Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics

Kaltashov, Igor A.; Bobst, Cedric E.; Abzalimov, Rinat R.; Wang, Guanbo; Baykal, Burcu; Wang, Shunhai

doi:10.1016/j.biotechadv.2011.05.006

Cited by 132 publications

(87 citation statements)

References 100 publications

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“…However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10,11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14,15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities.…”

mentioning

confidence: 99%

Origin and prediction of free-solution interaction studies performed label-free

Bornhop

Kammer

Kussrow

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for freesolution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R 2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.backscattering interferometry | assay methodology | molecular interactions | conformation change | hydration change C ontemporary assays enabling single-molecule detection (1,2) have accelerated the sequencing of the human genome (3) and facilitated imaging with extraordinary resolution without labels (4). To most closely approximate the natural state, an interaction assay methodology would interrogate the processes (reaction, molecular interaction, protein folding event, etc.) without perturbation. Label-free chemical and biochemical investigations (5, 6) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (7,8). However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10, 11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14, 15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities. Another increasingly popular free-solution approach is microscale thermophoresis (...

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mentioning

confidence: 99%

Origin and prediction of free-solution interaction studies performed label-free

Bornhop

Kammer

Kussrow

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Chromatography and mass spectrometry (MS)-based techniques for characterizing primary structure of mAbs have been well established and applied to study common posttranslational modifications such as phosphorylation, oxidation, or glycosylation, as well as sequence variation. 10,11 Understanding secondary and tertiary structures of these therapeutic mAbs is also important because their activity and stability are considerably influenced by their conformation. In addition, characterization of such proteins and their antigen complexes is essential to define their epitopes, which represent fundamental elements of intellectual property protection for companies.…”

Section: Introductionmentioning

confidence: 99%

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

Kaur

Tomechko

Kiselar

et al. 2014

mAbs

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Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs. IntroductionMethods to describe structures of proteins and their higher order complexes continue to evolve and various advances have increased speed, efficiency, and resolution of structure determination.1 Most proteins behave according to the functiondictated-by-structure principle, and their proper conformation is central to their role in processes such as enzyme catalysis, cell signaling, or ligand binding. [2][3][4] Protein drugs (biologics) are one of the most important and fastest growing classes of drugs in the commercial marketplace today. The function and efficacy...

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“…These typical studies include antibody primary structure by chemical sequencing (Edman degradation) as well as peptide mapping by means of ESI-MS and MALDI-MS [26][27][28]; higher order structure by RP-HPLC-ESI-MS, Ellman's assay CD, FTIR, hydrogen deuterium exchange (HDX)-MS and X-ray [28,29]; post-translational modifications (PTM) and charge variants by MSion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), capillary electrophoresis (CE) and isoelectric focusing (IEF) [30]; glycan profile and variants and size heterogeneity by HPLC-fluorescence, size-exclusion chromatography (SEC), native gel, capillary electrophoresis, RP-HPLC-MS and native intact MS [31], as well as solubility measurement by ultrafiltration or PEG-induced precipitation methods [32,33]. Moreover, the immunogenicity should be evaluated for both antibody and ADCs [34,35], but unnecessary for SMs.…”

Section: General Differences Between Sms and Lms (Mab And Adc) In Admmentioning

confidence: 99%

An Overall Comparison of Small Molecules and Large Biologics in ADME Testing

Wan

2016

ADMET DMPK

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Biologics mainly monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as new therapeutics are becoming increasingly important biotherapeutics. This review is intended to provide an overall comparison between small molecules (SMs) and biologics or large molecules (LMs) concerning drug metabolism and pharmacokinetic (DMPK) or associated with absorption, distribution, metabolism and elimination (ADME) testing from pharmaceutical industry drug discovery and development points of view, which will help design and conduct relevant ADME testing for biologics such as mAbs and ADCs. Recent advancements in the ADME for testing biologics and related bioanalytical methods are discussed with an emphasis on ADC drug development as an example to understand its complexity and challenges from extensive in vitro characterization to in vivo animal PK studies. General non-clinical safety evaluations of biologics in particular for ADC drugs are outlined including drug-drug interaction (DDI)and metabolite/catabolite assessments. Regulatory guidance on the ADME testing and safety evaluations including immunogenicity as well as bioanalytical considerations are addressed for LMs. In addition, the preclinical and human PK data of two marked ADC drugs (ADCETRIS, as examples are briefly discussed with regard to PK considerations and PK/PD perspectives.

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Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics

Cited by 132 publications

References 100 publications

Origin and prediction of free-solution interaction studies performed label-free

Origin and prediction of free-solution interaction studies performed label-free

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

An Overall Comparison of Small Molecules and Large Biologics in ADME Testing

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