Analytical Characterization for the Comparability Evaluation of TUR03 Drug Substance and Innovator Lots by Mass Spectrometry

3rd International Congress on Analytical and Bioanalytical Chemistry, İstanbul, Türkiye, 22 - 26 Mart 2021, ss.77-78

Yayın Türü: Bildiri / Tam Metin Bildiri
Basıldığı Şehir: İstanbul
Basıldığı Ülke: Türkiye
Sayfa Sayıları: ss.77-78
Acıbadem Mehmet Ali Aydınlar Üniversitesi Adresli: Evet

Özet

Therapeutic monoclonal antibodies (mAbs) are fastest growing class of biopharmaceutical and used against various oncological, autoimmune and inflammatory diseases. A biosimilar is defined as a similar version of the licensed biotherapeutic reference product (innovator) in terms of quality, safety, and efficacy. To demonstrate the biosimilarity, high-resolution mass spectrometry systems have emerged the greatest interest in recent years.

TUR03 is a proposed biosimilar candidate for one of the top-selling drugs and developed by Turgut Pharmaceuticals. In this study, a comprehensive mass spectrometric characterization of the TUR03 biosimilar was performed for intact and deglycosylated-reduced intact mass, peptide mapping, and N-glycan analysis. The obtained results were compared with the results of innovator lots.

Chromatographic and mass spectrometric analysis were performed using a Waters Acquity H-Class Bio UPLC system coupled with Xevo G2-XS QToF mass spectrometer (Waters Corporation, Milford, USA), equipped with an ESI source. The system was controlled by UNIFI scientific information system software (v1.9.4).

Intact mass analysis revealed that the major components are N-terminal pyroQ, C-terminal Lys truncation, and G0F N-glycosylation on both heavy chains. The major components detected by deglycosylated-reduced intact mass analysis were reduced mAb light chain and deglycosylated/reduced mAb heavy chain having N-terminal pyroQ and C-terminal Lys truncation. The results for intact and deglycosylated-reduced intact mass analysis were consistent with the expected structure of mAb and demonstrated comparability between TUR03 and tested innovator lots.

The primary structures (amino acid sequence) of innovator and TUR03 were determined by peptide mapping data dependent acquisition (DDA) analysis. Peptide-level sequence coverage was 100% when trypsin and Lys-C enzymes were used sequentially. Post-translation modifications (PTMs) detected by peptide mapping included N-terminal pyroQ, Met oxidation, Asp succinimide, and Asn deamidation. The major clip detected by peptide mapping was C-terminal Lys truncation of the heavy chain. The calculated PTM levels are comparable between TUR03 and tested innovator lots.

The released N-glycan analysis were carried out by using Waters GlycoWorks RapiFluor-MS N-glycan kit. First, the sample was denatured and deglycosylated. The released glycans were then labeled with the RapiFluor-MS solution. Samples were cleaned using solid-phase extraction on a GlycoWorks HILIC elution plate. The glycans were then separated on an UPLC system and the results were integrated and labeled based on retention times. The major detected N-glycan was the complex biantennary fucosylated N-glycan G0F, which ranged from ~60% to ~70%. The levels of fucosylated complex N-glycans were more than 90% and were comparable across TUR03 and innovator lots. The results for N-glycan profiling demonstrated comparability between TUR03 biosimilar and its innovator.

The overall results have revealed that TUR03 is highly similar to its innovator in terms of critical quality attributes (CQAs) measured by mass spectrometry.