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Pharmaceutical Review 3 Review Industrial application of impurity flocculation to streamline antibody purification processes Pharm. Bioprocess. Flocculation technologies offer significant benefits to industrial mammalian cell Michael Felo1, Yun Kenneth culture processes, including increased clarification efficiency, impurity removal and Kang*,2, James Hamzik1, Paul process simplification. In this paper, the application and performance of flocculation Balderes2 & Dale L Ludwig2 1 technologies employed in the harvest process of monoclonal antibody Chinese hamster Process Solutions, EMD Millipore, 290 Concord Road, Billerica, MA 01821, ovary cell culture are reviewed. Attention has been placed on technologies enhancing USA the removal of cells, cellular debris and reduction in impurities while maintaining the 2Bioprocess Sciences, Eli Lilly & Company, antibody in the product stream. Many flocculants are systematically evaluated with 450 East 29th Street, New York, respect to their mechanism of action, impact on downstream processing and product NY 10016, USA quality, and potential disadvantages. Practical considerations and future directions *Author for correspondence: Tel.: +1 646 638 5109 for application of flocculation in antibody manufacturing are discussed. Fax: +1 212 213 4785 [email protected] Flocculation is a process that has been time of harvest have dramatically increased, widely implemented in the chemical and though typically with corresponding eleva- food industries, as well as in waste water tions in cell density, cellular debris and treatment. To the contrary, it has only been related cellular impurities [5,6]. As a result, relatively recently that flocculation-based the challenges of separating cells and pretreatment of bacterial fermentation and debris now more closely parallel those of mammalian cell culture has been explored microbial fermentation processes. It is and, in several cases, implemented in order therefore not surprising that the evalua- to circumvent both harvest and purifica- tion of flocculation to enhance the perfor- tion challenges experienced in therapeutic mance of this step has become an area of protein production [1–3] . Flocculation has intense investigation. Specifically, floccu- been slow to gain acceptance in the manu- lants such as simple acids, divalent cations, facture of monoclonal antibodies (mAbs), polycationic polymers, caprylic acid and 2 where mammalian cell culture processes stimulus-responsive polymers have been are typically utilized. Until recently, com- evaluated for their ability to enhance cell mercial processes employing cell lines such culture clarification and reduce the levels of as CHO, NS0 and SP2/0 have only rarely DNA, host cell proteins (HCP) and viruses. 2015 reached cell densities where removal of sol- In this paper, the application and perfor- ids or cell-related soluble impurities during mance of various flocculation method in the the harvest step becomes excessively burden- harvest process of CHO cell culture produc- some. Standard unit operations such as cen- tion of mAbs are reviewed. Focus has been trifugation, depth filtration and microfiltra- placed on methodsthat enhance the removal tion, in most cases, have efficiently produced of cells, cellular debris and reduce impurity harvested cell culture fluid (HCCF) suitable levels while maintaining the antibody within for d ownstream purification [4]. the product stream. Though the process Through advances in mammalian cell cul- goals are similar for each of these flocculants, ture over the past 10–15 years, particularly the mechanisms of action, impact on product those in fed-batch and product-retaining quality and potential toxicity from residual perfusion processes, the mAb titers at the flocculant vary significantly. part of 10.4155/PBP.15.2 © 2015 Future Science Ltd Pharm. Bioprocess. (2015) 3(2), 115–125 ISSN 2048-9145 115 Review Felo, Kang, Hamzik, Balderes & Ludwig Key terms lead to further product loss and increases in process Flocculation: Flocculation is the agglomeration of costs for both filters and housings [10] . particles (cells, cell debris or colloids in mammalian cell In recent years, flocculation-based pretreatment culture) caused by a bridging effect induced by the of cell cultures for mAb production has been inves- flocculant. tigated [2,3,9,12–17]. Many flocculation agents, or floc- Stimulus-responsive polymer: Stimulus-responsive culants, ranging from simple electrolytes to synthetic polymers are high-performance polymers that change polyelectrolytes (or polymers) have been evaluated physical properties according to their environment. Such (Table 1). Flocculation is the agglomeration of particles materials can be sensitive to a number of factors, such as temperature, pH, conductivity and specific stimulus (cells, cell debris or colloids in mammalian cell culture) reagents. caused by a bridging effect induced by the flocculant. Flocculation agents bind to particles largely through Although the effectiveness of flocculation has been electrostatic charge interaction, although additional frequently demonstrated, adoption remains slow in bio- interactive forces such as hydrophobic interaction or pharmaceutical industrial applications. Critical issues hydrogen bonding may be involved [3,4,18]. Flocculated including platform process fit, flocculant clearance cell cultures have higher mean particle sizes compared downstream and regulatory concerns over implementa- with their untreated counterparts (Figure 1), which has tion have hindered routine incorporation of the technol- been observed to consistently correlate with improved ogy. Still, process and industry trends toward high cell centrate quality and filterability. Notably, positively density cell cultures, continuous processes, single-use charged flocculants, such as polyamines [14], cal- technologies and lower culture production volumes are cium chloride/potassium phosphate [16], chitosan [15], anticipated to continue to drive the development and polydiallyldimethyl ammonium chloride (PDAD- implementation of these flocculation technologies into MAC) [2,9,19] and stimulus-responsive polymers [3] have industrial biopharmaceutical m anufacturing processes. been shown to be successful in inducing flocculation, resulting in improved clarification efficiency, process Advances in cell culture processes drive yield and clearance of impurities during the primary development of flocculation for mAb mAb recovery process from mammalian cell culture. production process Advances in cell culture processes have significantly Performance, limitations & application of increased mAb titers to levels as high as 30 g/l. These flocculation methods for mAb production improvements have led to more challenging conditions processes for downstream processing, including cell densities (up Acid precipitation to 108 cells per ml), packed cell volume (up to 40% v/v) Reduction of cell culture pH at the time of harvest and increased levels of soluble impurities such as HCP is a straightforward approach to induce flocculation and high molecular weight (HMW) species [2,3,7–9]. and precipitation. This is accomplished through the Advances in mAb downstream processing are required introduction of concentrated acid solutions including in order to overcome the significant challenges posed acetic, phosphoric and citric acids. The acid solution by increased cell culture productivity. typically drives the cell culture fluid from a neutral pH Increases in cell mass, cell debris and colloids from to between pH 5.5 and 4.0, most commonly between high cell density culture, place a greater burden on the pH 5.0 and 4.5. This change in pH forces a transition clarification train, specifically on continuous disk-stack across the isoelectric points of some HCP impurities, centrifugation and depth filtration, which continue to rendering them insoluble through charge neutraliza- be routinely employed in the harvest step [10] . Cul- tion. However, this impurity reduction must be bal- ture suspensions containing high solid content with a anced with the stability and quality of the product wide particle size distribution and a high percentage under tested conditions during the harvest operation. of small particles further complicate the harvest unit The shift in particle size caused by acid precipita- operation, requiring significantly higher depth filter tion/flocculation is generally less pronounced than surface area and, in some cases, exceeding the existing that induced by polymer-based flocculation methods. filtration train capacity [11] . During the centrifugation Still, particle size increase induced by lower pH has process, high solid content in the cell culture feed leads been reported, leading to improved efficiency of con- to increases in pellet ejection interval and decreases tinuous disk-stack centrifugation [11] or microfiltration in product recovery due to volume losses. Submicron harvest processes [20]. For harvest processes utilizing particles are not easily removed by centrifugation and centrifugation, the larger particulates are more easily are left to the depth filtration, and perhaps sterilizing- removed from the cell culture, and the capacity of the grade filter, for removal. Increases in filtration areacan secondary depth filtration step has been increased by 116 Pharm. Bioprocess. (2015) 3(2) future science group Table 1. Comparison of different flocculants used to streamline cell culture harvest process and enhance the removal of impurities. Flocculant Mechanism