Analytical and functional similarity of biosimilar ABP 798 with rituximab reference product.

ABP 798 is a biosimilar candidate to rituximab reference product (RP). This comprehensive analytical similarity assessment was designed to assess the structural and functional similarity of ABP 798, rituximab (US), and rituximab (EU) using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. The similarity assessment was performed to evaluate product quality attributes associated with Fab, Fab/Fc, and Fc domains, including those known to affect the mechanisms of action. ABP 798 has the same amino acid sequence and exhibits similar secondary and tertiary structures, similar glycan and post-translational modification profiles, and biological activities as rituximab RP. There are minor differences in biochemical attributes, which are not considered clinically meaningful. The results of the analytical and functional similarity assessment demonstrate that ABP 798 is highly analytically similar to rituximab RP. These results support the totality of evidence and the scientific justification for extrapolation of ABP 798 to all therapeutic indications approved for rituximab.

In Situ Quantification of Polysorbate in Pharmaceutical Samples of Therapeutic Proteins by Hydrodynamic Profiling by NMR Spectroscopy.

Polysorbates are nonionic surfactants often used at variable levels in various formulations of protein therapeutics. Their quantification in pharmaceutical samples has posed an analytical challenge. Here we present an approach based on 1H NMR spectroscopy which can accurately estimate the concentration of polysorbate 80 (PS80) in intact pharmaceutical samples of an arbitrary formulation. The method, HAP-NMR (hydrodynamic profiling by NMR), is an extension of the protein fingerprint by line shape enhancement method (PROFILE) approach ( Poppe , L. ; Jordan , J. B. ; Lawson , K. ; Jerums , M. ; Apostol , I. ; Schnier , P. D. Anal. Chem. 2013 , 85 (20) , 9623 - 9629 ) and is based on the 1D 1H pulsed field gradient stimulated echo (PFGSTE) NMR experiment, which allows for the rectification of the 1D 1H NMR spectrum to a level suitable for a quantitative hydrodynamic analysis. Here we describe the methodology as applied to an antibody sample formulated in 9% (w/v) sucrose and with variable levels of PS80, ranging from 0.01% to 0.20% (w/v) sample concentrations. Equally important, we present evidence and propose a novel mechanism of how polysorbate stabilizes protein in pharmaceutical formulations.

Subvisible (2-100 µm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

Analytical and Functional Similarity of Aflibercept Biosimilar ABP 938 with Aflibercept Reference Product.

INTRODUCTION: ABP 938 is being developed as a biosimilar candidate to aflibercept reference product (RP), a biologic used for certain angiogenic eye disorders. This study was designed to provide a comparative analytical assessment of the structural and functional attributes of ABP 938 and aflibercept RP sourced from the United States (US) and the European Union (EU). METHODS: Structural and functional characterization studies were performed using state-of-the-art analytical techniques that were appropriate to assess relevant quality attributes and capable of detecting qualitative and quantitative differences in primary structure, higher-order structure and biophysical properties, product-related substances and impurities, general properties, and biological activities. RESULTS: ABP 938 had the same amino acid sequence and exhibited similar secondary and tertiary structures, and biological activity as aflibercept RP. There were minor differences in a small number of biochemical attributes which are not expected to impact clinical performance. In addition, aflibercept RP sourced from the US and EU were analytically similar. CONCLUSIONS: ABP 938 was structurally and functionally similar to aflibercept RP. Since aflibercept RP sourced from the US and EU were analytically similar, this allows for the development of a scientific bridge such that a single-source RP can be used in nonclinical and clinical studies.

Eylea^® (aflibercept) is a biologic medication approved for the treatment of patients with certain eye diseases that can result in low vision or blindness. Biosimilars are biologic medications that are highly similar to an existing approved biologic medication, often called a reference product. Biosimilars have the potential to reduce medication costs despite having no clinically significant differences in quality, efficacy, and safety from their reference products. ABP 938 is currently being developed as a biosimilar to aflibercept reference product. We have conducted similarity studies to compare multiple batches of ABP 938 and aflibercept reference product sourced from both the United States and the European Union, using state-of-the-art analytical methods. The results demonstrated that ABP 938 had the same amino acid sequence and similar structural and biological activities as aflibercept reference product. Before biosimilars can be used as medicines, studies such as this one are required by the Food and Drug Administration and other regulatory authorities to ensure that biosimilars are as safe and effective as their reference products.

Evaluating Clinical Safety and Analytical Impact of Subvisible Silicone Oil Particles in Biopharmaceutical Products.

Silicone oil is a commonly used lubricant in pre-filled syringes (PFSs) and can migrate over time into solution in the form of silicone oil particles (SiOPs). The presence of these SiOPs can result in elevated subvisible particle counts in PFS drug products compared to other drug presentations such as vials or cartridges. Their presence in products presents analytical challenges as they complicate quantitation and characterization of other types of subvisible particles in solution. Previous studies have suggested that they can potentially act as adjuvant resulting in potential safety risks for patients. In this paper we present several analytical case studies describing the impact of the presence of SiOPs in biotherapeutics on the analysis of the drug as well as clinical case studies examining the effect of SiOPs on patient safety. The analytical case studies demonstrate that orthogonal techniques, especially flow imaging, can help differentiate SiOPs from other types of particulate matter. The clinical case studies showed no difference in the observed patient safety profile across multiple drugs, patient populations, and routes of administration, indicating that the presence of SiOPs does not impact patient safety.

Analytical and Functional Similarity of the Biosimilar Candidate ABP 654 to Ustekinumab Reference Product.

BACKGROUND AND OBJECTIVE: ABP 654 is a proposed biosimilar to ustekinumab reference product (RP), a human immunoglobulin isotype class G subclass 1 kappa monoclonal antibody that acts as an antagonist of interleukin (IL)-23 and IL-12. Ustekinumab RP is indicated for the treatment of some forms of plaque psoriasis, active psoriatic arthritis, Crohn's disease, and ulcerative colitis. ABP 654 and ustekinumab RP utilize different expression systems, and the purpose of this study was to assess analytical similarity between ABP 654 and ustekinumab RP sourced from the United States (US) and the European Union (EU). METHODS: The analytical testing plan included general properties, primary structure, higher-order structure, product-related substances and impurities, particles and aggregates, biological activity, and thermal stability and degradation studies. RESULTS: ABP 654 was found to be analytically similar to ustekinumab RP with respect to physicochemical and biological properties, including structure, function, purity, and potency. CONCLUSIONS: Based on a comprehensive similarity assessment, ABP 654 was found to be similar to ustekinumab RP, notwithstanding minor physicochemical differences that are not expected to have a clinically meaningful effect on safety or efficacy.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

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.

Method to Determine Syringe Silicone Oil Layer Heterogeneity and Investigation of its Impact on Product Particle Counts.

Prefilled syringes (PFSs) are commonly used for parenteral delivery of protein therapeutics. In PFSs, the inner surface of the syringe barrel is typically coated with silicone oil for lubrication. The total amount of silicone oil as well as its distribution can impact syringe functionality and particle formation. However, methods to non-destructively characterize the silicone oil distribution are limited. In this paper, we developed a method to visualize and quantify the relative distribution of silicone oil in unfilled syringes using a custom-built multi-color interferometric imaging system. We then applied the system in a preliminary study to investigate the impact of the silicone oil distribution on the number of particles formed in solution after filling and extrusion for two different types of syringes. The syringe type with significantly lower particle counts also exhibited significantly more homogeneous silicone oil distributions. Within syringe types, no significant association was found between silicone oil distribution and particle formation. Our method can be used in further studies that investigate the impact of syringe siliconization on PFS functionality and particle formation.

Characterization of Subvisible Particles in Biotherapeutic Prefilled Syringes: The Role of Polysorbate and Protein on the Formation of Silicone Oil and Protein Subvisible Particles After Drop Shock.

Subvisible particles (SbVPs) are a critical quality attribute for biotherapeutics. Particle content in prefilled syringes (PFSs) of a biotherapeutic can include protein particles and silicone oil particles (SiOP). Here, a real-world protein therapeutic PFS shows that although polysorbate is effective in preventing protein particle formation, it also leads to the formation of SiOP. PFSs of protein and buffer formulations in the presence and absence of polysorbate are subjected to a drop shock to generate SbVP and the effect of polysorbate and protein in generating SbVP is investigated. Particle characterization by light obscuration and flow imaging shows that polysorbate prevents protein particle formation as intended, but the presence of polysorbate substantially increases the formation of SiOP. The protein itself also acts as a surfactant and leads to increased SiOP, but to a lesser degree compared to polysorbate. In a separate companion study by Joh et al., the risk of immunogenicity was assessed using in vivo and in vitro models. Flow imaging distinguishes between SiOP and protein particles and enables risk assessment of the natures of different SbVP in PFSs.

Silicone Oil Particles in Prefilled Syringes With Human Monoclonal Antibody, Representative of Real-World Drug Products, Did Not Increase Immunogenicity in In Vivo and In Vitro Model Systems.

Silicone oil is a lubricant for prefilled syringes (PFS), a common primary container for biotherapeutics. Silicone oil particles (SiOP) shed from PFS are a concern for patients due to their potential for increased immunogenicity and therefore also of regulatory concern. To address the safety concern in a context of manufacturing and distribution of drug product (DP), SiOP was increased (up to ∼25,000 particles/mL) in PFS filled with mAb1, a fully human antibody drug, by simulated handling of DP mimicked by drop shock. These samples are characterized in a companion report (Jiao N et al. J Pharm Sci. 2020). The risk of immunogenicity was then assessed using in vitro and in vivo immune model systems. The impact of a common DP excipient, polysorbate 80, on both the formation and biological consequences of SiOP was also tested. SiOP was found associated with (1) minimal cytokine secretion from human peripheral blood mononuclear cells, (2) no response in cell lines that report NF-κB/AP-1 signaling, and (3) no antidrug antibodies or significant cytokine production in transgenic Xeno-het mice, whether or not mAb1 or polysorbate 80 was present. These results suggest that SiOP in mAb1, representative of real-world DP in PFS, poses no increased risk of immunogenicity.

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 µm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.

Analytical and functional similarity of Amgen biosimilar ABP 215 to bevacizumab.

ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.

Characterizing Reversible Protein Association at Moderately High Concentration Via Composition-Gradient Static Light Scattering.

Analysis of weakly self-associating macromolecules at concentrations beyond a few g/L is challenging on account of the confounding effect of thermodynamic nonideality on the association signal. When the reversible association comprises only 1 or 2 oligomeric species in equilibrium with the monomer, the nonideality may be accounted for in a relatively rigorous manner, but if more association states are involved, the analysis becomes quite complex. We show that under reasonable assumptions, the nonideality in a composition-gradient static light scattering measurement may be accounted for in a simple fashion. The correction is applied to determining the stoichiometry and binding affinity of a protein previously characterized via sedimentation equilibrium and dynamic light scattering. The results of the new analysis are remarkably self-consistent and in line with the expectations for the form of self-association predicted previously from analysis of the surface residuals, establishing composition-gradient multi-angle static light scattering with nonideality corrections as a critical technology for characterizing associative interactions in concentrated solutions.

Assessing Analytical Similarity of Proposed Amgen Biosimilar ABP 501 to Adalimumab.

BACKGROUND: ABP 501 is being developed as a biosimilar to adalimumab. Comprehensive comparative analytical characterization studies have been conducted and completed. OBJECTIVE: The objective of this study was to assess analytical similarity between ABP 501 and two adalimumab reference products (RPs), licensed by the United States Food and Drug Administration (adalimumab [US]) and authorized by the European Union (adalimumab [EU]), using state-of-the-art analytical methods. METHODS: Comprehensive analytical characterization incorporating orthogonal analytical techniques was used to compare products. Physicochemical property comparisons comprised the primary structure related to amino acid sequence and post-translational modifications including glycans; higher-order structure; primary biological properties mediated by target and receptor binding; product-related substances and impurities; host-cell impurities; general properties of the finished drug product, including strength and formulation; subvisible and submicron particles and aggregates; and forced thermal degradation. RESULTS: ABP 501 had the same amino acid sequence and similar post-translational modification profiles compared with adalimumab RPs. Primary structure, higher-order structure, and biological activities were similar for the three products. Product-related size and charge variants and aggregate and particle levels were also similar. ABP 501 had very low residual host-cell protein and DNA. The finished ABP 501 drug product has the same strength with regard to protein concentration and fill volume as adalimumab RPs. ABP 501 and the RPs had a similar stability profile both in normal storage and thermal stress conditions. CONCLUSION: Based on the comprehensive analytical similarity assessment, ABP 501 was found to be similar to adalimumab with respect to physicochemical and biological properties.

Classification of glass particles in parenteral product vials by visual, microscopic, and spectroscopic methods.

Glass vials have been used as primary containers for parenteral drugs including biopharmaceuticals. Different types of glass-related particles, although in low occurrence rate, may be adventitiously introduced in these parenterals. Proper classification and investigations of these glass-related particles may help to understand their formation, improve process control, reduce glass-related particles, and deliver safe parenteral drugs to patients. In this article, we introduced a classification scheme, and identification procedures and methods, for the glass-related particles. We propose to classify them as glass chip, glass lamella/flake, and silica gel. Eight characteristics for each glass particle type have been identified and described for the visual inspection method. The limitations of the visual method and the need to correlate visual results with forensic analysis are discussed. Using representative examples from each type of glass particle, this study summarized their forensic differentiations based on microscopic methods of optical microscopy, scanning electron microscopy, micro-flow imaging, and spectroscopic methods of dnergy-dispersive spectroscopy and Fourier transform infrared spectroscopy. The mechanisms of glass particle formation are listed as references for drug development scientists to investigate the root causes and improve process control on visible glass particles in parenteral vials. LAY ABSTRACT: Glass vials have been used as primary containers for parenteral drugs including biopharmaceuticals. Different types of glass-related particles, although in low occurrence rate, may be adventitiously introduced in these parenterals. Proper classification and investigations of these glass-related particles may help to understand their formation, improve process control, reduce glass-related particles, and deliver safe parenteral drugs to patients. In this article, we introduced a classification scheme, and identification procedures and methods, for the glass-related particles. We propose to classify them as glass chip, glass lamella/flake, and silica gel. Using representative examples from each type of glass particle, this study summarized their forensic differentiations based on microscopic and spectroscopic methods. The mechanisms of glass particle formation are listed as references for drug development scientists to investigate the root causes and improve process control on visible glass particles in parenteral vials.

Monitoring ceramic hydroxyapatite media degradation using dynamic image analysis and uniaxial confined bulk compression.

Ceramic hydroxyapatite (CHT) is a multimodal chromatographic medium widely used in the pharmaceutical industry for the purification of biomolecules. CHT is a sintered form of hydroxyapatite crystals with moderate stability at acidic conditions. This moderate stability may lead to underperformance of CHT packed bed lifetime, especially under acidic conditions, which should be monitored by diagnostic tools to design optimal buffer systems for the step. This study presents the application of dynamic image analysis (DIA) and uniaxial confined bulk compression (UCBC) to monitor CHT particle degradation as a function of buffer composition. DIA was used to evaluate changes in solidity and morphology, while UCBC was used to evaluate changes in resistance to uniaxial compression. All properties were studied as a function of bed position and operational parameters. Results show that when CHT is exposed to acidic pH, adding phosphate and/or calcium at concentrations of 1 mM minimizes changes in particle solidity and mechanical strength. Changes in CHT morphological properties (i.e., convexity, aspect ratio) are also affected by the presence of calcium and/or phosphate in the inlet buffers. Furthermore, calcium and phosphate have a positive effect on the mechanical behavior of CHT, which is related to changes in the CHT particle solidity.

Separation and characterization of protein aggregates and particles by field flow fractionation.

Field flow fractionation (FFF) is a technique that holds great promise for the analysis and characterization of protein aggregates and particles, due to its wide dynamic range and matrix-free separation mechanism. FFF can be routinely used to achieve good monomer-oligomer separation and quantification for a variety of protein types, and is a reasonable choice for an orthogonal method for size exclusion chromatography and analytical ultracentrifugation. Quantifying sub-micrometer particles in protein therapeutics is a potential of the FFF technique that is yet to be realized, due to the lack of detection with sufficient sensitivity. In this article the effect of several important parameters on the optimization of FFF analyses are explored, and the strengths, weaknesses, and potential new applications of the technique are discussed.

A critical review of analytical methods for subvisible and visible particles.

The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.