Stabilization of adalimumab Fab through the introduction of disulfide bonds between the variable and constant domains.

Fab is a promising format for antibody drug. Therefore, efforts have been made to improve its thermal stability for therapeutic and commercial use. So far, we have attempted to introduce a disulfide bond into the Fab fragment to improve its thermal stability and demonstrated that it is possible to do this without sacrificing its biochemical function. In this study, to develop a novel stabilization strategy for Fab, we attempted to introduce a disulfide bond between the variable and constant domains and prepared three variants of Fab; H:G10C + H:P210C, L:P40C + L:E165C, and H:G10C + H:P210C + L:P40C + L:E165C. Differential scanning calorimetry measurements showed that each of these variants had improved thermal stability. In addition, the variants with two disulfide bonds demonstrated a 6.5 °C increase in their denaturation temperatures compared to wild-type Fab. The introduction of disulfide bonds was confirmed by X-ray crystallography, and the variants retained their antigen-binding activity. The variants were also found to be less aggregative than the wild type. Our results demonstrate that the introduction of a disulfide bond between the variable and constant domains significantly improves the thermal stability of Fab.

Demonstration of Physicochemical and Functional Similarity of Biosimilar Adalimumab-aqvh to Adalimumab.

BACKGROUND: Adalimumab-aqvh/CHS-1420 (YUSIMRYTM) (hereafter referred to as adalimumab-aqvh) was recently approved by the US Food and Drug Administration as a biosimilar for adalimumab. OBJECTIVE: The current study was conducted to investigate the analytical similarity of adalimumab-aqvh and the reference product, adalimumab. METHODS: The structural, functional, and stability attributes of adalimumab-aqvh and adalimumab were compared using state-of-the-art assays. The primary structure, disulfide structure, glycan profile, secondary and tertiary structures, molar mass, size variants, free thiol, charge variants, hydrophobic variants, post-translational modifications, subvisible particles, host cell proteins, and protein concentration were investigated. The functional similarity between adalimumab-aqvh and adalimumab was demonstrated by comparing fragment antigen-binding (Fab)-associated and fragment crystallizable (Fc)-associated biological activities. The stability of adalimumab-aqvh and of adalimumab was compared through forced degradation. RESULTS: The structural attributes of adalimumab-aqvh were identical to those of adalimumab or met the similarity criteria, with a few exceptions. Adalimumab-aqvh and adalimumab exhibited comparable stability profiles and functional activities. Any observed differences in the physiochemical attributes did not impact the conclusion of similarity because they did not influence any functional activities related to the adalimumab mechanism of action. CONCLUSION: The structural, functional, and stability data provide convincing evidence of biosimilarity between adalimumab-aqvh and the reference product, adalimumab.

Cation exchange chromatography on a monodisperse 3 µm particle enables extensive analytical similarity assessment of biosimilars.

Biosimilarity assessment requires extensive characterization and comparability exercises to investigate product quality attributes of an originator product and its potential biosimilar(s) and to highlight any differences between them. Performing a thorough comparison allows a shortened approval path, which also eliminates lengthy and expensive clinical trials, ensuring comparable product quality and efficacy but at lower drug prices. The wide variety of analytical methods available for biosimilar assessment ranges from biological to analytical assays, each providing orthogonal information to fully characterize biosimilar candidates. Intact native mass spectrometry (MS) has been shown to be an excellent tool for detection and monitoring of important quality attributes such as N-glycosylation, deamidation, sequence truncation and higher order structures. When combined with efficient upfront separation methods, simplification of the proteoform heterogeneity and associated complexity prior to MS analysis can be achieved. Native mass spectrometry can provide robust and accurate results within short analysis times and requires minimal sample preparation. In this study we report the use of a monodisperse strong cation exchange chromatography phase hyphenated with Orbitrap mass spectrometry (SCX-MS) to compare the best-selling biopharmaceutical product Humira® with 7 commercially approved biosimilar products. SCX-MS analysis allowed for the identification of previously described as well as so far unreported proteoforms and their relative quantitation across all samples, revealing differences in N-glycosylation and lysine truncation, as well as unique features for some products such as sialylation and N-terminal clipping. SCX-MS analysis, powered by a highly efficient separation column, enabled deep and efficient analytical comparison of biosimilar products.

Multi-attribute method (MAM) to assess analytical comparability of adalimumab biosimilars.

Adalimumab drug product (Humira ®), the first fully human monoclonal antibody (mAb) approved by FDA in 2002, led the top ten list of best-selling mAbs in 2018 and has been the most profitable drug in the world. With the expiration of patent protection in Europe in 2018 and in United States by 2023, the landscape is changing as up to 10 adalimumab biosimilars are expected to enter the market in the US. Biosimilars offer the potential to lower costs on health care systems and increase patient accessibility. The analytical similarity of seven different adalimumab biosimilars was accomplished in the present study using the multi-attribute method (MAM), a LC-MS based peptide mapping technique that allows for primary sequence assessment and evaluation of multiple quality attributes including deamidation, oxidation, succinimide formation, N- and C- terminal composition and detailed N-glycosylation analysis. In the first step, characterization of the most relevant post-translational modifications of a reference product was attained during the discovery phase of MAM. During the second step, as part of the MAM targeted monitoring phase, adalimumab batch-to batch variability was evaluated to define statistical intervals for the establishment of similarity ranges. The third step describes biosimilarity evaluation of predefined quality attributes and new peak detection for the assessment of any new or modified peak compared to the reference product. This study highlights a new perspective of the MAM approach and its underlying power for biotherapeutic comparability exercises in addition to analytical characterization. MAM offers a streamlined comparability assessment workflow based on high-confidence quality attribute analysis using high-resolution accurate mass mass spectrometry (HRAM MS) and the capability to detect any new or modified peak compared to the reference product.

Long-term data on the proposed adalimumab biosimilar BCD-057 in patients with moderate to severe psoriasis: A randomized controlled trial.

INTRODUCTION: The objective of this study was to demonstrate that BCD-057 is similar to innovator adalimumab (iADA) in terms of efficacy, safety, and pharmacokinetics in steady state in the target population of patients with moderate to severe plaque psoriasis (NCT02762955). METHODS: Patients were randomized in 1:1 ratio to receive 80 mg of BCD-057 or iADA at week 0 and 40 mg thereafter every other week from week 1. At week 24 patients from iADA group were re-randomized (1:1) to continue iADA or to be switched to BCD-057. The primary efficacy endpoint was 75% improvement in Psoriasis Area and Severity Index from baseline (PASI 75), secondary endpoints included PASI percent improvement and relative change in affected Body Surface Area (BSA) from baseline at weeks 16, 24, 33, and 55. Safety was assessed through monitoring of adverse events (AEs) and antidrug antibodies. Pharmacokinetics was evaluated at steady state. RESULTS: Overall, 346 adult patients were included in the study (174/172 patients in BCD-057/iADA arms, respectively). At week 16 PASI 75 was achieved by 60.34% and 63.37% of patients in BCD-057 and iADA arms, respectively (p = 0.5622). Bounds of the calculated 95% confidence interval (CI) for the difference between PASI 75 responses in arms [-13.26%; 7.2%] fall within the equivalence margin [-15% to 15%] demonstrating equivalent efficacy of BCD-057 and iADA. At week 55 81.61%, 85.56%, and 80.49% of patients in BCD-057, iADA and iADA/BCD-057 arms achieved PASI 75. Comparison of the secondary endpoints did not show significant differences between arms. A comparable pharmacokinetics was shown at steady state. Safety profiles and proportions of patients with antidrug antibodies were similar between arms. The switch from the iADA to BCD-057 did not affect the immunogenicity profile. CONCLUSION: Obtained data demonstrate that BCD-057 and iADA are highly similar in clinical efficacy, pharmacokinetics, safety, and immunogenicity in patients with moderate to severe plaque psoriasis.

Physicochemical and Functional Characterization of HS016, a Biosimilar of Adalimumab (Humira).

The characterization of a biosimilar drug HS016, the reference product adalimumab (Humira), and their biosimilarities were determined using physical chemistry and functional similarity tests. The primary and higher order structures, size and charge variants, glycosylation profiles, and in vitro potency of both antibodies were characterized both for unstressed and stability samples. Slight differences were observed in the relative levels of methionine oxidation, low molecular weight components, terminal lysine variant, high mannoses and galactosylated glycans between HS016 and Humira. However, no differences in antigen binding activity, Fc receptor affinity, antibody-dependent cell-mediated cytotoxicity or complemented-dependent cytotoxicity were found. The primary and higher order structures, physicochemical properties, and biological activity of HS016 and adalimumab were similar.

A novel method for the storage and transport of biological samples of therapeutic proteins prior to the detection of analytes using ELISA.

Therapeutic proteins have exhibited promising clinical applications in the diagnosis and treatment of some diseases. Prior to the detection of analytes using enzyme-linked immunosorbent assay, biological samples of therapeutic proteins are conventionally frozen at temperatures ranging from - 20 to - 80 °C to increase the stability of analytes. However, therapeutic proteins destabilization and aggregation may occur during the frozen storage or the freeze-thawing step. In this work, an effective method was proposed to freeze-dry therapeutic protein samples to allow subsequent storage or transport of samples without freezing them. This new method was validated with quality control samples of adalimumab and etanercept, and it was also used in the bioanalysis of adalimumab and etanercept in pharmacokinetic (PK) studies. Adalimumab and etanercept were stable for 14 days at 4 °C after being prepared and stored using the new method, with detection that was accurate and repeatable. Studies of adalimumab and etanercept in animals and humans showed that the PK parameters of the analytes stored with the new method were consistent with those of analytes stored using the conventional method. This effective method will be attractive for facilitating the storage and transport of plasma samples containing therapeutic proteins.

Size-based characterization of adalimumab and TNF-α interactions using flow induced dispersion analysis: assessment of avidity-stabilized multiple bound species.

The understanding and characterization of protein interactions is crucial for elucidation of complicated biomolecular processes as well as for the development of new biopharmaceutical therapies. Often, protein interactions involve multiple binding, avidity, oligomerization, and are dependent on the local environment. Current analytical methodologies are unable to provide a detailed mechanistic characterization considering all these parameters, since they often rely on surface immobilization, cannot measure under biorelevant conditions, or do not feature a structurally-related readout for indicating formation of multiple bound species. In this work, we report the use of flow induced dispersion analysis (FIDA) for in-solution characterization of complex protein interactions under in vivo like conditions. FIDA is an immobilization-free ligand binding methodology employing Taylor dispersion analysis for measuring the hydrodynamic radius (size) of biomolecular complexes. Here, the FIDA technology is utilized for a size-based characterization of the interaction between TNF-α and adalimumab. We report concentration-dependent complex sizes, binding affinities (Kd), kinetics, and higher order stoichiometries, thus providing essential information on the TNF-α-adalimumab binding mechanism. Furthermore, it is shown that the avidity stabilized complexes involving formation of multiple non-covalent bonds are formed on a longer timescale than the primary complexes formed in a simple 1 to 1 binding event.

Fucosylation and Sialylation of Fc-Fragment of anti-Tumour Necrosis Factor Alpha Antibodies do not Influence Their Immunogenicity in Monocyte-Derived Dendritic Cells.

BACKGROUND AND AIMS: Antibodies targeting tumor necrosis factor-alpha [TNF-alpha] are a mainstay in the treatment of inflammatory bowel disease. However, they fail to demonstrate efficacy in a considerable proportion of patients. On the other hand, glycosylation of antibodies might influence not only their immunogenicity but also their structure and function. We investigated whether specific glycosylation patterns of the Fc-fragment would affect the immunogenicity of anti-TNF-alpha antibody in monocyte-derived dendritic cells. METHODS: The effect of a specific Fc-glycosylation pattern on antibody uptake by monocyte-derived dendritic cells [mo-DCs] and how this process shapes the immunologic profile of mo-DCs was investigated. Three N-glycoforms of the anti-TNF-alpha antibody adalimumab, that differed in the content of fucose or sialic acid, were tested: [1] mock treated Humira, abbreviated 'Fuc-G0', where the N-glycan mainly consist of fucose and N-acetylglucosamine [GlcNAc], without sialic acid; [2] 'Fuc-G2S1/G2S2' with fucose and alpha 2,6 linked sialic acid; and [3] 'G2S1/G2S2' with alpha 2,6 linked sialic acid, without fucose. RESULTS: Our data demonstrated that neither fucosylation nor sialylation of anti-TNF-Abs [Fuc-G0, FucG2S1/G2S2, G2S1/G2S2] influence their uptake by mo-DCs. Additionally, none of the differentially glycosylated antibodies altered CD80, CD86, CD273, CD274 levels on mo-DCs stimulated in with lipopolysaccharide in the presence of antibodies. Next, we evaluated the levels of cytokines in the supernatant of mo-DCs stimulated with lipopolysaccharide in the presence of Fuc-G0, Fuc-G2S1/G2S2 or G2S1/G2S2-glycosylated anti-TNF antibodies. Only IL-2 and IL-17 levels were downregulated, and IL-5 production was upregulated by uptake of Fuc-G0 antibodies, as compared to control without antibodies. CONCLUSIONS: The specific modification in the Fc-glycosylation pattern of anti-TNF-alpha Abs does not affect their immunogenicity under the tested conditions. As this study was limited to mo-DCs, further investigation is required to clarify whether Ab uptake into mo-DCs might change the immunological profile of T- and B-cells, in order to ultimately reduce the formation of anti-drug antibodies and to improve the patient care.

Comparison of consistency and complementarity of reporting biosimilar quality attributes between regulatory and scientific communities: An adalimumab case study.

Biosimilar approval relies on the comparability of quality attributes (QAs), for which information can be derived from regulatory or scientific communities. Limited information is known about whether these sources are consistent with or complementary to each other. The consistency and complementarity of QA reporting in biosimilarity assessments for adalimumab biosimilars approved by the European Medicines Agency in European public assessment reports (EPARs) and scientific publications was assessed. A classification of 77 different QAs (53 structural and 24 functional attributes) was used to assess the types of and information on QAs reported. Six adalimumab biosimilars were analyzed, for which the number of QAs reported in EPARs and publications varied (range = 47 [61%]-60 [78%]). The proportion of QAs consistently reported in both sources varied (range = 28%-75%) among biosimilars; functional QAs (mean = 21 QAs [88%]; range = 19-23) were more consistently reported than structural QAs (mean = 33 QAs [62%]; range = 27-34). The EPARs frequently reported biosimilarity interpretation without providing test results (9-57 QAs in EPARs versus 0-8 QAs in publications), whereas publications frequently reported both test results and interpretations (13-40 QAs in publications versus 0-3 QAs in EPARs). Both sources provided information on the biosimilarity of QAs in a complementary manner and the same biosimilarity interpretation of test results for reported QAs (mean = 90%; range = 78%-100%), with a small discrepancy in biosimilarity interpretations of a few clinically relevant QAs related to post-translation modifications and biological activity. Comprehensive reporting of QAs can contribute to an improved understanding of the role of structural and functional attributes in establishing biosimilarity and the mechanism of action of biological substances in general.

Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry.

The biopharmaceutical industry continues to develop mAb-based biotherapeutics in increasing numbers. Due to their complexity, there are several critical quality attributes (CQAs) that need to be measured and controlled to guarantee product safety and efficacy. Charge variant analysis is a widely used method to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs) and, together with a bottom-up peptide centred approach, acts as a key analytical platform to fulfil regulatory requirements. Native MS measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact proteins such as mAbs. The resulting native mass spectrum of a mAb is characterized by a narrower charge-state envelope that simplifies the spectra and also condenses the ion signals into fewer peaks, increasing the signal-to-noise ratio. Algorithmic spectral deconvolution is needed for routine accurate and rapid molecular weight determination, and consequently, multiple deconvolution algorithms have evolved over the past decade. Here, we demonstrate the utility of the sliding window algorithm as a robust and powerful deconvolution tool for comprehensive characterisation of charge variant analysis data for mAbs. Optimum performance is evaluated by studying the impact of critical software parameters on detection, identification and relative quantitation of protein isoforms. By combining molecular mass and retention time information, it was possible to identify multiple modifications on adalimumab and trastuzumab, both IgG1 mAbs, including lysine truncation, deamidation and succinimide formation, along with the N-glycan distribution of each of the identified charge variants. Sliding window deconvolution also provides a key benefit of low abundant variant detection in a single analysis and the ability to detect co-eluting components with different relative abundances. The studied mAbs demonstrate the algoritms applicability for efficient data processing of both simple and complex mAbs analysed using pH gradient cation exchange chromatography coupled to native mass spectrometry.

New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept.

TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9 M and >10-8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation.

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.

Physicochemical analysis and biological characterization of FKB327 as a biosimilar to adalimumab.

FKB327 was approved by the European Medicines Agency as a biosimilar to European-authorized adalimumab (Humira® ; AbbVie Inc). Adalimumab is a monoclonal antibody, binding and inhibiting tumor necrosis factor (TNF)-α with use indicated for several immune-mediated, chronic, and inflammatory disorders. The approval is based on high similarity in the physicochemical properties between FKB327 and adalimumab. The objective of this study is to assess the biological similarity, with regard to Fab- and Fc-associated functions, and describe the relationship between physicochemical and biological characterization and functional activity. State-of-the-art orthogonal techniques were implemented to assess the structure and function of FKB327. Peptide mapping with liquid chromatography and mass spectrometry, capillary electrophoresis-sodium dodecyl sulfate, ultraviolet circular dichroism, size-exclusion high-performance liquid chromatography (HPLC), and cation exchange HPLC were the techniques used to assess structure. Functional activity was assessed with enzyme-linked immunosorbent assay, surface plasmon resonance, and cell-based assays. The polypeptide sequence of FKB327 was identical to that of adalimumab. FKB327 also was demonstrated to have a similar secondary and tertiary structure to adalimumab. Posttranslational heterogeneities, along with size and charge variants, were not clinically meaningful. FKB327 binds to TNF-α, FcγR, the neonatal Fc receptor, and C1q, and induces apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. The binding and activity of FKB327 were similar to that of adalimumab. FKB327 shares similar structure and activity with adalimumab. Based on characterization of physicochemical and biological properties, FKB327 is expected to have a similar safety, immunogenicity, and efficacy profile to adalimumab.

Identification and Quantification of Oxidation Products in Full-Length Biotherapeutic Antibodies by NMR Spectroscopy.

Therapeutic proteins are an indispensable class of drugs and often therapeutics of last resort. They are sensitive to oxidation, which is of critical concern, because it can affect drug safety and efficacy. Protein oxidation, with methionine and tryptophan as the most susceptible moieties, is mainly monitored by HPLC-MS techniques. However, since several oxidation products display the same mass difference, their identification by MS is often ambiguous. Therefore, an alternative analytical method able to unambiguously identify and, ideally, also quantify oxidation species in proteins is highly desired. Here, we present an NMR-based approach to monitor oxidation in full-length proteins under denaturing conditions, as demonstrated on two biotherapeutic monoclonal antibodies (mAbs). We show that methionine sulfoxide, methionine sulfone, N-formylkynurenine, kynurenine, oxindolylalanine, hydroxypyrroloindole, and 5-hydroxytryptophan result in characteristic chemical shift correlations suited for their identification and quantification. We identified the five most abundant oxidation products in forced degradation studies of two full-length therapeutic mAbs and can also unambiguously distinguish oxindolylalanine from 5-hydroxytryptophan, which are undistinguishable by MS due to the same mass shift. Quantification of the abundant methionine sulfoxide by NMR and MS gave highly comparable values. These results underline the suitability of NMR spectroscopy for the identification and quantification of critical quality attributes of biotherapeutics.

Quality by Design-Based Assessment for Analytical Similarity of Adalimumab Biosimilar HLX03 to Humira®.

Quality by design (QbD) is an efficient but challenging approach for the development of biosimilar due to the complex relationship among process, quality, and efficacy. Here, the analytical similarity of adalimumab biosimilar HLX03 to Humira® was successfully established following a QbD quality study. Quality target product profile (QTPP) of HLX03 was first generated according to the public available information and initial characterization of 3 batches of Humira®. The critical quality attributes (CQAs) were then identified through risk assessment according to impact of each quality attribute on efficacy and safety. The anticipated range for each CQA was derived from similarity acceptance range and/or the corresponding regulatory guidelines. Finally, a panel of advanced and orthogonal physicochemical and functional tests and comparison of 6 batches of HLX03 and 10 batches of the reference standard demonstrated high similarity of HLX03 to Humira®, except for slightly lower percentage of high mannosylated glycans (%HM) in HLX03 which had no effect on FcγRIII binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity in human peripheral blood mononuclear cell (PBMC). All above demonstrated the feasibility and efficiency of QbD-based similarity assessment of a biosimilar monoclonal antibody (mAb).

Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting.

Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM-MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab')2 and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab')2 domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab')2 regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab')2 and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab')2 domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.

Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions.

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)2 fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8-10.5 with PL 3-10 for variants of intact rituximab and of F(ab´)2 fragments, respectively, whereas PL 6.7-7.7 in combination with PL 3-10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)2 . In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.

Comparative nonclinical assessments of the biosimilar PF-06410293 and originator adalimumab.

Adalimumab, a recombinant fully human monoclonal antibody targeting tumor necrosis factor (TNF), is approved in the United States and Europe to treat various inflammatory and autoimmune indications. Biosimilars are approved biologics highly similar, but not identical, to approved biotherapeutics. To support clinical development of PF-06410293, an adalimumab biosimilar, nonclinical studies evaluated the structural, functional, toxicologic, and toxicokinetic similarity to originator adalimumab sourced from the United States (adalimumab-US) and European Union (adalimumab-EU). Structural similarity was assessed by peptide mapping. Biologic activity was measured via inhibition of TNF-induced apoptosis and Fc-based functionality assessments. In vivo nonclinical similarity was evaluated in a toxicity study in cynomolgus monkeys administered subcutaneous PF-06410293 or adalimumab-EU (0 or 157 mg/kg/week). Peptide mapping demonstrated PF-06410293, adalimumab-US, and adalimumab-EU had identical amino acid sequences. Comparative functional and binding assessments were similar. Effects of PF-06410293 and adalimumab-EU were similar and limited to pharmacologically mediated decreased cellularity of lymphoid follicles and germinal centers in spleen. Toxicokinetics were similar; maximum plasma concentration and area-under-the-concentration-time curve ratio of PF-06410293:adalimumab-EU ranged from 1.0 to 1.2. These studies supported PF-06410293 entry into clinical development. Many regulatory agencies now only request nonclinical in vivo testing if there is residual uncertainty regarding biosimilarity after in vitro analytical studies.

Assessment of Antibody Self-Interaction by Bio-Layer-Interferometry as a Tool for Early Stage Formulation Development.

PURPOSE: To speed up the drug development process in the biopharmaceutical industry, high throughput methods are indispensable for assessing drug candidates and potential lead formulations, in particular during late stages of discovery and early phases of development. This study aimed to establish a bio-layer-interferometry based high throughput assay for assessing formulation dependent mAb self-interaction (SI-BLI) and to compare the results with kD values obtained by dynamic light scattering (DLS). METHODS: Self-interaction of proprietary and commercially available mAbs was analyzed by SI-BLI and dynamic light scattering (DLS). RESULTS: We found significant correlations of the SI-BLI results and kD-values obtained by DLS for both, different mAbs in one platform formulation and for mAbs formulated in several buffer compositions. In total, we assessed self-interaction propensity of different mAbs in 58 formulations and found significant Pearson correlation (p < 0.05) between kD and results of SI-BLI. CONCLUSIONS: The SI-BLI results correlate with kD and enable fast ranking of both different drug candidates and potential lead formulations. Thus, SI-BLI might decrease the risk to lose potent mAb candidates during transition from discovery to development, and help to accelerate the development of high concentration liquid formulations.