Berrill, A.; (2009) Protein mass spectrometry for bioprocess development & monitoring. Doctoral thesis , UCL (University College London).

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Abstract

Bioprocesses for therapeutic protein production typically require significant resources to be invested in their development. This development could be improved with technologies that can elucidate the physicochemical properties of process stream components with small sample volumes in a rapid and readily performed manner. This is especially true in early phase development when material and established analytical methods are limiting. This thesis has investigated various process materials but was focussed mainly on those existing in an ApolipoproteinA-IM (ApoA-IM) process, produced using an Eschericia coli (E. coli) host. Using a mass spectrometric technique this project began by monitoring the product and contaminant during the ApoA-IM process and how this analytical approach compares to traditional analytical methods such as high performance liquid chromatography (HPLC). Results showed that, unlike many other analytical methods, surface enhanced laser desorption ionisation mass spectrometry (SELDI-MS) can handle early process samples that contain complex mixtures of biological molecules with limited sample pre-treatment and thereby provide meaningful process-relevant information. The change in material during the flocculation/centrifugation stage of the process was then examined. When only a change in cellular debris was observed an existing methodology developed at University College London (UCL) was implemented to maximise cellular debris removal. The predictive scale down methodology enabled rapid optimization of the operating conditions for a flocculation followed with a centrifugation step using only small volumes (20mL) of a high solids (~20% w/w) E. coli heat extract. These experiments suggested that adding a higher level of a cationic polymer could substantially increase the strength of the flocculated particles produced, thereby enhancing overall clarification performance in a large scale centrifuge. This was subsequently validated at pilot scale.The proteins remaining from this flocculation/centrifugation stage were then compared using the mass spectrometric technique to calculate the difficulty of removing each protein contaminant from the ApoA-IM product and suggested conditions for future sorbent scouting runs.

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