Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison

Abstract

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter-and cross-platform measurement comparability and assess analytical performance as appropriate.

Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation

“…Protein structure is inherently a four-dimensional phenomenon, and the dynamic aspects of a protein can be just as important as the mostly static structures typically associated with conventional high-resolution structural biology methods, such as X-ray crystallography. In recent years, gas-phase methods, such as native mass spectrometry and ion mobility spectrometry, have been developed that lack the high resolution of crystallography, nuclear magnetic resonance, or cryo-electron microscopy, but can complement these methods such that the combination of different techniques yields a better understanding of the structural ensemble [1][2][3][4][5][6][7]. A benefit of MS-based methods is their near-universal applicability and relatively high throughput.…”

Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section

“…In samples with a lowly populated conformational state minimal differences that this adds to the bulk measurement may not be visible to the analyst but may be captured by MS methods, both protein centric as discussed in section as well as peptide centric such as hydrogen–deuterium exchange mass spectrometry (HDX-MS). − In addition, bulk biophysical methods are often unable to provide insights regarding the effects of any given modification to the biomolecule structure. By contrast MS methods, both protein and peptide centric, require small volumes of sample, they are highly sensitive to structural changes, have short acquisition times, can isolate modified (mutated, oxidation, acetylation, methylation, PTM) regions on the sequence, and can easily relate such regions to the stability, dynamicity, or even aggregation propensity ,− of proteins.…”

“…They are of high molecular weight, are inherently polydisperse due to post translational modifications, and can present batch to batch variation, and often are prone to aggregation at the therapeutic dose level. − A great advantage of native CIU IM-MS experiments is that they are both quick and highly sensitive to small structural changes that can lead to small stability shifts . Consequently, this method is highly suitable to examine the structures and stabilities of biological therapeutics. ,,,− In a pioneering study, Tian et al used CIU IM-MS to examine mAb–biotin conjugates, and while native IM-MS with no activation step could not differentiate between the small conjugates, CIU experiments could. , Similar benefits were reported by Hernandez-Alba et al, who were able to distinguish closely related IgG4 mAbs species by generating CIU IM-MS heatmaps . These act as fingerprints and provide structural signatures for the IgG4 subfamily, which included a wild-type, a hinge-stabilized (hs), a IgG4 mab with a S228P mutation, and a bispecific IgG4 mAb (bsAb).…”

DichroPipeline: A suite of online and downloadable tools and resources for protein circular dichroism spectroscopic data analyses, interpretations, and their interoperability with other bioinformatics tools and resources

“…In recent years CD has become a standard biophysical analytical method, especially following the development of quality control standards and new methodologies that have enabled the examination of environmental and substrate‐binding effects on proteins in solution (Wallace, 2020; Miles et al, 2021), in membranes (Miles and Wallace, 2016), and in films (Yoneda et al, 2017), and for identifying structural changes associated with binding of substrates and effector molecules (Wallace and Janes, 2009). New structural analysis algorithms and consistent formatting styles (which enable facile comparisons between data collected under a variety of conditions and in different environments) (Ramalli et al, 2022), novel analytical methods that enable cross‐comparisons with a wide range of biophysical methods (Groves et al, 2021; Miles and Wallace, 2020), and new methods for sequence‐based structure predictions (i.e., Jumper et al, 2021), have enhanced the interoperability and complementarity of CD spectroscopy with other protein characterization methods.…”