Analytical Similarity Assessment of ABP 959 in Comparison with Eculizumab Reference Product.

BACKGROUND: ABP 959 is one of the first proposed biosimilars to eculizumab reference product (RP), a recombinant IgG2/4Ƙ monoclonal antibody (mAb) that binds human C5 complement protein and inhibits C5 cleavage to C5a and C5b, preventing the generation of the terminal complement complex C5b-9. Eculizumab RP is approved for the treatment of paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, myasthenia gravis in patients who are anti-acetylcholine receptor antibody positive, and neuromyelitis optica spectrum disorder in patients who are anti-aquaporin-4 antibody positive. OBJECTIVES: The objective of this work was to comparatively assess analytical (structural and functional) similarity between ABP 959 and eculizumab RP using sensitive, state-of-the art analytical methods capable of detecting minor differences in product quality attributes. METHODS: Comprehensive analytical (structural and functional) characterization utilizing orthogonal techniques was performed using multiple lots of ABP 959 and eculizumab RP over several years applying > 40 state-of-the-art assays. Comparisons were performed to investigate the primary structure and post-translational modifications including glycans, higher-order structure, particles and aggregates, product-related structures and impurities, thermal stability and forced degradation, general properties, and biological properties mediated by target binding. RESULTS: Results confirmed that ABP 959 had the same amino acid sequence, similar primary structure, higher-order structure, post-translational profiles, and the same protein content and concentration (e.g., ABP 959: 9.4-10.0; eculizumab EU: 9.4-10.0; eculizumab US: 9.3-10.3 mg/mL) as well as biological activity as eculizumab RP. CONCLUSIONS: Based on these results, it can be concluded that ABP 959 is analytically similar to eculizumab RP.

Analytical and Functional Similarity Assessment of ABP 710, a Biosimilar to Infliximab Reference Product.

PURPOSE: ABP 710 has been developed as a biosimilar to infliximab reference product (RP). The objective of this study was to assess analytical similarity (structural and functional) between ABP 710 and infliximab RP licensed by the United States Food and Drug Administration (infliximab [US]) and the European Union (infliximab [EU]), using sensitive, state-of-the-art analytical methods capable of detecting minor differences in product quality attributes. METHODS: Comprehensive analytical characterization utilizing orthogonal techniques was performed with 14 to 28 unique lots of ABP 710 or infliximab RP, depending on the assay. Comparisons were used to investigate the primary structure related to amino acid sequence; post-translational modifications (PTMs) including glycans; higher order structure; particles and aggregates; primary biological properties mediated by target and receptor binding; product-related substances and impurities; and general properties. RESULTS: ABP 710 had the same amino acid sequence, primary structure, higher order structure, PTM profiles and biological activities as infliximab RP. The finished drug product had the same strength (protein content and concentration) as infliximab RP. CONCLUSIONS: Based on the comprehensive analytical similarity assessment, ABP 710 was found to be highly analytically similar to infliximab RP for all biological activities relevant for clinical efficacy and safety.

A Comparison Between Emerging and Current Biophysical Methods for the Assessment of Higher-Order Structure of Biopharmaceuticals.

The higher-order structure (HOS) of protein therapeutics is a critical quality attribute directly related to their function. Traditionally, the HOS of protein therapeutics has been characterized by methods with low to medium structural resolution such as Fourier-transform infrared (FTIR), circular dichroism (CD), and intrinsic fluorescence spectroscopy, and differential scanning calorimetry (DSC). Recently, high-resolution nuclear magnetic resonance (NMR) methods have emerged as powerful tools for HOS characterization. NMR is a multi-attribute method with unique capabilities to provide information about all the structural levels of proteins in solution. We have in this study compared 1 D 1H Profile NMR with the established biophysical methods for HOS assessments using a set of blended samples of the monoclonal antibodies belonging to the subclasses IgG1 and IgG2. The study shows that Profile NMR can distinguish between most sample combinations (93%), DSC can differentiate 61% of the sample combinations, and near-ultraviolet CD spectroscopy can differentiate 52% of the sample combinations, whereas no significant distinction could be made between any samples using FTIR or intrinsic fluorescence. Our data therefore show that NMR has superior ability to address differences in HOS, a feature that could be directly applicable in comparability and similarity assessments.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.