An ultra scale‐down methodology to characterize aspects of the response of human cells to processing by membrane separation operations

Tools that allow cost‐effective screening of the susceptibility of cell lines to operating conditions which may apply during full scale processing are central to the rapid development of robust processes for cell‐based therapies. In this paper, an ultra scale‐down (USD) device has been developed for the characterization of the response of a human cell line to membrane‐based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. The cell line used to develop the measurements was a clinically relevant human fibroblast cell line. The impact was evaluated by cell damage on completion of membrane processing as assessed by trypan blue exclusion and release of intracellular lactate dehydrogenase (LDH). Similar insight was gained from both methods and this allowed the extension of the use of the LDH measurements to examine cell damage as it occurs during processing by a combination of LDH appearance in the permeate and mass balancing of the overall operation. Transmission of LDH was investigated with time of operation and for the two disc speeds investigated (6,000 and 10,000 rpm or ? _max ≈ 1.9 and 13.5 W mL^?1, respectively). As expected, increased energy dissipation rate led to increased transmission as well as significant increases in rate and extent of cell damage. The method developed can be used to test the impact of varying operating conditions and cell lines on cell damage and morphological changes. Biotechnol. Bioeng. 2017;114: 1241–1251. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

An ultra scale‐down analysis of the recovery by dead‐end centrifugation of human cells for therapy

An ultra scale‐down method is described to determine the response of cells to recovery by dead‐end (batch) centrifugation under commercially defined manufacturing conditions. The key variables studied are the cell suspension hold time prior to centrifugation, the relative centrifugal force (RCF), time of centrifugation, cell pellet resuspension velocities, and number of resuspension passes. The cell critical quality attributes studied are the cell membrane integrity and the presence of selected surface markers. Greater hold times and higher RCF values for longer spin times all led to the increased loss of cell membrane integrity. However, this loss was found to occur during intense cell resuspension rather than the preceding centrifugation stage. Controlled resuspension at low stress conditions below a possible critical stress point led to essentially complete cell recovery even at conditions of extreme centrifugation (e.g., RCF of 10000 g for 30 mins) and long (~2 h) holding times before centrifugation. The susceptibility to cell loss during resuspension under conditions of high stress depended on cell type and the age of cells before centrifugation and the level of matrix crosslinking within the cell pellet as determined by the presence of detachment enzymes or possibly the nature of the resuspension medium. Changes in cell surface markers were significant in some cases but to a lower extent than loss of cell membrane integrity. Biotechnol. Bioeng. 2015;112: 997–1011. © 2014 Wiley Periodicals, Inc.     

Ultra scale‐down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths

This article describes how a combination of an ultra scale‐down (USD) shear device feeding a microwell centrifugation plate may be used to provide a prediction of how mammalian cell broth will clarify at scale. In particular a method is described that is inherently adaptable to a robotic platform and may be used to predict how the flow rate and capacity (equivalent settling area) of a centrifuge and the choice of feed zone configuration may affect the solids carry over in the supernatant. This is an important consideration as the extent of solids carry over will determine the required size and lifetime of a subsequent filtration stage or the passage of fine particulates and colloidal material affecting the performance and lifetime of chromatography stages. The extent of solids removal observed in individual wells of a microwell plate during centrifugation is shown to correlate with the vertical and horizontal location of the well on the plate. Geometric adjustments to the evaluation of the equivalent settling area of individual wells (Σ_M) results in an improved prediction of solids removal as a function of centrifuge capacity. The USD centrifuge settling characteristics need to be as for a range of equivalent flow rates as may be experienced at an industrial scale for a machine of different shear characteristics in the entry feed zone. This was shown to be achievable with two microwell‐plate based measurements and the use of varying fill volumes in the microwells to allow the rapid study of a fivefold range of equivalent flow rates (i.e., at full scale for a particular industrial centrifuge) and the effect of a range of feed configurations. The microwell based USD method was used to examine the recovery of CHO‐S cells, prepared in a 5 L reactor, at different points of growth and for different levels of exposure to shear post reactor. The combination of particle size distribution measurements of the cells before and after shear and the effect of shear on the solids remaining after centrifugation rate provide insight into the state of the cells throughout the fermentation and the ease with which they and accumulated debris may be removed by continuous centrifugation. Hence bioprocess data are more readily available to help better integrate cell culture and cell removal stages and resolve key bioprocess design issues such as choice of time of harvesting and the impact on product yield and contaminant carry over. Operation at microwell scale allows data acquisition and bioprocess understanding over a wide range of operating conditions that might not normally be achieved during bioprocess development. Biotechnol. Bioeng. 2009; 104: 321–331 © 2009 Wiley Periodicals, Inc.     

Ultra scale‐down of protein refold screening in microwells: Challenges,solutions and application

Steps for the refolding of proteins from solubilized inclusion bodies or misfolded product often represent bottlenecks in process development, where optimal conditions are typically derived empirically. To expedite refolding optimization, microwell screening may be used to test multiple conditions in parallel. Fast, accurate, and reproducible assays are required for such screening processes, and the results derived must be representative of the process at full scale. This article demonstrates the use of these microscale techniques to evaluate the effects of a number of additives on the refolding of IGF‐1 from denatured inclusion bodies, using an established HPLC assay for this protein. Prior to this, microwell refolding was calibrated for scale‐up using hen egg‐white lysozyme (HEWL) as an initial model protein, allowing us to implement and compare several assays for protein refolding, including turbidity, enzyme activity, and chromatographic methods, and assess their use for microwell‐based experimentation. The impact of various microplate types upon protein binding and loss is also assessed. Solution mixing is a key factor in protein refolding, therefore we have characterized the effects of different methods of mixing in microwells in terms of their impact on protein refolding. Our results confirm the applicability and scalability of microwell screening for the development of protein refolding processes, and its potential for application to new inclusion body‐derived protein products. Biotechnol. Bioeng. 2009;103: 329–340. © 2008 Wiley Periodicals, Inc.     

Ultra scale‐down device to predict dewatering levels of solids recovered in a continuous scroll decanter centrifuge

During centrifugation operation, the major challenge in the recovery of extracellular proteins is the removal of the maximum liquid entrapped within the spaces between the settled solids–dewatering level. The ability of the scroll decanter centrifuge (SDC) to process continuously large amounts of feed material with high concentration of solids without the need for resuspension of feeds, and also to achieve relatively high dewatering, could be of great benefit for future use in the biopharmaceutical industry. However, for reliable prediction of dewatering in such a centrifuge, tests using the same kind of equipment at pilot‐scale are required, which are time consuming and costly. To alleviate the need of pilot‐scale trials, a novel USD device, with reduced amounts of feed (2 mL) and to be used in the laboratory, was developed to predict the dewatering levels of a SDC. To verify USD device, dewatering levels achieved were plotted against equivalent compression (Gt_comp) and decanting (Gt_dec) times, obtained from scroll rates and feed flow rates operated at pilot‐scale, respectively. The USD device was able to successfully match dewatering trends of the pilot‐scale as a function of both Gt_comp and Gt_dec, particularly for high cell density feeds, hence accounting for all key variables that influenced dewatering in a SDC. In addition, it accurately mimicked the maximum dewatering performance of the pilot‐scale equipment. Therefore the USD device has the potential to be a useful tool at early stages of process development to gather performance data in the laboratory thus minimizing lengthy and costly runs with pilot‐scale SDC. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1494–1502, 2013     

Ultra scale-down studies of the effect of flow and impact conditions during E. coli cell processing

The ability to recover cells from a fermentation broth in an intact form can be an important criterion for determining the overall performance of a recovery and purification sequence. Disruption of the cells can lead to undesired contamination of an extracellular product with intracellular components and vice versa loss of intracellular products may occur. In particular, the value of directed location of a product in the periplasmic space of say Escherichia coli (E. coli) would be diminished by such premature non-selective cell disruption. Several options exist for cell recovery/removal; namely centrifugation, in batch or continuous configuration, filtration or membrane operations, and in selected cases expanded beds. The choice of operation is dependant on many variables including the impact on the overall process sequence. In all cases, the cells are exposed to shear stresses of varying levels and times and additionally such environments exist in ancillary operations such as pumping, pipe flow, and control valves. In this study, a small-scale device has been designed to expose cells to controlled levels of shear, time and impact in a way that seeks to mimic those effects that may occur during full-scale processes. The extent of cell breakage was found to be proportional to shear stress. An additional level of breakage occurred due to the jet impacting on the collecting surface. Here it was possible to correlate the additional breakage with the impact velocity, which is a function of the distance that the jet travels before meeting the collection surface and the initial jet velocity.     

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. © 2010 Wiley Periodicals, Inc.     

Mimic of a large‐scale diafiltration process by using ultra scale‐down rotating disc filter

Ultra scale‐down (USD) approach is a powerful tool to predict large‐scale process performance by using very small amounts of material. In this article, we present a method to mimic flux and transmission performance in a labscale crossflow operation by an USD rotating disc filter (RDF). The Pellicon 2 labscale system used for evaluation of the mimic can readily be related to small pilot and industrial scale. Adopted from the pulsed sample injection technique by Ghosh and Cui (J Membr Sci. 2000;175:5‐84), the RDF has been modified by building in inserts to allow the flexibility of the chamber volume, so that only 1.5 mL of processing material is required for each diafiltration experiment. The reported method enjoys the simplicity of dead‐end mode operation with accurate control of operation conditions that can mimic well the crossflow operation in large scale. Wall shear rate correlations have been established for both the labscale cassette and the USD device, and a mimic has been developed by operating both scales under conditions with equivalent averaged shear rates. The studies using E. coli lysate show that the flux vs. transmembrane pressure profile follows a first‐order model, and the transmission of antibody fragment (Fab′) is independent of transmembrane pressure. Predicted flux and transmission data agreed well with the experimental results of a labscale diafiltration where the cassette resistance was considered. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Ultra scale‐down approaches for clarification of mammalian cell culture broths in disc‐stack centrifuges

Ultra‐scale down (USD) methodology developed by University College London for cell broth clarification with industrial centrifuges was applied to two common cell lines (NS0 and GS‐CHO) expressing various therapeutic monoclonal antibodies. A number of centrifuges at various scales were used with shear devices operating either by high speed rotation or flow‐through narrow channels. The USD methodology was found effective in accounting for both gravitational and shear effects on clarification performance with three continuous centrifuges at pilot and manufacturing scales. Different shear responses were observed with the two different cell lines and even with the same cell line expressing different products. Separate particle size analysis of the treated broths seems consistent with the shear results. Filterability of the centrifuged solutions was also evaluated to assess the utility of the USD approach for this part of the clarification operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

An ultra scale‐down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product‐related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell‐based ultra scale‐down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non‐hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (～10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand. Biotechnol. Bioeng. 2013; 110: 1973–1983. © 2013 Wiley Periodicals, Inc.     

Ultra scale‐down approach to correct dispersive and retentive effects in small‐scale columns when predicting larger scale elution profiles

Ultra scale‐down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale‐down mimics must predict large‐scale process performance accurately. For example, one application of a scale‐down model involves using it to predict large‐scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large‐scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale‐down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale‐down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory‐ and 18.3 L pilot‐scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger‐scale elution profiles. These data then provide the justification to adjust small‐scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large‐scale and which can then be used for subsequent ultra scale‐down operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Ultra scale‐down characterization of the impact of conditioning methods for harvested cell broths on clarification by continuous centrifugation—Recovery of domain antibodies from rec E. coli

The processing of harvested E. coli cell broths is examined where the expressed protein product has been released into the extracellular space. Pre‐treatment methods such as freeze–thaw, flocculation, and homogenization are studied. The resultant suspensions are characterized in terms of the particle size distribution, sensitivity to shear stress, rheology and solids volume fraction, and, using ultra scale‐down methods, the predicted ability to clarify the material using industrial scale continuous flow centrifugation. A key finding was the potential of flocculation methods both to aid the recovery of the particles and to cause the selective precipitation of soluble contaminants. While the flocculated material is severely affected by process shear stress, the impact on the very fine end of the size distribution is relatively minor and hence the predicted performance was only diminished to a small extent, for example, from 99.9% to 99.7% clarification compared with 95% for autolysate and 65% for homogenate at equivalent centrifugation conditions. The lumped properties as represented by ultra scale‐down centrifugation results were correlated with the basic properties affecting sedimentation including particle size distribution, suspension viscosity, and solids volume fraction. Grade efficiency relationships were used to allow for the particle and flow dynamics affecting capture in the centrifuge. The size distribution below a critical diameter dependant on the broth pre‐treatment type was shown to be the main determining factor affecting the clarification achieved. Biotechnol. Bioeng. 2014;111: 913–924. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

A scale‐down mimic for mapping the process performance of centrifugation,depth and sterile filtration

In the production of biopharmaceuticals disk‐stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot‐scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale‐down approach based upon the use of a shear device and a bench‐top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large‐scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934–1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

An ultra scale‐down approach to study the interaction of fermentation,homogenization, and centrifugation for antibody fragment recovery from rec E. coli

Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high‐pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale‐down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high‐pressure homogenization for recovery of an antibody fragment (Fab′) and the impact of fermentation harvest time. This approach is followed by microwell‐based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high‐pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab′ release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale. Biotechnol. Bioeng. 2013 9999:XX–XX. © 2013 Wiley Periodicals, Inc. Biotechnol. Bioeng. 2013; 110: 2150–2160. © 2013 Wiley Periodicals, Inc.