M. Hoare

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Rapid monitoring of recombinant protein products: a comparison of current technologies

Specific measurement of recombinant protein titer in a complex environment during industrial bioprocessing has traditionally relied on labor-intensive and time-consuming immunoassays. In recent years, however, developments in analytical technology have resulted in improved methods for protein product monitoring during bioprocessing. The choice of product-monitoring technology for a particular bioprocess will depend on a variety of assay factors and instrument-specific factors. In this article, we have compiled an overview of the advantages and disadvantages of the most commonly used technologies used: electrochemiluminescence, optical biosensors, rapid chromatography and nephelometry. The advantages of each technology for measuring both small and large recombinant therapeutic proteins are compared with a conventional enzyme-linked immunosorbent assay (ELISA) technique.

Micro biochemical engineering to accelerate the design of industrial-scale downstream processes for biopharmaceutical proteins.

The article examines how a small set of easily implemented micro biochemical engineering procedures combined with regime analysis and bioprocess models can be used to predict industrial scale performance of biopharmaceutical protein downstream processing. This approach has been worked on in many of our studies of individual operations over the last 10 years and allows preliminary evaluation to be conducted much earlier in the development pathway because of lower costs. It then permits the later large scale trials to be more highly focused. This means that the risk of delays during bioprocess development and of product launch are reduced. Here we draw the outcomes of this research together and illustrate its use in a set of typical operations; cell rupture, centrifugation, filtration, precipitation, expanded bed adsorption, chromatography and for common sources, E. coli, two yeasts and mammalian cells (GS‐NSO). The general approach to establishing this method for other operations is summarized and new developments outlined. The technique is placed against the background of the scale‐down methods that preceded it and complementary ones that are being examined in parallel. The article concludes with a discussion of the advantages and limitations of the micro biochemical engineering approach versus other methods. Biotechnol. Bioeng. 2008;100: 473–487.

Characterization of flow intensity in continuous centrifuges for the development of laboratory mimics

Predicting the recovery of “delicate” biological materials by centrifugation using laboratory centrifuges has been a major challenge to biochemical engineers partly because of the difficulty in accurately quantifying the shear stresses in continuous-flow industrial centrifuges and partly because the clarification and dewatering conditions in the laboratory units do not represent those occurring in industrial centrifuges. In this paper, the flow field in the feed zone of an industrial multichamber-bowl centrifuge is mapped and its profile of energy dissipation rate established using computational fluid dynamics (CFD). A small high-speed rotating-disc device is designed with the capacity to reproduce the CFD-predicted energy dissipation rates in the feed zone. Milliliter quantities of the process material are shear-treated in the device operating at a speed that mimics the local critical flow conditions in the industrial centrifuge. The results are used to assess the impact of flow conditions in the feed zone of the centrifuge on the physical properties of protein precipitates and on their predicted recovery using a laboratory centrifuge. This approach was used to explain the reduction in the performance of the centrifuge from the predicted 88% clarification to the observed 39% clarification of the precipitate particles. The combination of the small high-speed disc device and the laboratory centrifuge is particularly advantageous when dealing with biological products at the early stages of process development for which only small quantities of test material are often available.

Disruption of baker's yeast in a high-pressure homogenizer : new evidence on mechanism

The mechanism of disruption of bakers yeast in a high-pressure homogenizer was examined. A modified Bernoulli expression was used to define the flow velocities through the valve and valve seat for differing valve configurations. The distance between the exiting jet from the valve seat and the impact ring was varied by altering the impact ring diameter. The main disruption mechanism was shown to be due to impingement and the rate of cell breakage related to the stagnation pressure or maximum wall stress of the fluid jet. Decreased valve gap width, as estimated from the flow conditions in the valve, and decreased impact distance both contribute to an increased cell disruption rate, with the performance of the homogenizer related to the product of these two dimensions for the range of valve seats and impact distances studied.

Bioprocess Engineering Issues That Would Be Faced in Producing a DNA Vaccine at up to 100 m3 Fermentation Scale for an Influenza Pandemic

The risk of a pandemic with a virulent form of influenza is acknowledged by the World Health Organization (WHO) and other agencies. Current vaccine production facilities would be unable to meet the global requirement for vaccine. As a possible supplement a DNA vaccine may be appropriate, and bioprocess engineering factors bearing on the use of existing biopharmaceutical and antibiotics plants to produce it are described. This approach addresses the uncertainty of timing of a pandemic that precludes purpose‐built facilities. The strengths and weaknesses of alternative downstream processing routes are analyzed, and several gaps in public domain information are addressed. The conclusion is that such processing would be challenging but feasible.

The use of laboratory centrifugation studies to predict performance of industrial machines: Studies of shear‐insensitive and shear‐sensitive materials

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.

Nucleation and growth of microbial lipase crystals from clarified concentrated fermentation broths

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation.

SELECTIVE FLOCCULATION OF NUCLEIC-ACIDS, LIPIDS, AND COLLOIDAL PARTICLES FROM A YEAST-CELL HOMOGENATE BY POLYETHYLENEIMINE, AND ITS SCALE-UP

Nucleic acids, lipid, and colloidal particulate material can be selectively flocculated from a yeast cell homogenate by the cationic polymer polyethyleneimine (PEI). Flocculation can occur from a crude homogenate, a homogenate clarified centrifugally, or by the prior use of sodium tetraborate (borax). Flocculation from a homogenate previously clarified by the use of borax is best suited for large-scale operation. The supernatant obtained following centrifugation is effectively free of nucleic acid, lipid, and particulate material with essentially 100% soluble enzyme recovery. Enzyme specific activity increases by approximately 45% compared to a zero PEI control.

SELECTIVE FLOCCULATION OF CELLULAR CONTAMINANTS FROM SOLUBLE-PROTEINS USING POLYETHYLENEIMINE - A STUDY OF SEVERAL ORGANISMS AND POLYMER MOLECULAR-WEIGHTS

Saccharomyces cerevisiae, Escherichia coli, and Pseudomonas putida homogenates were treated with polyethyleneimine (PEI) of various molecular weights and concentrations, and the removal of cellular contaminants noted. The optimum doses were determined and the effects of ionic strength observed. Although there was little variation between the abilities of the various PEIs to remove cellular contaminants, the possibility of floc redissolution at higher polymer concentrations occurred more with increased molecular weight of the polymer. Increasing the ionic strength decreased the occurrence of floc redissolution. Both E. coli and P. putida required a higher concentration of PEI than S. cerevisiae to achieve significant removal of cellular contaminants. Loss of soluble protein by absorption to flocs with E. coli was influenced strongly by ionic strength.