Physiological responses of CHO cells to repetitive hydrodynamic stress.

A majority of the previous investigations on the hydrodynamic sensitivity of mammalian cells have focused on lethal effects as determined by cell death or lysis. In this study, we investigated the effect of hydrodynamic stress on CHO cells in a fed-batch process using a previously reported system which subjects cells to repetitive, high levels of hydrodynamic stress, quantified by energy dissipation rate (EDR). The results indicated that cell growth and monoclonal antibody production of the test cells were very resistant to the hydrodynamic stress. Compared to the control, no significant variation was observed at the highest EDR tested, 6.4 x 10(6) W/m(3). Most product quality attributes were not affected by intense hydrodynamic stress either. The only significant impact was on glycosylation. A shift of glycosylation pattern was observed at EDR levels at or higher than 6.0 x 10(4) W/m(3), which is two orders of magnitude lower than the EDR where physical cell damage, as measured by lactate dehydrogenase release, was observed. While not as extensively investigated, a second monoclonal antibody produced in a different CHO clone exhibited the same glycosylation change at an intensive EDR, 2.9 x 10(5) W/m(3). Conversely, a low EDR of 0.9 x 10(2) W/m(3) had no effect on the glycosylation pattern. As 6.0 x 10(4) W/m(3), the lowest EDR that triggers the glycosylation shift, is about one order of magnitude higher than the estimated, maximum EDR in typically operated, large-scale stirred tank bioreactors, further studies in a lower EDR range of 1 x 10(3)-6.0 x 10(4) W/m(3) are needed to assess the glycosylation shift effect under typical large-scale bioreactor operation conditions. Follow-up studies in stirred tanks are also needed to confirm the glycosylation shift effect and to validate the repetitive hydrodynamic stress model.

On-line coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product.

A methodology based on on-line coupling of size exclusion chromatography (SEC) with mixed-mode liquid chromatography (LC) has been developed. The method allows for simultaneous measurement of a wide range of components in biopharmaceutical drug products. These components include the active pharmaceutical ingredient (protein) and various kinds of excipients such as cations, anions, nonionic hydrophobic surfactant and hydrophilic sugars. Dual short SEC columns are used to separate small molecule excipients from large protein molecules. The separated protein is quantified using a UV detector at 280 nm. The isolated excipients are switched, online, to the Trinity P1 mixed-mode column for separation, and detected by an evaporative light scattering detector (ELSD). Using a stationary phase with 1.7 µm particles in SEC allows for the use of volatile buffers for both SEC and mix-mode separation. This facilitates the detection of different excipients by ELSD and provides potential for online characterization of the protein with mass spectrometry (MS). The method has been applied to quantitate protein and excipients in different biopharmaceutical drug products including monoclonal antibodies (mAb), antibody drug conjugates (ADC) and vaccines.