Hydrogen/deuterium exchange-mass spectrometry analysis of high concentration biotherapeutics: application to phase-separated antibody formulations

Abstract

High concentration biotherapeutic formulations are often required to deliver large doses of drugs to achieve a desired degree of efficacy and less frequent dose. However, highly concentrated protein-containing solutions may exhibit undesirable therapeutic properties, such as increased viscosity, aggregation, and phase separation that can affect drug efficacy and raise safety issues. The characterization of high concentration protein formulations is a critical yet challenging analytical task for therapeutic development efforts, due to the lack of technologies capable of making accurate measurements under such conditions. To address this issue, we developed a novel dilution-free hydrogen/deuterium exchange (HDX) mass spectrometry (MS) method for the direct conformational analysis of high concentration biotherapeutics. Here, we particularly focused on studying phase separation phenomenon that can occur at high protein concentrations. First, two aliquots of monoclonal antibodies (mAbs) were dialyzed in either hydrogen- or deuterium-containing buffers at low salt and pH. Phases that separated were then discretely sampled and subjected to dilution-free HDX-MS analysis through mixing the non-deuterated and deuterated protein aliquots. Our HDX-MS results analyzed at a global protein level reveal less deuterium incorporation for the protein-enriched phase compared to the protein-depleted phase present in high concentration formulations. A peptide level analysis further confirmed these observed differences, and a detailed statistical analysis provided direct information surrounding the details of the conformational changes observed. Based on our HDX-MS results, we propose possible structures for the self-associated mAbs present at high concentrations. Our new method can potentially provide useful insights into the unusual behavior of therapeutic proteins in high concentration formulations, aiding their development.

Developments in Hydrogen/Deuterium Exchange Mass Spectrometry

“…The way the samples are introduced to deuterium and manipulations of the sample in terms of mixing, ligands, temperature, additives, and so forth are embedded in such a paper. A study published in 2019 described an HDX MS method for studying protein conformation at very high concentrations, an important condition (particularly for biopharmaceuticals) that had also been investigated using HDX MS prior to 2017. , Finally, various ways to study lipid-associated proteins, including both peripheral and transmembrane proteins, were reported along with studies that vary the format of the membrane mimetic. , An example (Figure ) of using HDX MS to study the transmembrane protein rhomboid protease (GlpP) in three different types of nanodiscs with various native lipid compositions shows that HDX can be different and biologically relevant depending on the lipid environment in which the protein finds itself. When performing HDX MS on membrane proteins, depending on the exchange format and membrane mimetic, it could be necessary to remove lipid molecules that were present during exchange prior to LC–MS analysis.…”

“…The way the samples are introduced to deuterium and manipulations of the sample in terms of mixing, ligands, temperature, additives, and so forth are embedded in such a paper. A study published in 2019 described an HDX MS method for studying protein conformation at very high concentrations, 68 an important condition (particularly for biopharmaceuticals) that had also been investigated using HDX MS prior to 2017. 69,70 Finally, various ways to study lipid-associated proteins, including both peripheral and transmembrane proteins, were reported along with studies that vary the format of the membrane mimetic.…”

HDX-MS: An Analytical Tool to Capture Protein Motion in Action

“…This approach has been successfully used to study the self-association of antibodies at a high concentration [ 19 ], but the methodology has a limitation in that all proteins are not amicable to lyophilization without substantial structural perturbation. Another recent approach is microdialysis of protein sample in D 2 O buffer [ 20 ]. The advantage of this method is the same as earlier, but this method requires a large volume of D 2 O buffer, which can be expensive.…”

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry

“…The deuterium uptake results as a function of HDX labeling time is shown in Figure 6 for five representative peptides: four mAb1 CDR peptides (HC CDR1 30-33, HC CDR2 50-54, HC CDR3 101-104, and LC CDR2 48-53) and one mAb1 non-CDR peptide (HC non-CDR 36-47) as a comparison. The deuterium uptakes of the five corresponding peptides at the same regions in mAb2 (HC CDR1 31-34, HC CDR2 50-53, HC CDR3 99-103, LC CDR2 47-52, and HC non-CDR [36][37][38][39][40][41][42][43][44][45][46][47] are also shown as a comparison (Figure 6). The deuterium uptakes of these peptides increased as the HDX reaction time increased until an equilibrium was reached at the 24-hour timepoint.…”

“…Using these dialysis-coupled HDX-MS approaches, they were able to analyze the conformation and dynamics of a mAb at a high concentration of 200 mg/mL and a low concentration of 3 mg/mL. Tian et al developed an alternative HDX-MS approach to analyze the structural consequence of liquid-liquid phase separation 43 . In this approach, the authors dialyzed two mAb solutions to H 2 O buffer and D 2 O buffer at the same concentration, allowed mAbs to separate into a higher-density phase of 150 mg/mL and a lower-density phase of 28 mg/mL, and then initiated the HDX reactions by mixing the two protein phases at a 1:1 ratio.…”