Clarification of recombinant proteins from high cell density mammalian cell culture systems using new improved depth filters

Abstract

Increasingly high cell density, high product titer cell cultures containing mammalian cells are being used for the production of recombinant proteins. These high productivity cultures are placing a larger burden on traditional downstream clarification and purification operations due to higher product and impurity levels. Controlled flocculation and precipitation of mammalian cell culture suspensions by acidification or using polymeric flocculants have been employed to enhance clarification throughput and downstream filtration operations. While flocculation is quite effective in agglomerating cell debris and process related impurities such as (host cell) proteins and DNA, the resulting suspension is generally not easily separable solely using conventional depth filtration techniques. As a result, centrifugation is often used for clarification of cells and cell debris before filtration, which can limit process configurations and flexibility due to the investment and fixed nature of a centrifuge. To address this challenge, novel depth filter designs were designed which results in improved primary and secondary direct depth filtration of flocculated high cell density mammalian cell cultures systems feeds, thereby providing single-use clarification solution. A framework is presented here for optimizing the particle size distribution of the mammalian cell culture systems with the pore size distribution of the gradient depth filter using various pre-treatment conditions resulting in increased depth filter media utilization and improved clarification capacity. Feed conditions were optimized either by acidification or by polymer flocculation which resulted in the increased average feed particle-size and improvements in throughput with improved depth filters for several mammalian systems.

A modular approach for the ultra-scale-down of depth filtration

“…Particle characterization can be used to interrogate depth filtration and provide insight on scalability [3,15,31,32]. To assess the utility of particle characterization for clarity measurements, particle characteristics were compared with Hach turbidity measurements for a set of filtrate samples collected from four depth filters (Fig.…”

“…pH, conductivity, etc.). Screening of filtration conditions could be integrated with existing high throughput harvest, particle conditioning, and clarification workflows [3,[10][11][12][13][14][15][16]. Laboratory qualification of clinical production processes could be expedited and concluded with less material.…”

“…Depth filters are commonly employed for clarification of cellular debris, such as host cell proteins and DNA, or for capture of solid product [1][2][3]. Although often used for steric retention, the adsorptive properties of certain filters offer the potential for targeted removal of impurities at any location in a purification train and have utility as polishing steps [4].…”

Technology advancements in antibody purification

“…However, depth filtration was tested by Singh et al 112 as a method for primary recovery. The authors suggest that it is a method that could be used to purify recombinant proteins that are currently being produced in high titer numbers in mammalian cells.…”

“…The authors suggest that it is a method that could be used to purify recombinant proteins that are currently being produced in high titer numbers in mammalian cells. 111 By optimizing particle size and pore size and testing various pretreatment conditions, Singh et al 112 have designed new and improved "Clarisolve TM " (Merck Millipore, Billerica, MA, USA) depth filters that could be used for primary clarification of flocculated material or as disposable filters for single-step filtration. 111 …”