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

The availability of material for experimental studies is a key constraint in the development of full-scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale-down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modelling, and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only millilitre quantities before embarking on highly demanding full-scale studies. In this study the performance of a pilot-scale tangential flow filtration (TFF) system based on a membrane flat-sheet cassette using pumped flow was predicted by devising an USD device comprising a stirred cell using a rotating disc. The USD device operates with just 2.1 cm2 of membrane area and, for example, just 1.7 mL of feed for diafiltration studies. The novel features of the design involve optimisation of the disc location and the membrane configuration to yield an approximately uniform shear rate. This is characterised using computational fluid dynamics for a defined layer above the membrane surface. A pilot-scale TFF device operating at ~500-fold larger feed volume and membrane area was characterised in terms of the shear rate derived from flow rate-pressure drop relationships for the cassette. Good agreement was achieved between the USD and TFF devices for the flux and resistance values at equivalent average shear rates for a monoclonal antibody diafiltration stage.

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 dependent on the broth pre-treatment type was shown to be the main determining factor affecting the clarification achieved.

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli.

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.

Development of a high-yield live-virus vaccine production platform using a novel fixed-bed bioreactor.

The increasing importance of viral vaccine manufacturing has driven the need for high cell density process optimization that allows for higher production levels. Vero cells are one of the more popular adherent cell lines used for viral vaccine production. However, production is limited due to the logistical limitations surrounding adherent cell line processes, such as large equipment footprints, time and labor-intensive processes, and larger costs per dose. We have addressed this limitation with the establishment of a viral vaccine production system utilizing the novel single use scale-X™ carbo bioreactor. The unit is compact and is scalable and one of the novel features of the system is the continuous in-line downstream purification and concentration processes associated with the bioreactor vessel. We present the results from a campaign featuring a proprietary Vero cell line for production of a live recombinant Vesicular stomatitis virus vaccine that features the Lassa Fever virus glycoproteins. Metabolite analyses and viral yield comparison between traditional flasks, cell factories, and the scale-X carbo bioreactor were performed, and on average, the single use bioreactor produced 2-4 logs higher titers per surface area, approximately 5 × 1010 pfu/cm2, compared to classical flatstock, 2.67 × 106 pfu/cm2, and cell factories production, 5.77 × 108 pfu/cm2. Overall, we describe a novel bioreactor platform that allows for a cost-efficient and scalable process for viral vaccine production.

Evaluation of options for harvest of a recombinant E. Coli fermentation producing a domain antibody using ultra scale-down techniques and pilot-scale verification.

Ultra scale-down (USD) methods operating at the millilitre scale were used to characterise full-scale processing of E. coli fermentation broths autolysed to different extents for release of a domain antibody. The focus was on the primary clarification stages involving continuous centrifugation followed by depth filtration. The performance of this sequence was predicted by USD studies to decrease significantly with increased extents of cell lysis. The use of polyethyleneimine reagent was studied to treat the lysed cell broth by precipitation of soluble contaminants such as DNA and flocculation of cell debris material. The USD studies were used to predict the impact of this treatment on the performance and here it was found that the fermentation could be run to maximum productivity using an acceptable clarification process (e.g., a centrifugation stage operating at 0.11 L/m(2) equivalent gravity settling area per hour followed by a resultant required depth filter area of 0.07 m(2) /L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:382-392, 2016.