Christopher J. Hewitt

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

The scale-up of microbial batch and fed-batch fermentation processes

Publisher Summary The scale-up of single-celled aerobic microbial fermentation processes is complicated that can lead to unpredictable process performance. However, this is not due to the introduction of fluid dynamic generated stresses (or so-called “shear damage”), whether arising from agitator generated turbulence or bursting bubbles, rather it is because the large-scale fed-batch bioreactor provides a very dynamic environment with large spatial and temporal heterogeneities. Such environmental heterogeneities can induce multiple physiological responses in cells. These responses consume energy and resources such that biomass concentration and product yields can be reduced. These phenomena are not observed in well-mixed homogeneous laboratory-scale reactors where much process development is done and their effects are difficult to model mathematically. Therefore, the ability to obtain data on how a recombinant laboratory process may perform at the large scale, dependent on feeding regime employed or controlling action taken is invaluable for any detailed and informed development program.

The translation of cell-based therapies: clinical landscape and manufacturing challenges

Cell-based therapies have the potential to make a large contribution toward currently unmet patient need and thus effective manufacture of these products is essential. Many challenges must be overcome before this can become a reality and a better definition of the manufacturing requirements for cell-based products must be obtained. The aim of this study is to inform industry and academia of current cell-based therapy clinical development and to identify gaps in their manufacturing requirements. A total of 1342 active cell-based therapy clinical trials have been identified and characterized based on cell type, target indication and trial phase. Multiple technologies have been assessed for the manufacture of these cell types in order to facilitate product translation and future process development.

Large-scale expansion and exploitation of pluripotent stem cells for regenerative medicine purposes: beyond the T flask

Human pluripotent stem cells will likely be a significant part of the regenerative medicine-driven healthcare revolution. In order to realize this potential, culture processes must be standardized, scalable and able to produce clinically relevant cell numbers, whilst maintaining critical biological functionality. This review comprises a broad overview of important bioprocess considerations, referencing the development of biopharmaceutical processes in an effort to learn from current best practice in the field. Particular focus is given to the recent efforts to grow human pluripotent stem cells in microcarrier or aggregate suspension culture, which would allow geometric expansion of productive capacity were it to be fully realized. The potential of these approaches is compared with automation of traditional T-flask culture, which may provide a cost-effective platform for low-dose, low-incidence conditions or autologous therapies. This represents the first step in defining the full extent of the challenges facing bioprocess engineers in the exploitation of large-scale human pluripotent stem cell manufacture.

Effect of sterilisation by gamma irradiation on the ability of polycaprolactone (PCL) to act as a scaffold material

This paper investigates the effect of sterilisation by gamma irradiation (dose 2.5Mrad) on the following properties of polycaprolactone (PCL): (1) degradation rate (catalysed by lipase), (2) mechanical properties, (3) the ability of cells to attach and subsequently grow on its surface. Gel permeation chromatography (GPC) was used to determine the effects of gamma irradiation of weight average (M(w)) and number average (M(n)) molecular weights. Gamma irradiation significantly decreased the rate of degradation, although the rates depended on the initial mass of polymer; it also affected the appearance of the degraded specimens when they were examined by scanning electron microscopy. Irradiation also significantly increased the mechanical yield stress but not the failure stress of PCL. It caused a significant increase in M(w) and decrease in M(n) that could be attributed to chain scission and cross-linking. Chondrocyte attachment and growth on PCL was not significantly affected by gamma irradiation.

The use of flow cytometry to study the impact of fluid mechanical stress on Escherichia coli W3110 during continuous cultivation in an agitated bioreactor

Continuous culture fermentations of Escherichia coli W3110 have been carried out at controlled dissolved oxygen levels of 40% and 10% of saturation. Satisfactory and reproducible results were obtained. Agitation speeds of 400 and 1200 rpm at an aeration rate of 1 vvm have been used as well as an aeration rate of 3 vvm at 400 rpm. The upper levels of these variables represent much higher agitation and aeration intensities than those normally used in practical fermentations. The fermentations were monitored by mass spectrometry and optical density, and cell samples were studied by flow cytometry, SEM, and TEM. Protocols were developed so the state of both cell membranes and cell size could be measured by flow cytometry. Under all the conditions of agitation and aeration, flow cytometric analysis indicated that both cell membranes were intact and that a cytoplasmic membrane potential existed; also the cell size did not change, results confirmed by SEM and TEM. There were no detectable changes in off-gas analysis or optical density during the continuous fermentation nor in the cell structure as revealed by SEM or TEM, except at the highest agitation intensity. Under the latter conditions, after 7 h, the outer polysaccharide layer on the cell was stripped away. It is concluded that any changes in biological performance of this E. coli cell line due to variations in agitation or aeration intensity or scale of operation cannot be attributed to fluid dynamic stresses associated with the turbulence generated by impellers or with bursting bubbles.

The application of multi-parameter flow cytometry to the study of recombinant Escherichia coli batch fermentation processes.

Multi-parameter flow cytometric techniques coupled with dual colour fluorescent staining were used to study the physical and metabolic consequences of inclusion body formation in batch cultures of the recombinant Escherichia coli strain MSD3735. This strain contains a plasmid coding for the isopropylthiogalactopyranoside-inducible model eukaryotic protein AP50. It is known that the synthesis of foreign proteins at high concentrations can exert a severe metabolic stress on the host cell and that morphological changes can occur. In this work, using various points of induction, it was shown that inclusion body formation is followed immediately by measurable changes in the characteristic intrinsic light scatter patterns for the individual cell (forward scatter, 90° side scatter) and a concomitant progressive change in the individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability. This work establishes flow cytometry as a potentially valuable tool for monitoring recombinant fermentation processes, providing important information for scale-up. Further, we discuss the possibility of optimising inclusion body formation by manipulating the fermentation conditions based on these rapid “real-time” measurements.

Studies related to antibody fragment (Fab) production in Escherichia coli W3110 fed‐batch fermentation processes using multiparameter flow cytometry

Microbiology is important to industry therefore rapid and statistically representative measurements of cell physiological state, proliferation, and viability are essential if informed decisions about fermentation bioprocess optimization or control are to be made, because process performance will depend largely on the number of metabolically active viable cells. Samples of recombinant Escherichia coli W3110, containing the gene for the D1.3 anti‐lysozyme Fab fragment under the control of the lac‐based expression system, were taken at various stages from fed‐batch fermentation processes and stained with a mixture of bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI/BOX). Where appropriate, measurements of dissolved oxygen tension (DOT), OD600nm and Fab concentration were made. Depending on time of induction the maximum amount of Fab accumulating in the supernatant varied quite markedly from 1 to 4 μg ml−1 as did subsequent cell physiological state with respect to PI/BOX staining with a concomitant drop in maximum biomass concentration. Depending on point of induction a fourfold increase in Fab production could be achieved accompanied by a ∼50% drop in maximum biomass concentration but with a higher proportion of viable cells as measured by multiparameter flow cytometry.

The production of α-amylase (E.C.3.2.1.1.) byBacillus amyloliquefaciens, in a complex and a totally defined synthetic culture medium

The production of α-amylase byBacillus amyloliquefaciens in both complex and synthetic culture media was examined at a laboratory fermenter scale. In a complex medium which supports fast growth rates, enzyme production occurred only when the growth rate declined, principally in the stationary phase. By contrast, in a synthetic culture medium with lactose as the carbon source supporting much lower growth rates, enzyme formation occurred simultaneously with cell growth. The repression of enzyme formation during rapid growth may be due either to catabolite repression or to the low level of mRNA synthesis concerned with the production of exoproteins.

Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins

A C-terminal green fluorescent protein (GFP) fusion to a model target protein, Escherichia coli CheY, was exploited both as a reporter of the accumulation of soluble recombinant protein, and to develop a generic approach to optimize protein yields. The rapid accumulation of CheY∷GFP expressed from a pET20 vector under the control of an isopropyl-β-d-thiogalactoside (IPTG)-inducible T7 RNA polymerase resulted not only in the well-documented growth arrest but also loss of culturability and overgrowth of the productive population using plasmid-deficient bacteria. The highest yields of soluble CheY∷GFP as judged from the fluorescence levels were achieved using very low concentrations of IPTG, which avoid growth arrest and loss of culturability postinduction. Optimal product yields were obtained with 8 μM IPTG, a concentration so low that insufficient T7 RNA polymerase accumulated to be detectable by Western blot analysis. The improved protocol was shown to be suitable for process scale-up and intensification. It is also applicable to the accumulation of an untagged heterologous protein, cytochrome c(2) from Neisseria gonorrhoeae, which requires both secretion and extensive post-translational modification.