Michael Hoare

Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells

Chinese hamster ovary cells producing recombinant human interferon‐γ were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized‐bed bioreactor, reaching a maximum cell density in excess of 3 × 107 cells (mL microcarrier)−1 at a specific growth rate (μ) of 0.010 h−1. During establishment of the culture, the N‐glycosylation of secreted recombinant IFN‐γ was monitored by capillary electrophoresis of intact IFN‐γ proteins and by HPLC analysis of released N‐glycans. Rapid analysis of IFN‐γ by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN‐γ (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN‐γ by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN‐γ proteins increased after 210 h of culture, indicative of an increase in N‐glycan sialylation. This was confirmed by cation‐exchange HPLC analysis of released, fluorophore‐labeled N‐glycans, which showed an increase in the proportion of tri‐ and tetrasialylated N‐glycans associated with IFN‐γ during culture, with a concomitant decrease in the proportion of monosialylated and neutral N‐glycans. Comparative analyses of IFN‐γ produced by CHO cells in stirred‐tank culture showed that N‐glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.

Steady‐State Unimolecular Processes in Multilevel Systems

The problem of reaction efficiencies in steady‐state multilevel systems is analyzed using a matrix formulation. Several special cases are treated with particular attention to the existence of a low‐pressure anomaly. The results apply equally well to the calculation of unimolecular reaction efficiencies, fluorescence yields and the analogous quantities in any process involving competitive pressure‐dependent transitions between two subsets of states.

Adapted Ultra Scale-Down Approach for Predicting the Centrifugal Separation Behavior of High Cell Density Cultures

The work presented here describes an ultra scale‐down (USD) methodology for predicting centrifugal clarification performance in the case of high cell density fermentation broths. Existing USD approaches generated for dilute systems led to a 5– to 10‐fold overprediction of clarification performance when applied to such high cell density feeds. This is due to increased interparticle forces, leading to effects such as aggregation, flocculation, or even blanket sedimentation, occurring in the low shear environment of a laboratory centrifuge, which will not be apparent in the settling region of a continuous‐flow industrial centrifuge. A USD methodology was created based upon the dilution of high solids feed material to ∼2% wet wt/vol prior to the application of the clarification test. At this level of dilution cell‐cell interactions are minimal. The dilution alters the level of hindered settling in the feed suspensions, and so mathematical corrections are applied to the resultant clarification curves to mimic the original feed accurately. The methodology was successfully verified: corrected USD curves accurately predicted pilot‐scale clarification performance of high cell density broths of Saccharomyces cerevisiae and Escherichia coli cells. The USD method allows for the rapid prediction of large‐scale clarification of high solids density material using millilitre quantities of feed. The advantages of this method to the biochemical engineer, such as the enabling of rapid process design and scale‐up, are discussed.

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale-down study of the effect of harvesting by centrifugation on the post-translational modification profile

With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post‐translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale‐down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG4 monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C‐terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C‐terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)2 20–35%), with an associated decrease in the proportion of long glycans with culture age (e.g., (G2F)2 7–4%, and G1F/G2F from 15.2% to 7.8%). Ultra scale‐down mimics showed that subsequent processing of these cultures did not change the post‐translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale‐down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e.g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product. Biotechnol. Bioeng. 2010;107: 85–95.

Prediction of shear damage of plasmid DNA in pump and centrifuge operations using an ultra scale-down device

Supercoiled circular (SC) plasmid DNA is often subjected to fluid stress in large‐scale manufacturing processes. It is thus important to characterize the engineering environment within a particular unit operation as well as within the associated ancillary equipment during process design for plasmid DNA manufacture so as to avoid shear‐induced degradation of the SC isoform, which would compromise product efficacy in therapeutic applications. In the past few years, ultra scale‐down (USD) tools were developed within our laboratory to mimic the engineering environments experienced by biomolecules within a range of manufacturing‐scale ancillary, primary recovery, and purification operations, using milliliter quantities of material. Through the use of a USD shear device, the effect of elongational strain rate on SC plasmid DNA degradation was studied in this paper, and from that, the impact of a centrifugal pump, a Mono pump, and a disk‐stack centrifuge feed zone on SC plasmid DNA degradation was predicted and experimentally verified at scale. Model predictions, over the range of conditions studied, were in good agreement with experimental values, demonstrating the potential of the USD approach as a decisional tool during bioprocess design.

Selective Flocculation and Precipitation for the Improvement of Virus-Like Particle Recovery from Yeast Homogenate

The purification of an intracellular product from a complex mixture of contaminants after cell disruption is a common problem in processes downstream of fermentation systems. This is particularly challenging for the recovery of particulate (80 nm in diameter) multimeric protein products, named virus‐like particles (VLPs), from cell debris and other intracellular components. Selective flocculation for debris removal followed by selective precipitation of the target protein can be used as a preclarification step to aid purification. In this paper, selective borax flocculation of cell debris in yeast homogenate, followed by selective poly(ethylene glycol) precipitation of VLPs are defined with a view to demonstrating their potential in aiding the initial clarification stages of the purification sequence. The translation from laboratory scale to pilot scale operation is addressed, demonstrating the challenge of scale‐up of solid‐liquid separation stages for biological particle processing.

A Kalman filter algorithm and monitoring apparatus for at-line control of fractional protein precipitation.

Downstream processing operations are often carried out blind in the process timescale since product monitoring on-line is not common. Knowledge of the location and concentration of the product and key contaminants is complementary to other process information for process development and, if available on-line in conjunction with a suitable model, control. This article sets out to demonstrate a model describing a two-cut fractional protein precipitation process and how this may be used for control of the process to maximize yield in the face of variable process stream conditions. Estimation of the model parameters is achieved by means of data-fitting by least squares and in comparison prediction by a Kalman filter algorithm. A description and error analysis of equipment for at-line monitoring of the soluble product in a pilot plant environment is presented which includes a micro-centrifuge necessary to clarify small volumes of sample prior to analysis. Finally, an account of the successful implementation of this equipment and the Kalman filter algorithm for control at bench scale is given where conditions in the process stream are deliberately disturbed to test the control operation. (c) 1997 John Wiley & Sons, Inc.

The Monitoring and Control of Protein Purification and Recovery Processes

A novel method for the on-line monitoring of fractional precipitation of proteins has been developed using a combination of an automated microcentrifuge and flow injection analysis for enzyme and total protein analysis. The optimisation of a fractional precipitation process is discussed as is also the use of the on-line monitoring equipment for control to predetermined set-points of the fraction of the enzyme remaining soluble. The on-line measurement of precipitate concentration and size distribution using a combination of turbidity and light diffraction measurements and its application to improving centrifugal recovery of precipitates is discussed. One such improvement is the use of these measurement techniques to control the in-line low-frequency conditioning of the precipitate to improve particle characteristics such as size distribution, density and resistance to shear-related or mechanical break-up.

Relationship between preparation of cells for therapy and cell quality using artificial neural network analysis

OBJECTIVEnThe successful preparation of cells for therapy depends on the characterization of causal factors affecting cell quality. Ultra scale-down methods are used to characterize cells in terms of their response to process engineering causal factors of hydrodynamic shear stress and time. This response is in turn characterized in terms of causal factors relating to variations as may naturally occur during cell preparation, i.e., passage number, generation number, time of the final passage stage and hold time in formulation medium.nnnMETHODSnTo investigate the influence of all of these causal factors we have adopted a non-linear, multivariate predictive artificial neural network (ANN) based modeling approach to help create clearer insights into their effect on cell membrane integrity and surface marker content. A prostate cancer cell line candidate for cancer therapy (P4E6) was used and cell surface markers CD9, CD147 and HLA A-C were investigated.nnnRESULTSnAll causal factors studied were found to be significant in establishing an ANN model for the prediction of cell quality parameters with the extent of exposure to shear stress being the most significant and then passage number (range 57-66) and generation number (range 10-19) determining most strongly the cells resistance to shear stress. Both the operation of the final cell passage and the hold time of the cells in a formulation buffer also determine the cells resistance to shear stress. The processing parameters related to cell handling after preparation, i.e., shear stress and time of exposure were found to be the most influential affecting cell quality.nnnCONCLUSIONnCD9 surface marker loss was the most sensitive indicator of the effects of shear stress followed by loss of membrane integrity and then HLA A-C, while CD147 remained unaffected by shear stress or even prone to increase. Also greater stability of cell surface marker presence was noted for cells generated at greater passage numbers or generation numbers or for reduction in hold time in formulation buffer.

Windows of operation for bioreactor design for the controlled formation of tissue‐engineered arteries

The availability of large numbers of units of artificial arteries would offer significant benefits to the clinical management of bypass surgery. Tissue engineering offers the potential of providing vessels that can mimic the morphology, function, and physiological environment of native vessels. Ideally this would involve culturing stem cells in vitro within a biodegradable tubular scaffold so as to construct tissue for implantation. Essential to establishing a robust process for the production of tissue‐engineered arteries is the understanding of the impact of changes in the operating conditions and bioreactor design on the construct formation. In this article, models of transport phenomena were developed to predict the critical flow rates and mass transfer requirements of a prototype bioreactor for the formation of tissue‐engineered arteries. The impact of the cell concentration, tube geometry, oxygen effective diffusivity in alginate, substrate and metabolite concentration levels, feed rate, and recycle rate on the design of the bioreactor was visualized using windows of operation and contour plots. The result of this analysis determined the best configuration of the bioreactor that meets the cellular transport requirements as well as being reliable in performance while seeking to reduce the amount of nutrients to be used.