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Development of a new hydrogen exchange mass spectrometry (HX-MS) method to assess reversible self-association (RSA) of high concentration proteins without dilution
Weerasinghe, Mihiri Shyamali
Weerasinghe, Mihiri Shyamali
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Abstract
Monoclonal antibodies (mAbs) are one of the fastest growing classes of biologic drugs and have been used to treat many diseases. Subcutaneous self-administration of high concentration mAb formulations has become popular since it decreases the number of hospital visits for patients. Since mAbs are protein-based drugs, they can face physical and chemical instabilities in solution. In addition, highly concentrated mAbs can show undesired solution properties such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation. RSA causes mAbs to form non-covalently associated networks in solution and can make the formulations extremely viscous, potentially diminishing their therapeutic value. Hydrogen exchange-mass spectrometry (HX-MS) has proven useful to map the sites that mediate RSA interactions. Traditional HX requires that mAbs be diluted in deuterium oxide (D2O). However, this dilution can cause mAb dissociation, so traditional HX has limited utility for the direct analysis of highly concentrated protein solutions. Previously, we resolved this challenge by reconstituting lyophilized mAbs directly into D2O. Although lyophilization is a good strategy for HX, the lyophilization method is undesirable for pharmaceuticals formulated as liquids. Therefore, there is a need to develop a new dilution-free HX-MS method to better characterize the nature of concentration-dependent RSA of mAbs. The primary objective of this dissertation is to develop a new workflow to exchange self-associated mAbs formulated at high concentrations in water (H2O) into D2O without dilution. Three approaches were investigated to achieve this goal.
In the first approach, conventional dialysis was used for HX without dilution. Previous experiments have established that conventional dialysis is unsuitable for non-dilution HX because the rate of hydrogen/deuterium (H/D) transfer across dialysis membranes is too slow.
In the second approach, gel filtration was employed to rapidly (~30 seconds) exchange mAbs formulated in H2O-based buffers into D2O-based buffers with minimum or no dilution. Preliminary results with a small protein cytochrome c indicated the approach could be adapted to an HX-MS workflow. To demonstrate proof-of-concept in mAbs, an IgG1 monoclonal antibody (mAb-C) was studied. When mAb-C was formulated at a low concentration (e.g. 5 mg/mL), the gel filtration approach showed minimal dilution. However, when very highly concentrated mAb-C solutions were used (e.g. 60 mg/mL), the gel filtration approach showed significant dilution. Therefore, the gel filtration HX method was also unsuitable for performing HX-MS experiments without dilution.
In the third approach, a new thin film dialysis method to exchange proteins into D2O without dilution was developed using a novel thin film micro dialysis device capable of labeling proteins directly at high protein concentrations. The device passively exchanges proteins formulated in H2O into D2O without dilution. To demonstrate proof-of-concept in mAbs, an IgG1 monoclonal antibody (mAb-J) known to undergo RSA at high protein concentrations was studied. Using the novel device and workflow, the RSA interface of mAb-J was identified. To validate the process, the RSA of mAb-J was confirmed by comparing the results to the results obtained using traditional lyophilization-based HX. Additionally, the dialysis efficiency of this novel thin film micro dialysis device was compared with the dialysis efficiency of the conventional dialysis method. The results suggest that the dialysis efficiency of thin film micro dialysis is much higher than conventional dialysis methods.
The novel thin film dialysis HX method was also applied to reveal the protein interfaces formed during the RSA of an IgG4 monoclonal antibody (mAb-Z) at different protein concentrations. Establishment of this new analytical method would represent an important advancement in HX at high protein concentrations since dilution of protein stock upon addition of D2O has always been a fundamental problem for assessing RSA. This new method can be used for any HX study at high protein concentration without diluting the protein sample. Therefore, this approach will be a significant advancement to aid formulation development of highly concentrated therapeutic proteins in the pharmaceutical industry.
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2022-01-01
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University of Kansas
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Keywords
highly concentrated therapeutic proteins, Hydrogen Exchange, monoclonal antibody, Protein-protein interactions, reversible self-association