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.

Advancement of microfluidic modulation spectroscopy as a highly sensitive protein characterization technique for the detection of small structural changes.

The higher order structure (HOS) of a protein is vital to its function and activity, making it a critical component in the development of protein-based therapeutics. Characterization of HOS can be performed using various biophysical techniques, but many of these methods have limitations such as low throughput, complicated workflow, narrow concentration range, and low sensitivity. Microfluidic Modulation Spectroscopy (MMS) is an emerging technology that addresses these limitations, offering high sensitivity and automated analysis for protein secondary structure. This study evaluates and compares the different well plate formats and scan modes of two MMS instruments. The newer Apollo system features a high throughput 96-well plate format and sweep scan mode that allows a 50% reduction in sample volume consumption and measurement time compared to the previous system. By measuring two proteins with drastically different secondary structures, the results demonstrated that the measurements were highly repeatable (>99% repeatability by area of overlap) regardless of the well plate formats or the scan modes. The limit of quantitation (LOQ) for determining structural impurity using the sweep scan mode was 3.2% and significantly better than that of FTIR at 23% from previous studies. This work highlighted the advancement of MMS as a highly sensitive technique to detect small changes of protein structures due to aggregation or misfolding.

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.

State-of-the-art and emerging trends in analytical approaches to pharmaceutical-product commercialization.

The biopharmaceutical landscape continues to evolve rapidly, and associated modality complexity and the need to improve molecular understanding require concomitant advances in analytical approaches used to characterize and release the product. The Product Quality Attribute Assessment (PQAA) and Quality Target Product Profile (QTPP) frameworks help catalog and translate molecular understanding to process and product-design targets, thereby enabling reliable manufacturing of high-quality product. The analytical target profile forms the basis of identifying best-fit analytical methods for attribute measurement and continues to be successfully used to develop robust analytical methods for detailed product characterization as well as release and stability testing. Despite maturity across multiple testing platforms, advances continue to be made, several with the potential to alter testing paradigms. There is an increasing role for mass spectrometry beyond product characterization and into routine release testing as seen by the progress in multi-attribute methods and technologies, applications to aggregate measurement, the development of capillary zone electrophoresis (CZE) coupled with mass spectrometry (MS) and capillary isoelectric focusing (CIEF) with MS for measurement of glycans and charged species, respectively, and increased application to host cell protein measurement. Multitarget engaging multispecific modalities will drive advances in bioassay platforms and recent advances both in 1- and 2-D NMR approaches could make it the method of choice for characterizing higher-order structures. Additionally, rigorous understanding of raw material and container attributes is necessary to complement product understanding, and these collectively can enable robust supply of high-quality product to patients.

Advancing secondary structure characterization of monoclonal antibodies using Microfluidic Modulation Spectroscopy.

Infrared (IR) spectroscopy is rapidly gaining traction for monitoring biotherapeutic critical quality attributes. Microfluidic Modulation Spectroscopy (MMS), a novel automated IR technology, has been shown to be an effective technique for generating high quality, reproducible secondary structure data for protein therapeutics including monoclonal antibodies. In this study, monoclonal antibodies (mAbs) at concentrations ranging from 0.5 to 50 mg/mL were analyzed and high-quality data was obtained by optimizing two critical acquisition parameters (a) sample modulation frequency and (b) detector dwell time settings. The ability to generate reproducible data with high sensitivity at low formulation concentrations indicates that MMS is a reliable method for evaluating the secondary structure of low concentration biotherapeutic formulations and modalities.

A Mass Spectrometric Characterization of Light-Induced Modifications in Therapeutic Proteins.

During the development of a therapeutic protein, its quality attributes that pertain to the primary structure must be appropriately characterized, commonly by LC-MS/MS peptide mapping experiments. Extracting attribute information from LC-MS/MS data requires knowledge of the attribute of interest. Therefore, it is important to understand all potential modifications on the therapeutic proteins. In this work, we performed UV and visible light irradiation experiments on several therapeutic proteins, with or without the presence of a photosensitizer. Light-induced modifications were detected and characterized by tryptic digestion followed by LC-MS/MS analysis. A list of potential light-induced modifications, with their respective mass changes, was obtained. These modifications are primarily on methionine, tryptophan, histidine, cysteine, tyrosine and phenylalanine residues. Many of these modifications have not been previously reported on therapeutic proteins. Our findings therefore provide a database of potential light-induced modifications that would enable the routine characterization of light-induced modifications on therapeutic proteins.

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.

Principal Component Analysis of 1D 1H Diffusion Edited NMR Spectra of Protein Therapeutics.

The one-dimensional (1D) diffusion edited proton NMR method, Protein Fingerprint by Lineshape Enhancement (PROFILE) has been demonstrated to be suitable for higher order structure (HOS) characterization of protein therapeutics including monoclonal antibodies. Recent reports in the literature have demonstrated its advantages for HOS characterization over traditional methods such as circular dichroism and Fourier-transform infrared spectroscopy. Previously, we have demonstrated that the PROFILE method is complementary with high resolution 2D methyl correlated NMR methods and how both may be deployed as a multi-modal platform to further the utility of NMR for HOS characterization. A major limitation of the PROFILE method remains its need for high signal to noise data due to its reliance on convolution difference processing and linear correlation metrics to assess spectral similarity. Here we present an alternative method for analyzing 1D diffusion edited spectra, which overcomes this limitation by using nonlinear iterative partial least squares (NIPALS) principal component analysis, and which we dub PROtein Fingerprint Observed Using NIPALS Decomposition (PROFOUND). We demonstrate that results from the PROFOUND method are robust with respect to instrument, operator and in the presence of high experimental noise and how it may be employed to provide quantitative assessment of spectral similarity.

Use of the 2D 1H-13C HSQC NMR Methyl Region to Evaluate the Higher Order Structural Integrity of Biopharmaceuticals.

The higher-order structure (HOS) of protein therapeutics is directly related to the function and represents a critical quality attribute. Currently, the HOS of protein therapeutics is characterized by methods with low to medium structural resolution, such as Fourier transform infrared (FTIR), circular dichroism (CD), intrinsic fluorescence spectroscopy (FLD), and differential scanning calorimetry (DSC). High-resolution nuclear magnetic resonance (NMR) methods have now been introduced, representing powerful approaches for HOS characterization (HOS by NMR). NMR is a multi-attribute method with unique abilities to give information on all structural levels of proteins in solution. In this study, we have compared 2D 1H-13C HSQC NMR with two established biophysical methods, i.e., near-ultraviolet circular dichroism (NUV-CD) and intrinsic fluorescence spectroscopy, for the HOS assessments for the folded and unfolded states of two monoclonal antibodies belonging to the subclasses IgG1 and IgG2. The study shows that the methyl region of the 1H-13C HSQC NMR spectrum is sensitive to both the secondary and tertiary structure of proteins and therefore represents a powerful tool in assessing the overall higher-order structural integrity of biopharmaceutical molecules.

Vaccine-induced, but not natural immunity, against the Streptococcal inhibitor of complement protects against invasive disease.

Highly pathogenic emm1 Streptococcus pyogenes strains secrete the multidomain Streptococcal inhibitor of complement (SIC) that binds and inactivates components of the innate immune response. We aimed to determine if naturally occurring or vaccine-induced antibodies to SIC are protective against invasive S. pyogenes infection. Immunisation with full-length SIC protected mice against systemic bacterial dissemination following intranasal or intramuscular infection with emm1 S. pyogenes. Vaccine-induced rabbit anti-SIC antibodies, but not naturally occurring human anti-SIC antibodies, enhanced bacterial clearance in an ex vivo whole-blood assay. SIC vaccination of both mice and rabbits resulted in antibody recognition of all domains of SIC, whereas naturally occurring human anti-SIC antibodies recognised the proline-rich region of SIC only. We, therefore, propose a model whereby natural infection with S. pyogenes generates non-protective antibodies against the proline-rich region of SIC, while vaccination with full-length SIC permits the development of protective antibodies against all SIC domains.

Determination of the experimental extinction coefficient of therapeutic proteins using the Edelhoch method.

The objective of this study was to determine how the theoretical values of the extinction coefficient (EC) compares to the experimentally determined extinction coefficient for a large set of biotherapeutic proteins measured by the Edelhoch method. We have performed extensive analysis based on over 176 observations covering 19 different types of molecules from different structural classes covering mAbs, bispecific antibodies, fusion proteins and BiTE molecules. Precision was measured by assessing the repeatability of the measurements for each molecule and determining the relative standard deviation (%RSD). The maximum RSD observed for any given molecule was 1.7% with an average RSD of 0.9%. Deviation from the theoretical extinction coefficient was determined by calculating the experimental bias first, which is the difference between the mean experimental extinction coefficient and the theoretical extinction coefficient. The percent bias (%bias) was then calculated as (bias ÷ theoretical EC) × 100. The maximum %bias observed for any given molecule was 5.3% with an average %bias of 2.6%. Our results indicate that the Edelhoch method is highly reliable with significant improvement in execution efficiency with reduction in cost, time and improvements in safety when compared to the commonly used methods such as amino acid analysis (AAA) technique.

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.

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.

Comparative Analysis of One-Dimensional Protein Fingerprint by Line Shape Enhancement and Two-Dimensional 1H,13C Methyl NMR Methods for Characterization of the Higher Order Structure of IgG1 Monoclonal Antibodies.

The use of NMR spectroscopy has emerged as a premier tool to characterize the higher order structure of protein therapeutics and in particular IgG1 monoclonal antibodies (mAbs). Due to their large size, traditional 1H-15N correlation experiments have proven exceedingly difficult to implement on mAbs, and a number of alternative techniques have been proposed, including the one-dimensional (1D) 1H protein fingerprint by line shape enhancement (PROFILE) method and the two-dimensional (2D) 1H-13C methyl correlation-based approach. Both 1D and 2D approaches have relative strengths and weaknesses, related to the inherent sensitivity and resolution of the respective methods. To further increase the utility of NMR to the biopharmaceutical community, harmonized criteria for decision making in employing 1D and 2D approaches for mAb characterization are warranted. To this end, we have conducted an interlaboratory comparative study of the 1D PROFILE and 2D methyl methods on several mAbs samples to determine the degree to which each method is suited to detect spectral difference between the samples and the degree to which results from each correlate with one another. Results from the study demonstrate both methods provide statistical data highly comparable to one another and that each method is capable of complementing the limitations commonly associated with the other, thus providing a better overall picture of higher order structure.

Nano differential scanning fluorimetry for comparability studies of therapeutic proteins.

Differential scanning calorimetry (DSC) has been extensively used in the biopharmaceutical industry to characterize protein thermal stability and domain folding integrity. Recently, nano differential scanning fluorimetry (nanoDSF) has emerged as a powerful tool for thermal stability analysis and studies of protein domain unfolding. Due to increased interests in the qualification of characterization methods, we are in this study presenting the qualification results for the comparability studies of thermal stability analysis using nanoDSF. The results show that nanoDSF is able to detect thermal transition signals for mAbs, BiTE® molecules, and cytokines at a wide concentration range with high precision, clearly indicating that nanoDSF is suitable for characterization including comparability studies of therapeutic proteins. Compared to the current recognized industry standard DSC, the nanoDSF method enables thermal stability analysis over a much wider concentration range, consumes considerably less materials, and provides significantly higher throughput.

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.

A quantitative Streptococcus pyogenes-human protein-protein interaction map reveals localization of opsonizing antibodies.

A fundamental challenge in medical microbiology is to characterize the dynamic protein-protein interaction networks formed at the host-pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein-protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host-pathogen interactions involving other bacterial species.

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.

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.

Streptococcal inhibitor of complement (SIC) modulates fibrinolysis and enhances bacterial survival within fibrin clots.

Some strains of the bacterial pathogen Streptococcus pyogenes secrete protein SIC (streptococcal inhibitor of complement), including strains of the clinically relevant M1 serotype. SIC neutralizes the effect of a number of antimicrobial proteins/peptides and interferes with the function of the host complement system. Previous studies have shown that some S. pyogenes proteins bind and modulate coagulation and fibrinolysis factors, raising the possibility that SIC also may interfere with the activity of these factors. Here we show that SIC interacts with both human thrombin and plasminogen, key components of coagulation and fibrinolysis. We found that during clot formation, SIC binds fibrin through its central region and that SIC inhibits fibrinolysis by interacting with plasminogen. Flow cytometry results indicated that SIC and plasminogen bind simultaneously to S. pyogenes bacteria, and fluorescence microscopy revealed co-localization of the two proteins at the bacterial surface. As a consequence, SIC-expressing bacteria entrapped in clots inhibit fibrinolysis, leading to delayed bacterial escape from the clots as compared with mutant bacteria lacking SIC. Moreover, within the clots SIC-expressing bacteria were protected against killing. In an animal model of subcutaneous infection, SIC-expressing bacteria exhibited a delayed systemic spread. These results demonstrate that the bacterial protein SIC interferes with coagulation and fibrinolysis and thereby enhances bacterial survival, a finding that has significant implications for S. pyogenes virulence.