Comparative Analytical Characterization of Selected Biosimilar Formulations Available in India

Abstract

The significance of performing a comparative analytical similarity assessment cannot be overstated in the process of evaluating the quality of a biosimilar product and its regulatory submission. This is due to the fact that biosimilar products are not identical to the innovator product. Moreover, it is considered essential to establish appropriate shelf-life and storage conditions for a drug product to ensure the safety and efficacy of the product over its intended shelf-life, following its market authorization. In this thesis, analysis of critical product attributes of marketed Bevacizumab biosimilar-1, and biosimilar-2 (Bio-1, and Bio-2) and Erythropoietin-1, Erythropoietin-2, and Erythropoietin-3 (Epo-1, Epo-2, and Epo-3) biosimilar products were carried out in comparison to their reference product using state-of-the-art analytical techniques. Quantitative estimation of native protein was performed using UV-Visible spectrophotometer at 280 nm (UV280) & reverse phase high performance liquid chromatography (RP-HPLC) method, high order structure characterization was done using attenuated total reflectance-fourier transform infrared spectrophotometer (ATR-FTIR) & Far-UV circular dichroism (Far-UV CD) method, charge & size variant analysis was carried out using imaged capillary isoelectric focusing (iCIEF) & capillary electrophoresis coupled with sodium dodecyl sulfate (CE-SDS) technique in both reduced and non-reduced condition. Matrix assisted laser desorption ionization coupled with time of flight detector (MALDI-TOF) & liquid chromatography coupled with electrospray ionization and quadrupole time of flight detector (LC-ESI-QTOF) techniques were used to carry out intact mass analysis for Bevacizumab products. Furthermore, the stability of Bevacizumab marketed formulations after exposure to thermal, chemical, and mechanical stress conditions is also discussed in this thesis. The profile of aggregates, other product-related impurities, and any alterations to high order structure triggered as a function of thermal, chemical, and mechanical stress conditions were evaluated using advanced analytical techniques such as size exclusion chromatography coupled with multi angle laser scattering (SEC-MALS) & Far-UV CD. The results for comparative analytical assessment of Bevacizumab biosimilar products demonstrated similar intact mass profiles using MALDI-TOF with experimental mass values of 149,785 Da (Bio-1), 149,095 Da (Bio-2), and 149,189 Da (Avastin (Avt) reference product). Using LC-ESI-QTOF similar total ion chromatograph profiles were observed for Bevacizumab biosimilar products, with experimental retention time (Rt) of 3.380±0.2 min (Bio-1), 3.378±0.2 min (Bio-2), and 3.628±0.2 min (Avt). The most abundant peaks of the deconvoluted mass spectrum obtained using LC-ESI-QTOF corresponded to the experimental mass values of 149,168 Da for Bio-1 and Bio-2 formulations, and 149,167 Da for Avt formulation. Similar ATR-FTIR spectra were observed for Bio-1, Bio-2, and Avt formulations, with peaks at 1617/cm, 1635/cm, 1668/cm, 1670/cm, and 1684/cm. The prominent secondary structure of Bio-1 (40.9±2.0%), Bio-2 (46.5±2.0%), and Avt (44.1±2.0%) control formulations were confirmed to be antiparallel β-sheet using Far-UV CD technique. The CE-SDS analysis was employed to investigate the conformational states of intact monoclonal antibody (mAb), light chain, non-glycosylated heavy chain, as well as high and low molecular weight species in Bio-1 and Bio-2 formulations under both reduced and non-reduced conditions. The concentration of Bio-1 and Bio-2 formulations, as determined using UV280 method, was found to be 24.4-25.1 mg/ml and 24.3-24.9 mg/ml, respectively, and both were within the preset concentration range of Avt, 24.1-25.3 mg/ml. High product purity of 96.0%±0.6% and 96.25%±1.6% were observed for Bio-1, Bio-2, and Avt formulations using RP-HPLC technique at 214 nm and 280 nm The forced degradation study of Bvz biosimilar products using Far-UV CD technique demonstrated changes in the secondary structure conformation with an average loss of 15% and 8.3% in all formulations due to elevated temperature and pH condition. SEC-MALS technique was used for isolating high molecular weight (HMW) aggregates at 11.2±0.2 min followed by monomer peak at 16.5±0.2 min for samples exposed to thermal and mechanical stress conditions, aggregation were observed, with increasing molecular weight (MW) and radius over time. Poly acrylamide gel electrophoresis with sodium dodecyl sulfate (SDS PAGE) technique was used for separating light molecular weight (LMW) aggregates from monomer band with reported MW of 150KDa. The results for analytical assessment of Erythropoietin biosimilar formulations demonstrated that the presence of human serum albumin (HSA) cause interference in analytical evaluation using; UV280, resorcinol assay for sialic acid estimation, icIEF, and CE-SDS therefore, making them applicable for qualitative estimation only. RP-HPLC method was developed and validated for the purity analysis of Erythropoietin-reference standard (Epo-RS) and formulations with reported average % assay of 96.41%±0.79 and 96.21%±0.61 at 214 nm and 280 nm. The application of the resorcinol assay indicated the presence of 13±1 moles of sialic acid per mole of Epo-RS. The icIEF analysis revealed the presence of 8 isoform bands in Epo RS, with isoelectric points ranging from 4.3 to 5.4. Under reduced conditions using CE-SDS, 12 distinct bands were observed for Epo-RS, with molecular weights ranging from 64 kDa to 149 kDa. Conversely, under non-reduced CE-SDS conditions, 11 bands were detected for Epo-RS, with molecular weights ranging from 71 kDa to 166 kDa. To summarize, in this research work, analytical characterization was performed for analysis of critical product attributes of Bevacizumab and Erythropoietin biosimilar and reference product, employing advanced analytical tools. Furthermore, the impact of diverse stress conditions on the structural stability and integrity of Bevacizumab biosimilar products was also investigated.