analysis
  • Linkage analysis
• Molecular weight
• Imaging Capillary Isoelectric Focusing (icIEF)
• CE-SDS
• RP-HPLC-UV
• Ion Exchange Chromatography (IEX)
• Hydrophobic Interaction Chromatography (HIC)
• Melting Temperature
  • Differential Scanning Calorimetry (DSC)
• Higher Order Structure
  • Circular Dichroism (CD)
  • Fourier Transform Infra-Red (FT-IR)
  • Intrinsic Fluorescence (IF)
  • Nuclear Magnetic Resonance (NMR)
• Protein aggregation analysis
  • Size Exclusion Chromatography (UPLC) with UV, Refractive Index and Multi-Angle Laser Light Scattering detection (SEC-MALS)
  • Sedimentation Velocity Ultra Centrifugation (SV-AUC)

TECHNICAL CONSIDERATIONS- N-LINKED OLIGOSACCHARIDES

One of the interesting structural features of Golimumab is the antennal composition of the N-linked oligosaccharides. More significant levels of sialylation (both N-acetyl and N-glycolylneuraminic acid; NeuAc and NeuGc) are observed on this monoclonal antibody, compared to other IgG1 monoclonal antibodies. Significant attention should be paid when characterizing the N-linked oligosaccharide profiles and levels of sialylation within Innovator/RMP and Biosimilar batches. Monosaccharide Composition Analysis, Sialic Acid Analysis, N-linked Oligosaccharide Population Analysis and Linkage (Methylation) Analysis should be performed to define the levels and types of Sialic Acids present as well as defining the Sialic Acid to Galactose linkages. These techniques also serve to give a detailed and thorough investigation into the overall N-glycan population on the sample.

CLIENT TESTIMONIAL

“As a biosimilar developer, it is important that we work with partners who are familiar with the molecule and can provide a quality service based on this experience. BioPharmaSpec scientists have been characterizing biosimilars for many years and we benefitted greatly from this level of expertise” – Project Manager