Biophysical Techniques for Characterizing the Higher Order Structure and Interactions of Monoclonal Antibodies

Gokarn, Yatin R.; Arthur, Kelly K.; Bepperling, Alexander; Day, Eric S.; Filoti, Dana I.; Greene, Daniel G.; Hayes, David; Kroe‐Barrett, Rachel; Laue, Thomas M.; Lin, Jasper C.; McGarry, Brian E.; Razinkov, Vladimir I.; Singh, Sanjaya; Taing, Rosalynn; Venkataramani, Sathyadevi; Weiss, William F.; Yang, Danlin; Zarraga, Isidro E.

doi:10.1021/bk-2015-1201.ch006

Cited by 28 publications

(32 citation statements)

References 54 publications

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“…Figure 5 f shows the second derivative FTIR spectrum of bevacizumab and its charge variants. The amide I frequency peaks corresponding to antiparallel β-sheet structure (1635 cm −1 and 1697 cm −1 ), intermolecular β-sheet (1617 cm −1 ), β-turns (1668 cm −1 and 1682 cm −1 ), and random coils (1650 cm −1 ) were chosen as per the literature, and a comparison was made between the charge variants and the unmodified bevacizumab product 30 . The main product's spectra, Basic variant 1, Basic variant 2, and acidic variants, were dominated by the band at the 1635 cm −1 , suggesting intramolecular native β-sheets as the major structural component.…”

Section: Resultsmentioning

confidence: 99%

LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

Singh

Kumar

Malani

et al. 2021

Sci Rep

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The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.

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Section: Resultsmentioning

confidence: 99%

LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

Singh

Kumar

Malani

et al. 2021

Sci Rep

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“…The thermodynamic nonideality term, BM, was constrained using the measured B (1 3 10 25 mL-mol/g 2 ) and M (144,000 g/mol) values from the NIST mAb. 38 In addition to k s , the meniscus position and the loading concentrations (c expected ) of the two components were allowed to float during the fitting process. In all cases, c fit /c expected 5 1.0 6 0.1, with one exception where c fit /c expected 5 0.8.…”

Section: Characterization Of Human Serum Iggs and Mab Iggsmentioning

confidence: 99%

Weak IgG self‐ and hetero‐association characterized by fluorescence analytical ultracentrifugation

et al. 2018

Self Cite

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Weak protein-protein interactions may be important to binding cooperativity. A panel of seven fluorescently labeled tracer monoclonal IgG antibodies, differing in variable (V) and constant (C) region sequences, were sedimented in increasing concentrations of unlabeled IgGs of identical, similar, and different backgrounds. Weak IgG::IgG attractive interactions were detected and characterized by global analysis of the hydrodynamic nonideality coefficient, k . The effects of salt concentration and temperature on k suggest the interactions are predominantly enthalpic in origin. The interactions were found to be variable in strength, affected by both the variable and constant regions, but indiscriminate with respect to IgG subclass. Furthermore, weak attractive interactions were observed for all the mAbs with freshly purified human poly-IgG. The universality of the weak interactions suggest that they may contribute to effector function cooperativity in the normal immune response, and we postulate that the generality of the interactions allows for a broader range of epitope spacing for complement activation. These studies demonstrate the utility of analytical ultracentrifuge fluorescence detection in measuring weak protein-protein interactions. It also shows the strength of global analysis of sedimentation velocity data by SEDANAL to extract hydrodynamic nonideality k to characterize weak macromolecular interactions.

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“…Methods to study the structural stability and aggregation profiles of proteins can be roughly categorized into three groups [47] , [48] , [49] : (i) separation methods such as electrophoresis, chromatography, or centrifugation; (ii) spectroscopy based methods such as fluorescence, absorbance, light-scattering, FTIR, MS, and NMR; and (iii) microscopy based methods such as TEM, SEM, AFM, and optical. Each technique has its advantages and disadvantages and there is no single method that would be appropriate for any given system since the stability and aggregation profile of any protein is a multifaceted problem.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…HPLC is the main method used in aggregation studies of proteins [49] , [56] . It can be operated in different modes; size-exclusion (SEC), ion-exchange, or hydrophobic-hydrophobic interaction chromatography.…”

Section: Experimental Methodsmentioning

confidence: 99%

Biobetters From an Integrated Computational/Experimental Approach

Kuyucak

Kayser

2017

Computational and Structural Biotechnology Journal

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Biobetters are new drugs designed from existing peptide or protein-based therapeutics by improving their properties such as affinity and selectivity for the target epitope, and stability against degradation. Computational methods can play a key role in such design problems—by predicting the changes that are most likely to succeed, they can drastically reduce the number of experiments to be performed. Here we discuss the computational and experimental methods commonly used in drug design problems, focusing on the inverse relationship between the two, namely, the more accurate the computational predictions means the less experimental effort is needed for testing. Examples discussed include efforts to design selective analogs from toxin peptides targeting ion channels for treatment of autoimmune diseases and monoclonal antibodies which are the fastest growing class of therapeutic agents particularly for cancers and autoimmune diseases.

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Biophysical Techniques for Characterizing the Higher Order Structure and Interactions of Monoclonal Antibodies

Cited by 28 publications

References 54 publications

LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

Weak IgG self‐ and hetero‐association characterized by fluorescence analytical ultracentrifugation

Biobetters From an Integrated Computational/Experimental Approach

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