Demonstration of Physicochemical and Functional Similarity of the Biosimilar BAT1806/BIIB800 to Reference Tocilizumab.

Science-based bioprocess design for filamentous fungi

Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities.

Use of Raman spectroscopy and PLS for the quantification of critical quality attributes in biopharmaceutical products.

The traditional drug manufacturing process involves numerous qualitative attributes that directly impact product quality. The Process Analytical Technology approach considers that to enhance process control, identifying critical process parameters and critical quality attributes that affect the manufacturing process is very much necessary. The Ayurvedic drug manufacturing process is more driven by fuzzy qualitative attributes. The present study was executed to identify the Critical Process Parameters and Critical Quality Attributes in the manufacturing process of a herbo-mineral formulation, viz. Arogyavardhini Rasa. Fuzzy set Qualitative Comparative Analysis (fsQCA) methodology was adopted to observe and identify the critical parameters. The study was executed in three steps, viz. Data collection, Data arrangement, and Analysis. The raw data collected was arranged and analyzed in the software R studio using the package QCA in four steps, viz. Calibration, Analysis of Necessity and Sufficiency, Truth Table construction, and Minimization. The results obtained show that Size reduction, Mardana, and Drying are the identified Critical Process Parameters that, in combination, lead to the outcome, i.e., good product quality. Thus, this study proves that Fuzzy Set Qualitative Comparative Analysis can be used as an efficient tool for the identification and measurement of the Critical Process Parameters that affect the Critical Quality Attributes and, thereby, the product quality in the manufacturing of Arogyavardhini Rasa.

ABSTRACTIn the biopharmaceutical environment, controlling the Critical Quality Attributes (CQA) of a product is essential to prevent changes that affect its safety or efficacy. Physico-chemical techniques and bioassays are used to screen and monitor these CQAs. The higher order structure (HOS) is a CQA that is typically studied using techniques that are not commonly considered amenable to quality control laboratories. Here, we propose a peptide mapping-based method, named native peptide mapping, which could be considered as straightforward for HOS analysis and applicable for IgG4 and IgG1 antibodies. The method was demonstrated to be fit-for-purpose as a stability-indicating assay by showing differences at the peptide level between stressed and unstressed material. The unfolding pathway induced by a heat stress was also studied via native peptide mapping assay. Furthermore, we demonstrated the structure–activity relationship between HOS and biological activity by analyzing different types of stressed samples with a cell-based assay and the native peptide mapping. The correlation between both sets of results was highlighted by monitoring peptides located in the complementary-determining regions and the relative potency of the biotherapeutic product. This relationship represents a useful approach to interrogate the criticality of HOS as a CQA of a drug.

Rapid and comprehensive monoclonal antibody Characterization using microfluidic CE-MS

QbD-guided pharmaceutical development of Pembrolizumab biosimilar candidate PSG-024 propelled to industry meeting primary requirements of comparability to Keytruda®

BackgroundBAT1706 is a proposed biosimilar of bevacizumab, a vascular endothelial growth factor A (VEGF-A)-targeting biologic used to treat several different cancers, including metastatic colorectal cancer. A comprehensive physicochemical and functional similarity assessment is a key component of demonstrating biosimilarity between a reference biologic and a proposed biosimilar. Here we report the physicochemical and functional similarity of BAT1706 and reference bevacizumab sourced from both the United States (US-bevacizumab) and the European Union (EU-bevacizumab).MethodA large range of product attributes, including primary and higher order structure, post-translational modifications, purity, stability, and potency, were characterized for BAT1706 and EU/US-bevacizumab using sensitive state-of-the-art analytical techniques. Up to 18 lots of US- and 29 lots of EU-bevacizumab, and 10 unique drug substance lots of BAT1706, were assessed.ResultBAT1706 was shown to have an identical amino acid sequence and an indistinguishable higher-order structure compared with EU/US-bevacizumab. BAT1706 and EU/US-bevacizumab also exhibited similar post-translational modifications, glycan profiles, and charge variants. Potency, assessed using a wide range of bioassays, was also shown to be comparable between BAT1706 and EU/US-bevacizumab, with statistical equivalence demonstrated for VEGF-A binding and neutralizing activity.ConclusionOverall, this extensive comparability exercise demonstrated BAT1706 to match EU/US-bevacizumab in terms of all physicochemical and functional attributes assessed.

BackgroundBAT1806/BIIB800 (Tofidence™/tocilizumab-bavi), a biosimilar of tocilizumab, demonstrated a high degree of analytical and functional similarity to reference tocilizumab (TCZ) in a comprehensive comparative analytical assessment. Minor differences with respect to TCZ were observed for some attributes and this study assessed the potential impact of these differences through structure activity relationship characterization.MethodsStructure activity relationship studies were conducted to assess glycation, glycosylation, charge variants, hydrophobicity, oxidation, and deamidation differences, using a range of investigative techniques. Structure activity relationship studies were performed on one lot each of BAT1806/BIIB800 and TCZ (European Union sourced only) except for glycation, where additional lots sourced from China and the USA were used.ResultsAverage total glycated protein content of BAT1806/BIIB800 was higher than TCZ (10.08% vs 1.19%); however, biological activity, including target binding and functional potency, was unaffected. Stress-induced glycation of BAT1806/BIIB800 and TCZ also did not affect the biological activity of the products despite up to 60% total glycation content. Minor differences were observed between BAT1806/BIIB800 and TCZ in glycosylation, charge variants, hydrophobicity, oxidation, and deamidation without a relevant impact on interleukin-6 receptor binding, Fc-receptor binding, and effector functions. In forced degradation studies, oxidation and deamidation trends were comparable between the two products.ConclusionsComparative structure activity relationship characterization of BAT1806/BIIB800 and TCZ indicated that there are no relevant differences in quality attributes between BAT1806/BIIB800 and reference TCZ. Observed differences between BAT1806/BIIB800 and TCZ had no functional impact on BAT1806/BIIB800. The results support the conclusion that BAT1806/BIIB800 is similar to TCZ.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40259-024-00698-7.

Background/Purpose:Tocilizumab (TCZ), an interleukin‐6 receptor (IL‐6R) inhibitor, is effective in systemic and polyarticular juvenile idiopathic arthritis (sJIA, pJIA). Body weight (BW)‐adjusted intravenous dosing regimens (TCZ 8 mg/kg Q2W for sJIA and Q4W for pJIA) were assessed in Japanese phase 3 trials. In Japanese patients (pts), BW adjustment led to lower TCZ exposure with lower BW; thus, higher doses were proposed for pts with BW <30 kg in the global TENDER (sJIA) and CHERISH (pJIA) trials. Herein, we describe the pharmacokinetics (PK), pharmacodynamics (PD), and exposure‐efficacy/‐safety relationships of adjusted BW‐based TCZ therapy in sJIA/pJIA pts.Methods:PK/PD results were summarized in TCZ‐treated pts from part 1 of TENDER and CHERISH. TENDER part 1 (n = 75) comprised a 12‐wk, double‐blind phase with pts randomised 1:1 to TCZ or placebo Q2W: TCZ 12 mg/kg if <30 kg and 8 mg/kg if >30 kg. CHERISH part 1 (n = 177) comprised a 16‐wk TCZ Q4W open‐label phase (TCZ 8 mg/kg if >30 kg and 8 mg/kg or 10 mg/kg if <30 kg). Pts were 2–17 y old with >5 active joints and >38°C fever for >5 days (sJIA) or >3 of >5 joints with limited motion (pJIA). Blood samples were analyzed for TCZ, IL‐6R blockade markers (IL‐6, soluble IL‐6R [sIL‐6R]), C‐reactive protein (CRP) levels, and erythrocyte sedimentation rate (ESR). Efficacy was measured as JIA ACR30/50/70/90 response rates. Population PK (popPK) modeling was used to further analyze serum TCZ concentration data in addition to descriptive summary statistics.Results:In sJIA pts, mean serum TCZ concentrations over time and steady state TCZ exposures at wk 12 were similar between TCZ 8 mg/kg and 12 mg/kg BW groups. Predose concentrations trended upward over time, stabilizing by wks 10–12. IL‐6, sIL‐6R, CRP, and ESR profiles were overlaid between BW groups, showing similar IL‐6R blockade. Consistently, primary efficacy outcomes by JIA ACR 70/90 response were also comparable. In pJIA pts, the <30 kg group taking TCZ 8 mg/kg had lower TCZ concentrations than the <30 kg group taking 10 mg/kg and the ≥30 kg taking 8 mg/kg, which were similar to each other. IL‐6, sIL‐6R, CRP, and ESR profiles indicated reduced IL‐6R blockade in pts <30 kg taking TCZ 8 mg/kg versus the other two groups. Consistent with this, quartile analysis showed lower JIA ACR30/50/70/90 response rates at wk 16 in the lowest TCZ exposure quartile. No clear trends in adverse events across exposure quartiles were seen in sJIA or pJIA pts. PopPK analysis showed similar linear clearance of TCZ in sJIA and pJIA pts, but the Michaelis‐Menten constant in target‐mediated clearance was 3–4× higher in sJIA pts.Conclusion:Results support adjusting BW‐based TCZ dosing in sJIA/pJIA pts with the optimal dosages: sJIA, TCZ 12 mg/kg Q2W for BW <30 kg, and 8 mg/kg Q2W for BW ≥30 kg; pJIA, TCZ 10 mg/kg Q4W for BW <30 kg and 8 mg/kg Q4W for BW ≥30 kg. Taking into consideration comparable linear clearances in sJIA/pJIA pts, the higher Michaelis‐Menten constant in target‐mediated clearance for sJIA pts may be due to higher cell surface IL‐6R levels. Observations are consistent with higher baseline IL‐6 signaling in sJIA pts and potentially explain the need for higher dose regimens to saturate IL‐6R for optimal therapy in sJIA pts.

Understanding process parameter interactions and their effects on mammalian cell cultivations is an essential requirement for robust process scale-up. Furthermore, knowledge of the relationship between the process parameters and the product critical quality attributes (CQAs) is necessary to satisfy quality by design guidelines. So far, mainly the effect of single parameters on CQAs was investigated. Here, we present a comprehensive study to investigate the interactions of scale-up relevant parameters as pH, pO2 and pCO2 on CHO cell physiology, process performance and CQAs, which was based on design of experiments and extended product quality analytics. The study used a novel control strategy in which process parameters were decoupled from each other, and thus allowed their individual control at defined set points. Besides having identified the impact of single parameters on process performance and product quality, further significant interaction effects of process parameters on specific cell growth, specific productivity and amino acid metabolism could be derived using this method. Concerning single parameter effects, several monoclonal antibody (mAb) charge variants were affected by process pCO2 and pH. N-glycosylation analysis showed positive correlations between mAb sialylation and high pH values as well as a relationship between high mannose variants and process pH. This study additionally revealed several interaction effects as process pH and pCO2 interactions on mAb charge variants and N-glycosylation pattern. Hence, through our process control strategy and multivariate investigation, novel significant process parameter interactions and single effects were identified which have to be taken into account especially for process scale-up.

Background:Blocking interleukin-6 (IL-6) receptor (IL-6R) with tocilizumab (TCZ) has demonstrated effectiveness in reducing Giant Cell Arteritis (GCA) flares and sparing glucocorticoids (GCs) [1,2] but little is known about the functional...

Background and ObjectiveTocilizumab is a recombinant, humanised monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which specifically targets the interleukin-6 receptor (IL-6R). The RGB-19 product was developed as a biosimilar to the reference medicinal product RoActemra® (authorised for use in the European Union [EU]). The current study focuses on the demonstration of structural, physico-chemical and functional similarity between RGB-19 (intravenous [IV] and subcutaneous [SC] presentations) and EU-sourced RoActemra® (IV and SC presentations).MethodsThe RGB-19 biosimilar tocilizumab product was comprehensively tested using an extensive state-of-the-art analytical and functional panel of 44 methods to demonstrate similarity to the EU-sourced RoActemra®. Biosimilarity was rigorously evaluated through an extensive array of orthogonal physico-chemical and functional assays, supplemented by a detailed comparative characterisation of the primary and higher order structures of the therapeutic proteins.ResultsExtensive structural analyses confirmed that the primary and higher order structures of tocilizumab proteins in RGB-19 IV and SC drug products are identical or exhibit a high degree of similarity to those of the RoActemra® reference products. The impurity profiles of RGB-19 and RoActemra® products were found to be highly comparable, as demonstrated by a series of physico-chemical techniques. A high level of similarity was shown for the most critical (soluble IL-6R binding and cell-based anti-proliferation assay) and for all other bioassay attributes. Based on the statistical evaluation, negligible differences could be detected for sialylation, glycation, fragments and charge variants, which do not affect the functional properties.ConclusionBased on the similarity study, RGB-19 and RoActemra® can be considered highly similar drug products. The minor differences found for some physico-chemical attributes do not affect the biological potency, binding and other critical attributes, and are therefore not considered clinically meaningful.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40259-025-00734-0.

Identification of a monoclonal antibody clipping variant by cross-validation using capillary electrophoresis – sodium dodecyl sulfate, capillary zone electrophoresis – mass spectrometry and capillary isoelectric focusing – mass spectrometry

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.