Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

A biosimilar is a biological medicinal product that is comparable to a reference medicinal product in terms of quality, safety, and efficacy. SB4 was developed as a biosimilar to Enbrel® (etanercept) and was approved as Benepali®, the first biosimilar of etanercept licensed in the European Union (EU). The quality assessment of SB4 was performed in accordance with the ICH comparability guideline and the biosimilar guidelines of the European Medicines Agency and Food and Drug Administration. Extensive structural, physicochemical, and biological testing was performed with state-of-the-art technologies during a side-by-side comparison of the products. Similarity of critical quality attributes (CQAs) was evaluated on the basis of tolerance intervals established from quality data obtained from more than 60 lots of EU-sourced and US-sourced etanercept. Additional quality assessment was focused on a detailed investigation of immunogenicity-related quality attributes, including hydrophobic variants, high-molecular-weight (HMW) species, N-glycolylneuraminic acid (NGNA), and α-1,3-galactose. This comprehensive characterization study demonstrated that SB4 is highly similar to the reference product, Enbrel®, in structural, physicochemical, and biological quality attributes. In addition, the levels of potential immunogenicity-related quality attributes of SB4 such as hydrophobic variants, HMW aggregates, and α-1,3-galactose were less than those of the reference product.

In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages roughly 30% because of the long exposure to H 2 O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability. olution-phase hydrogen/deuterium exchange (HDX) coupled with mass spectrometry is a non-perturbative method to study protein conformational changes in protein-ligand and protein-protein interactions [1,2]. The exchange reaction rate minimizes at pH 2.0 -3.0 and at a temperature of about 0°C. To minimize back exchange of amide backbone -ND to -NH, the procedures after quench are performed under quench conditions. However, back exchange still averages nearly 30% [3], depending on various factors, e.g., peptide sequence [4]. With matrix-assisted laser desorption/ionization, the back-exchange level is even higher [5].In addition to low pH and low temperature, the H 2 O concentration should be kept low and exposure to H 2 O should be minimized during and after quench. Thus, elution of peptide fragments should occur as fast as possible. Our standard reversedphase liquid chromatography (RPLC) C 5 column elutes peptide fragments in about 4.5 min. Supercritical fluid chromatography (SFC) reduces back exchange because its mobile phase is mainly CO 2 instead of H 2 O [6]. Dramatic reduction of back exchange has been achieved with SFC separation. However, short column life and reliability of the experiment still require improvement.Here we present results with a high flow rate reversedphase ProZap™ C 18 column to reduce back exchange, while also increasing sequence coverage and total fragment number. Compared to a conventional C 5 column, the ProZap C 18 column has a wider inside diameter, shorter length, smaller particle size, and larger pore size, enabling higher flow rate and lower back pressure. The retention time is thereby reduced from 4.5 min (Jupiter™ C 5 column) to 1.5 min, for a 3-fold reduction in H 2 O exposure time and concomitant reduction in back exchange for the fragment peptides. Besides reduction in back exchange, the ProZap column also provides much improved elution efficiency for increased fragment number and...

Key Points• Electron microscopy and hydrogen-deuterium exchange establish the C1 domain as the major binding site for the VWF D9D3 domain on FVIII.• Additional sites implicated in the FVIII-VWF interaction are located within the a3 acidic peptide and the A3 and C2 domains of FVIII.Association with the D9D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D9D3 domain. The EM averages show that FVIII interacts with the D9D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D9D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction. (Blood. 2015;126(8):935-938)