Therapeutic proteins can degrade upon administration as they are subjected to a variety of stresses in human body compartments. In vivo degradation may cause undesirable pharmacokinetic/pharmacodynamic profiles. Pre-clinical in vitro models have gained scientific interest as they enable one to evaluate the in vivo stability of monoclonal antibodies (mAbs) and ultimately can improve patient safety. We used a novel approach by stripping serum of endogenous proteins, which interfere with analytical test methods. This enabled the direct analysis of the target protein without laborious sample work-up procedures. The developed model retained the osmolality, conductivity, temperature, and pH of serum. We compared the impact of human, bovine, and artificial serum to accelerated stability conditions in histidine buffer. Target mAbs were assessed in regard to visible and sub-visible particles, as well as protein aggregation and fragmentation. Both mAbs degraded to a higher extent under physiological conditions compared to accelerated stability conditions. No relevant stability differences between the tested mAbs were observed. Our results reinforced the importance of monitoring protein stability in biological fluids or fluids emulating these conditions closely. Models enabling analysis in fluids directly allow high throughput testing in early pre-clinical stages and help in selecting molecules with increased in vivo stability.

Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities.

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Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Numerous reports indicate an oncogenic role for HBV-encoded HBsAg; however, the underlying mechanisms are not well understood.

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