Identification and tracking of problematic host cell proteins removed by a synthetic, highly functionalized nonwoven media in downstream bioprocessing of monoclonal antibodies.

The repertoire of complex proteins produced by the host cell during monoclonal antibody (mAb) production has generated a bottleneck in downstream bioprocessing. Low ppm levels of host cell proteins (HCPs) must be achieved at the downstream purification process stage to generate an end product suitable for use in humans. The increased demand for mAb drug products globally has driven research to focus on affordability of mAb production platforms. This has fuelled advancements in manufacturing R&D to deliver higher product titres with better economics without sacrificing product quality. This study highlights the beneficial effects of inclusion of the Emphaze™ AEX Hybrid Purifier, compared to a conventional clarification process, for removal problematic HCPs during downstream bioprocessing of mAbs. Advanced proteomic methods were used to track and identify known 'problematic' HCPs through a multi-cycle Protein A purification process. Removal of histone proteins was observed, along with an average total HCP reduction of 38-fold and an average reduction of 2.3 log in HCDNA concentration. Chromatographic clarification using the Emphaze™ AEX Hybrid Purifier in conjunction with Protein A chromatography resulted in the removal of problematic HCPs including 78 kD glucose-regulated protein, nidogen-1, heat shock proteins, actin, serine protease HTRA1 and matrix metalloproteinase-19. It is shown herein that the Emphaze™ AEX Hybrid Purifier, which is readily incorporated into a mAb purification process during the clarification stage, has the potential to increase Protein A resin lifetime and potentially reduce the number of subsequent polishing chromatographic steps needed to remove HCPs that have a tendency to co-purify with mAb products.

Staying alive! Sensors used for monitoring cell health in bioreactors.

Current and next generation sensors such as pH, dissolved oxygen (dO) and temperature sensors that will help drive the use of single-use bioreactors in industry are reviewed. The current trend in bioreactor use is shifting from the traditional fixed bioreactors to the use of single-use bioreactors (SUBs). However as the shift in paradigm occurs there is now a greater need for sensor technology to play 'catch up' with the innovation of bioreactor technology. Many of the sensors still in use today rely on technology created in the 1960's such as the Clark-type dissolved oxygen sensor or glass pH electrodes. This is due to the strict requirements of sensors to monitor bioprocesses resulting in the use of traditional well understood methods, making it difficult to incorporate new sensor technology into industry. A number of advances in sensor technology have been achieved in recent years, a few of these advances and future research will also be discussed in this review.

Monitoring leachables from single-use bioreactor bags for mammalian cell culture by dispersive liquid-liquid microextraction followed by ultra high performance liquid chromatography quadrupole time of flight mass spectrometry.

A method for the identification of leachables in chemically defined media for CHO cell culture using dispersive liquid-liquid microextraction (DLLME) and UHPLC-MS is described. A Box-Behnken design of experiments (DoE) approach was applied to obtain the optimum extraction conditions of the target analytes. Performance of DLLME as extraction technique was studied by comparison of two commercial chemically defined media for CHO cell culture. General extraction conditions for any group of leachables, regardless of their specific chemical functionalities can be applied and similar optimum conditions were obtained with the two media. Extraction efficiency and matrix effects were determined. The method was validated using matrix-matched standard calibration followed by recovery assays with spiked samples. Finally, cell culture media was incubated in 7 single use bioreactors (SUBs) from different vendors and analysed. TBPP was not detected in any of the samples, whereas DtBP and TBPP-ox were found in all samples, with bDtBPP detected in six SUBs. This method can be used for early identification of non-satisfactory SUB films for cultivation of CHO cell lines for biopharmaceutical production.

An LC-MS method for the determination of pharmaceutical compounds in wastewater treatment plant influent and effluent samples.

Pharmaceuticals are continually introduced into the environment as a result of industrial and domestic use. In recent years they have emerged as environmental pollutants. An analytical method has been developed allowing for simultaneous detection and identification of 20 pharmaceutical compounds from various therapeutic classes using solid phase extraction (SPE) followed by liquid chromatography-electrospray ionisation mass spectrometry (LC-MS/MS). The limits of detection and limits of quantitation for the method were in the ng/L-microg/L range. The method was applied to influent and effluent samples from three wastewater treatment plants (WWTPs). Fifteen compounds were identified in the sample matrix with salicylic acid and ibuprofen being the most abundant at 9.17 and 3.20 microg/L respectively.