RESUMEN
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.
Asunto(s)
Biotecnología/métodos , Filtración/métodos , Proteínas Recombinantes/aislamiento & purificación , Animales , Células CHO , Agregación Celular , Recuento de Células , Técnicas de Cultivo de Célula , CricetulusRESUMEN
The processing of recombinant proteins from high cell density, high product titer cell cultures containing mammalian cells is commonly performed using tangential flow microfiltration (MF). However, the increased cellular debris present in these complex feed streams can prematurely foul the membrane, adversely impacting MF capacity and throughput. In addition, high cell density cell culture streams introduce elevated levels of process-related impurities, which increase the burden on subsequent purification operations to remove these complex media components and impurities. To address this challenge, an evaluation of mammalian cell culture broth buffer properties was examined to determine if enhanced impurity removal and clarification performance could be achieved. A framework is presented here for establishing optimized mammalian cell culture buffer conditions, involving trade-offs between product recovery and purification and improved clarification at manufacturing-scale production. A reduction in cell culture broth pH to 4.7-5.0 induced flocculation and impurity precipitation which increased the average feed particle-size. These conditions led to enhanced impurity removal and improved MF throughput and filter capacity for several mammalian systems. Feed conditions were further optimized by controlling ionic composition along with pH to improve product recovery from high cell density/high product titer cell cultures.
Asunto(s)
Reactores Biológicos , Medios de Cultivo/química , Inmunoglobulina G/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Animales , Tampones (Química) , Células CHO , Cricetinae , Cricetulus , Concentración de Iones de Hidrógeno , Inmunoglobulina G/biosíntesis , Proteínas Recombinantes/biosíntesisRESUMEN
The dramatic advances in recombinant DNA and proteomics technology over the past decade have supported a tremendous growth in biologics applied to diagnostics, biomarkers, and commercial therapeutic markets. In particular, antibodies and fusion proteins have now become a main focus for a broad number of clinical indications, including neurology, oncology, and infectious diseases with projected increase in novel first-class molecules and biosimilar entities over the next several years. In line with these advances are the improved analytical, development, and small-scale preparative methods employed to elucidate biologic structure, function, and interaction. A number of established methods are used for solvent removal, including lyophilization, reverse extraction, solute precipitation, and dialysis (solvent exchange), ultrafiltration, and chromatographic techniques. Notably, advances in the miniaturization and throughput of protein analysis have been supported by the development of a plethora of microscale extraction procedures and devices that exploit a wide array of modes for small-scale sample preparation, including the concentration and desalting of protein samples prior to further analysis. Furthermore, advances in process handling and data monitoring at microscale have dramatically improved complex control and product recovery of small quantities of biologics using techniques such as lyophilization and precipitation. In contrast, the efficient concentration of feed streams during preparative chromatography has been enhanced by improvements to protein binding capacity achieved through advanced bead and ligand design. The objective of solvent removal may be to prepare or concentrate solutes for analysis, or to facilitate their production or modification. Here, we describe the most recent advances in these techniques, particularly focusing on improved capabilities for bench-scale preparative methods.