What Can Cell Culture Flocculation Offer for Antibody Purification Processes

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What Can Cell Culture Flocculation Offer for Antibody Purification Processes Editorial 2 Editorial What can cell culture flocculation offer for antibody purification processes “The eventual goal of the application of flocculation in monoclonal antibody processing is to develop a separation unit operation alternative to chromatography, with equivalent separation efficiency, Pharm. Bioprocess. high process capacity, good facility fit and sound process economics.” Keywords: antibody • flocculation • harvest • impurity removal • purification Recent advances in cell culture processes solid content with wide particle distribution Yun (Kenneth) Kang have significantly increased monoclonal anti- and a high percentage of small particles fur- Author for correspondence: body (mAb) titers to levels as high as 30 g/l. ther complicate the harvest unit operation, BioProcess Sciences, Eli Lilly Not unexpectedly, these improvements have requiring significantly higher depth filter & Company, 450 East 29th Street, New York, NY 10016, USA resulted in a new set of starting conditions surface area which may exceed the existing [email protected] for downstream processing, including: ele- filtration train capacity. Harvest operations vated cell density levels (up to 108 cells/ml), have become extremely challenging, if not Dale L Ludwig BioProcess Sciences, Eli Lilly increased solid content (packed cell volume, impossible because of these issues. & Company, 450 East 29th Street, up to 40%) and increased soluble impurity Flocculation has been widely used for New York, NY 10016, USA levels such as host cell proteins (HCP) and many years in the chemical and food indus- Paul Balderes high molecular weight species (HMW; up tries, as well as in wastewater treatment. In BioProcess Sciences, Eli Lilly to 40%) [1–4] . New advances in mAb down- recent years, flocculation-based pretreatment & Company, 450 East 29th Street, stream processing are critically needed, in of cell cultures to circumvent both harvest New York, NY 10016, USA order to meet the significant challenges posed and purification issues experienced in mAb by increased cell culture productivity. production has been explored and imple- Purification productivity can potentially mented [2–4,10–14], and is in effect a strategy be improved in a number of ways: through with the potential to ‘kill two birds with one process intensification by increasing chro- stone’. matography column dimensions, operating Many flocculation agents or flocculants, 6 in multiple production cycles, utilizing high ranging from simple electrolytes to synthetic capacity or mixed-mode chromatographic polyelectrolytes (or polymers) have been resins or membrane adsorbers or by reduc- investigated. Flocculation is the agglomera- ing the number of necessary purification tion of particles (cells, cell debris or colloids 2014 steps [5–8]. However, elevated levels of HCP in mammalian cell culture) caused by a bridg- and HMW in high cell density culture place ing effect induced by the flocculant. Floccu- greater demands on purification unit opera- lation agents are adsorbed to particles largely tions, often necessitating the use of novel or through electrostatic attraction although alternative technology to mitigate the higher additional interactive forces such as hydro- burden placed on downstream steps [1–4,9]. phobic interaction or hydrogen bonding may Additionally, increases in cell quantity, be involved [2,15–16]. Flocculated cell culture cell debris and colloids from high cell den- has a higher mean particle size compared sity culture pose significant challenges to the with untreated cell culture and increased harvest process, placing a greater burden on mean particle size has been observed to con- the centrifugation and/or clarification filter sistently correlate with improved filterability. train. Culture suspensions containing high Notably, positively charged flocculants, such 10.4155/PBP.14.33 © 2014 Future Science Ltd Pharm. Bioprocess. (2014) 2(6), 483–485 ISSN 2048-9145 483 Editorial Kang, Ludwig & Balderes as polyamines [12], calcium chloride/potassium phos- graphic resins, particularly Protein A. Third, removal phate [14], chitosan [13], polydiallyldimethyl ammo- of impurities prior to Protein A could reduce the nium chloride [3,4] and stimulus responsive polymer number of chromatographic steps and enable a two- [2] have been shown to be successful in inducing floc- column purification platform, with greater flexibility culation, resulting in improved clarification efficiency, in the selection of the second chromatographic step. process yield and clearance of impurities during the The second chromatography column could potentially primary mAb recovery process from mammalian cell be developed primarily for viral clearance purposes. culture. Membrane chromatography suitable for high produc- It has been shown that flocculation of cell culture tivity or continuous processing might also be explored can enhance the removal of cells and cell debris via [5,19]. Therefore, introduction of flocculant into the centrifugation, microfiltration or depth filtration and harvest process could result in a simplified and more increase filtration throughput or capacity[2, 13,17–18]. robust purification process and/or provide greater For example, chitosan used as a flocculant in mamma- process flexibility for mAb downstream processing, lian cell culture significantly improved centrifugation potentially paving the way for the implementation efficiency as indicated by reduced centrate turbidity, of a fully disposable and/or continuous purification resulting in a six- to seven-fold increase in the volumet- manufacturing process. ric throughput of the subsequent depth filtration step Finally, charged flocculants may serve as viral clear- [13] . In another case, depth filter capacity was increased ance agent through their ability to bind oppositely by more than eightfold when cell culture was floccu- charged viruses [10,17], thus providing an orthogo- lated with a smart polymer [2]. In each case, process nal viral removal step, again adding flexibility to the economics were improved due to reduction in filter manufacturing operation. costs and enhancement of process robustness, while In summary, flocculation may enable the develop- process yield and product quality were not adversely ment of a robust harvest process with the benefit of affected. removing potential obstacles to the downstream pro- cess. However, implementation of flocculation at com- mercial manufacturing scale should be assessed on a If flocculation is to be considered for use in “ case-by-case basis. The compatibility of flocculation to harvest operations, due diligence must be harvest unit operation, disk stack centrifugation, depth performed to verify its impact on downstream filtration or microfiltration, should first be evaluated chromatography operations. ” prior to implementation for an existing facility. Any cell culture, up to 2000 l, can be pretreated by floccu- Removal of process or product-related impurities lant directly in a bioreactor and then clarified by depth such as HCP, host DNA and HMW is an additional filter trains, making it attractive for high titer and low benefit that flocculation may provide. Introduction of volume cell culture processes. When cell culture scale a smart polymer directly into a bioreactor was demon- is larger than 2000 l, a high-capacity depth filtration strated to result in reduced impurity levels in Protein device should be evaluated in order to ensure com- A eluate, meeting the requirements of drug substance patibility with flocculation. Due to the high packed and, thereby, reducing the impurity load to subsequent cell volume, alternatively, settlement of cells and cell purification steps[2] . Furthermore, a synergistic effect debris for high cell density culture can be evaluated on impurity removal was observed when flocculation to increase filtration capacity, and decrease filtration was combined with acid precipitation [2]. The combi- area. However, the settling behaviors of the flocculated nation of flocculation and acid precipitation may there- culture need to be studied to ensure that the operation fore be beneficial and should be explored if the target can be performed within a reasonable timeframe and antibody is stable in tested acidic conditions. without negatively impacting product quality. Novel Separation of soluble impurities from antibody strategies may also be investigated to recover the mAb product during harvest can reduce the overall impu- product from settled cell pellets if needed. rity load to downstream purification steps, which may If flocculation is to be considered for use in harvest have several benefits. First, a higher product/impurity operations, due diligence must be performed to verify ratio could permit increased load on subsequent chro- its impact on downstream chromatography operations. matographic steps, resulting in higher capacity chro- Due to potential flocculant toxicity, residual flocculant matography unit operations. Second, a higher purity levels in the drug substance should be assessed and harvested culture fluid with lower centrate turbidity acceptable clearance must be reached to ensure the and soluble impurity levels can potentially increase the safety of the drug product [2,20]. The obvious advan- number of operation cycles and lifetime of chromato- tage of flocculation using stimulus responsive polymer 484 Pharm. Bioprocess. (2014) 2(6) future science group What can cell culture flocculation offer for antibody purification processes Editorial and precipitation
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