Evaluation of cell disruption effects on primary recovery of antibody fragments using microscale bioprocessing techniques

Abstract

Intracellular antibody Fab' fragments periplasmically expressed in Escherichia coli require the release of Fab' from the cells before initial product recovery. This work demonstrates the utility of microscale bioprocessing techniques to evaluate the influence of different cell disruption operations on subsequent solid-liquid separation and product recovery. Initially, the industrial method of Fab' release by thermochemical extraction was established experimentally at the microwell scale and was observed to yield Fab' release consistent with the larger scale process. The influence of two further cell disruption operations, homogenization and sonication, on subsequent Fab' recovery by microfiltration was also examined. The results showed that the heat-extracted cells give better dead-end microfiltration performance in terms of permeate flux and specific cake resistance. In contrast, the cell suspensions prepared by homogenization and sonication showed more efficient product release but with lower product purity and poorer microfiltration performance. Having established the various microscale methods the linked sequence was automated on the deck of a laboratory robotic platform and used to show how different conditions during thermochemical extraction impacted on the optimal performance of the linked unit operations. The results illustrate the power of microscale techniques to evaluate crucial unit operation interactions in a bioprocess sequence using only microliter volumes of feed.

Integrated Application of Bioprocess Engineering and Biotechniques for Quality & Bulk Drug Manufacturing

“…LC-MS is a powerful technique used for many applications which has very high sensitivity and selectivity. Generally its application is oriented towards the general detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture) [27]. The limitations of LC-MS in urine analysis drug screening are that it often fails to distinguish …”

A modular approach for the ultra-scale-down of depth filtration

“…pH, conductivity, etc.). Screening of filtration conditions could be integrated with existing high throughput harvest, particle conditioning, and clarification workflows [3,[10][11][12][13][14][15][16]. Laboratory qualification of clinical production processes could be expedited and concluded with less material.…”

An ultra scale‐down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes

“…USD methodologies are able to offer a wider understanding of the effect of individual parameters by decoupling dependencies, for example, shear stress in the entry zone of a continuous flow industrial‐scale centrifuge, sedimentation in the settling zone, and shear stress during sediment discharge (Boychyn et al, 2001, 2004; Chan et al, 2006). The use of USD techniques has been described for chromatography (Wenger, Dephillips, Price, & Bracewell, ; Willoughby, Martin, & Titchener‐Hooker, ), NFF (Lau et al, ; Reynolds et al, ), for membrane separations in normal‐flow mode (Jackson, Liddell, & Lye, ; Rayat, Micheletti, & Lye, ), in cross‐flow mode using pumped flow (Rayat et al, ), and in mechanically‐agitated mode using a rotating disc (Ma et al, ). This latter form of the USD device allows the flow over the membrane to be varied independently of the transmembrane flux or pressure.…”