A new CZE method for profiling human serum albumin and its related forms to assess the quality of biopharmaceuticals.

We present a new CZE method, which uses a polyethylene oxide-coated capillary to separate native HSA from more than five of its structural variants. These variants include oxidized, truncated, and cysteinylated forms of HSA which can all be found in biopharmaceutical products. Both CE and MS confirmed the high degree of heterogeneity of HSA preparations. Recovery studies demonstrated that adsorption of HSA on the capillary was significantly reduced under the conditions we developed, which led to a satisfactory repeatability (RSD for migration times and relative peak areas were less than 0.2 and 7.0%, respectively). Assignment of the main peaks was attempted using in vitro degraded/stressed HSA. We used our method to test batch-to-batch comparability and detected slight quantitative differences in the proportion of native HSA in batches produced from different fractionation methods.

A validated capillary electrophoresis method to check for batch-to-batch consistency during recombinant human glycosylated interleukin-7 production campaigns.

This work reports the validation of a simple CZE method to be used in quality control of recombinant human glycosylated interleukin-7 (rhIL-7) batches produced in Chinese Hamster Ovary (CHO) cells. The separation buffer was a 25mM sodium borate at pH 10 containing 12mM diaminobutane (DAB) used as a dynamic coating agent of the capillary. This method allowed the separation of seven peaks ranging from low to high sialylated glycoforms. An extensive study on conditioning methods of the capillary has been conducted to yield repeatable results. Excellent RSD of EOF mobility (less than 0.6%) was obtained when conditioning included capillary equilibration under virtual analyses and storage in 0.1M NaOH overnight. Method specificity has been demonstrated to be able to discriminate different rhIL-7 glycoforms produced in CHO from formulation matrix. Linearity was demonstrated between 0.5 and 4mg/mL. LOQ was 0.5mg/mL. Repeatability (RSD<1.4 and 3.3% for t(m) and A%, respectively), intermediate precision of inter-day (RSD<2.1 and 4.5), inter-analyst (RSD<2.0 and 3.0) and inter-equipment (RSD<3.8 and 3.7 for electrophoretic mobility and A%, respectively) were all very satisfactory. Evaluation of robustness revealed that pH and DAB concentration are critical parameters in the method while slight alteration of ionic strength of electrolyte or change of capillary source did not affect the results. Finally the method was shown to provide reliable informations to address comparability studies and batch-to-batch consistency of biomanufactured rhIL-7.

CZE for glycoform profiling and quality assessment of recombinant human interleukin-7.

The aim of the present work was to develop a simple high-resolution CZE method for glycoform profiling and quality assessment of recombinant human interleukin-7 (rhIL-7) produced in Chinese hamster ovary cells. Classical and isoelectric buffers and several monoamines used as alkaline component of BGE at very low concentration have been investigated in order to optimize the resolution. The best separation was achieved using a fused-silica capillary and a buffer composed of 25 mM citrate/triethanolamine at pH 2.6. Under these acidic conditions, triethanolamine was able to reverse EOF favorably and to limit rhIL-7 adsorption. This method allowed the separation of four groups of peaks ranging from low to high-sialylated glycoforms. An extensive study on inter-run rinsing procedures has been conducted. Rinsing with 50 mM SDS was retained to achieve the optimal repeatability. Excellent intermediate precision was obtained for migration time (RSD < 0.6%), while RSD for intraday studies were only less than 2.9%. Satisfactory inter and intraday repeatabilities were also observed for relative peak area. We finally demonstrated that reliable information could be obtained to address comparability studies and demonstrate batch-to-batch consistency of biomanufactured rhIL-7.