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Ultra Scale-Down Discovery of Low Shear Stress Processing For Selective Recovery of Next Generation Fusion Proteins Department of Biochemical Engineering, University College London: E. Lau (presenting author, e-mail: [email protected]), S. Kong, M. Hoare ImmunoBiology Limited, Cambridge, UK: S. McNulty, C. Entwisle, A. McIlgorm, K. Dalton Abstract Fusion protein products potentially pose increased challenges in preparation and purification. Of particular concerns are: (i) the impact of shear stress related effects on product integrity and (ii) the presence of product-related contaminants which could prove challenging to remove during the high resolution purification steps. USD studies demonstrated the value of low shear stress centrifugation or filtration in achieving selective recovery of the required molecular form of the product. USD studies were also used to predict improved removal of contaminants such as lipids and nucleic acids and cellular debris when using filtration. These USD predictions were verified at pilot scale. Introduction Fc-fusion proteins are molecules in which the immunoglobulin Fc region is fused to a protein of interest (Huang, 2009). One potential application of Fc-fusion proteins is as a vaccine. In this presentation, the processing of a Fcfusion protein vaccine candidate against the Hepatitis C virus (HCV) is presented. Generic processing platforms are in existence for the production of therapeutic grade antibody and antibody-like molecules in order to accelerate the process development of potential drug candidates. One advantage of the presence of a hIgG1-Fc region is that it enables purification using Protein A chromatography (Huang, 2009) once a suitable level of broth clarification is achieved using clarification platforms such as centrifugation (Hutchinson et al. 2006; Kelley et al. 2009) or depth filtration (Prashad and Tarrach, 2006; Yigzaw et al. 2006). Glossary Equations OD s − OD0 × 100% S = OD − OD f 0 1) % solid remaining t 1 1 = + * V * Q0* Vmax 2) Pore restriction model 3) Pore restriction model accounted for start-up errors (t − t0 ) (V * − V0 * ) = (V * t 1 (t − t0 ) + 1 * * − V0 Q0 ) max Product profile during fermentation Day A MW 4 6 7 8 10 12 188 kDa 98 kDa 98 kDa % solid remaining OD600 of sample OD600 of well spun sample OD600 of feed Filtration time (s) Time for start-up condition (s) USD prediction of centrifuge capacity (Q/∑) (m s-1) Total filtrate per filter area (L m-2) Total filtrate per area for the start-up phase (L m-2) Min. flux (L m-2 h-1) Ultra scale-down S ODs OD0 ODf t t0 V/t∑ V* V0* Q*min USD Pilot scale 5 L STR fermentation Initial flux post-start-up (LMH) Fully glycosylated fusion protein with binding efficacy to human CD64 receptor Alternative glycosylation form of mIB7 Dimer form of mIB7 Dimer form of mIB7* Monomer form of mIb7 Monomer form of mIB7* Q0 mIB7 mIB7* mIB7d mIB7d* mIB7m mIB7m* Ultra Scale-Down (USD) approach mIB7d* Non-reducing MW 4 6 B Day 7 8 10 12 mIB7m mIB7m* 38 kDa Reducing Western blots of bioreactor samples (Human-Fc detection). Day-4, 6, 7, 8, 10 and 12 samples are presented. Clarified cell culture samples were purified using Protein A. Two gel systems were utilised for Western blot analysis. (A) non-reducing and (B) reducing. mIB7d* is a different glycosylated variant with lower molecular weight than mIB7d. In order to reduce heterogeneity in process stream, the cell culture broth was harvested in day 6 in order to maximise recovery mIB7d relative to mIB7d*. Disc stack continuous centrifuge (CSA-1, Westfalia) with 0.6L bowl volume High-speed rotating disc shear device provides shearing Cuno ZetaPlus 05SP depth filter with 25 cm2 filter area Custom built filter media housing with 0.28 cm2 • Product-related contaminant: mIB7* V* (L m-2) 100 Filtration time (s) 150 200 % Solids remaining D Left: Effect of centrifugation and filtration on Fc-fusion protein mIB7 recovery and contaminants level. (A) The recovery levels of ELISA+ve material are marginally lower in filtration samples. (B) DNA and (C) Lipid levels in depth filtration are ~50% and ~40% lower than obtained for centrifugation respectively. (D) The % solid remaining in USD and pilot-scale depth filtration experiments are ~60% lower than those obtained for both centrifugation scales 2 (Note, the final broth load per filter area was similar for both filtration scales studied, i.e. 1.7 mL cm-2). 1 B io n ifu ga tio n ga t ri fu nt r d d ar ar nd nd ta n r sta ion n n er s tio io k e 7 at tio rk Ib7 ga at a a ar Ib ifug n n ug m m rifu n n ug t m d m tr tio tio trif ht ied ent tio atio trif a a g gh ifie cen a n n r i i r lt g e lt g e e rif c e r w pu ale tion e fi rifu e c w pu ale tion e fi rifu e c l t l c a al nt al c a ar ar c e c ul n A s iltr c a en c a ul n A s iltr ec ei ch f t-s c t-s ec ei ch f t-s c t-s ol rot en SD ilo SD ilo ol rot en SD ilo SD ilo M P B U M P B U P U P P U P sc al e ce nt n at io n ce P ilo t- ilo P U tsc al e fil tr tio tio fil tr a ifu ga D S ce nt r le SD n 0 191 kDa Day 4 USD prediction Day 6 USD prediction , Day 4 pilot , Day 6 pilot Predictions from pilot data and model mIB7dimer mIB7dimer* mIB7monomer mIB7monomer* 97 kDa 64 kDa 20 Non-reducing Reducing 51 kDa Above: Western blots showing supernatant and filtrate qualities at USD and pilot scale. The filtrates obtained from both USD and pilot scale are of comparable quality in terms of product molecular weight bands. They are also similar to those obtained for bench scale centrifugation (no shear) or low shear stress USD and pilot-scale centrifugation. 0 50 ELISA positive 0.0 3 10 0 0.5 70 (B) % solids remaining in supernatant based on OD600 measurements following centrifugal clarification at various equivalent V/t∑lab. Low shear treatment mimics hydro-hermetic feed zone found in CSA-1. Significant increases from 30% to 150% in solids carryover are observed as a result of exposure to low shear stress levels in USD studies. Day 4 USD Day 6 USD 1.0 -1 Option 2: Depth filtration A C U 60 (A) Western blot of a reducing gel showing sensitivity to shear stress of day 4 samples at (V/t∑)lab of 21.9, 43.4 and 63.7 ×10-9 m s-1. Cells were subjected to no shear stress, low shear stress (0.019×106 W kg-1) and high shear stress (0.37×106 W kg-1) environments. Lane F is the well spun sample subjected to no shear stress , , materials (mg L-1) Lipid (g L-1) 50 -9 10 1 sc a 40 V/tΣ Σlab (x10 m s ) 30 B en ch 30 Study of supernatant qualities observed during primary clarification of day 4 and day 6 CHO-S cultures. 30 0 2 0 1 20 40 A 6 DNA (mg L-1) 2 0 40 12 B % Solids remaining B Day 6 no shear Day 6 low shear Day 6 CSA-1 3 Tecan Freedom EVO® robotic liquid handling platform and data logging USD vs Pilot scale 4 20 96 deep square well plates provide high throughput V/tƉ screen using bench top centrifuge • Product: mIB7 (expressed as dimer) Option 1: Centrifugation V* (L m-2) * mIB7d 62 kDa 0 Max. volume filtered per filter area (L m-2) V*max 0 50 100 150 Conclusion 200 Filtration time (s) Effect of cell culture age on filter performance for USD and pilot-scale constant pressure depth filtration (at 100 mBar). (A) Raw data for day 4 and day 6 USD filtration. (B) Comparison of pilot-scale filtration profiles and prediction out of USD analysis. Pilot-scale data is modelled with resultant V*max values of 40 L m-2 (day 4) and 28 L m-2 (day 6). These compare with predicted values from USD analysis of 45 L m-2 (day 4) and 24 L m-2 (day 6). Data obtained from USD and pilot scale processes were comparable in terms of % solid carryover, product recovery, contaminating DNA and lipids and mIB7d quality. Product variations increased with culture age. Therefore, to recover a secreted product with minimal product-related variants, using a day 6 harvest combined with depth filtration as primary recovery step is recommended. In conclusion, the USD techniques employed in this study were predictive of their pilot scale counterpart. Publication: Eduardo Lau, Simyee Kong, Shaun McNulty, Claire Entwisle, Ann Mcilgorm, Kate Dalton, Mike Hoare, 2013, “An Ultra Scale-Down Characterization of Low Shear Stress Primary Recovery Stages to Enhance Selectivity of Fusion Protein Recovery From Its Molecular Variants”, Biotechnology and Bioengineering, Volume 110, Issue 7, pages 1973-1983 References: Chichi Huang, 2009, “Receptor-Fc fusion therapeutics, traps, and MIMETIBODYTM technology”, Current Opinion in Biotechnology, 20:692-699. Hutchinson et al., 2006, “ Shear stress analysis of mammalian cell suspension for prediction of industrial centrifugation and its verification”, Biotech. And Bioeng., 95:483-491. Kelley et al., 2009, “Downstream processing of monoclonal antibodies: Current practices and further opportunities”, In: Gottschalk U, editor. Process scale purification of antibodies. P1-23. Prashad et al., 2006, “Depth filtration: Cell clarification of bioreactor offloads”, Filtration & Separation, 43(7):28-30. Yigzaw et al., 2006, “Exploitation of the adsorption properties of depth filters for host cell protein removal during monoclonal antibody purification”, Biotechnol. Prog., 22:288-296.