During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.

Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

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Some monoclonal antibodies (mAbs) are reported to display concentration-dependent reversible self-association (RSA). There are multiple studies that investigate the effect of RSA on product characteristics such as viscosity, opalescence, phase separation and aggregation. This work investigates the effects of RSA on a bind-and-elute mode cation exchange chromatography (CEX) unit operation. We report a case study in which the RSA of an IgG2 (mAb X) resulted in significant peak splitting during salt gradient elution in CEX. Multiple factors including resin type, load challenge, residence time and gradient slope were evaluated and demonstrated little effect on the peak splitting of mAb X. It was determined that high NaCl concentrations in combination with high protein concentrations induced mAb X to form one RSA species that binds more strongly to the column, resulting in a large second elution peak. The finding of NaCl-induced RSA suggested that lower NaCl elution concentrations and different types of salts could mitigate RSA and thus eliminate peak splitting. Different salts were tested, showing that chaotropic salts such as CaCl2 reduced the second elution peak by inducing less RSA. The addition of a positively charged amino acid (such as 50mM histidine) into the CEX elution buffer resulted in elution at lower NaCl concentrations and also effectively reduced peak splitting. However, experiments that were intended to reduce salt concentration by increasing the elution buffer pH did not significantly mitigate peak splitting. This is because higher pH conditions also increase RSA. This work identifies salt-induced RSA as the cause of peak splitting of a mAb in CEX and also provides solutions to reduce the phenomenon.