CHO cell deposition during initial stages of clarification using the D0HC commercial depth filter in a Micro 20 capsule

Significant increases in cell density and product titer have led to renewed interest in the application of depth filtration for initial clarification of cell culture fluid in antibody production. The performance of these depth filters will depend on the local pressure and velocity distribution within the filter capsule, but these are very difficult to probe experimentally, leading to challenges in both process design and scale-up. We have used a combination of carefully designed experimental studies and computational fluid dynamics (CFD) to examine these issues in both lab-scale (SupracapTM 50) and pilot-scale (StaxTM ) depth filter modules, both employing dual-layer lenticular PDH4 media containing diatomaceous earth. The SupracapTM 50 showed a more rapid increase in transmembrane pressure and a more rapid DNA breakthrough during filtration of a Chinese Hamster Ovary cell culture fluid. These results were explained using CFD calculations which showed very different flow distributions within the modules. CFD predictions were further validated using measurements of the residence time distribution and dye binding in both the lab-scale and pilot-plant modules. These results provide important insights into the factors controlling the performance and scale-up of these commercially important depth filters as well as a framework that can be broadly applied to develop more effective depth filters and depth filtration processes.

Depth filtration is widely used in downstream bioprocessing to remove particulate contaminants via depth straining and is therefore applied to harvest clarification and other processing steps. However, depth filtration also removes proteins via adsorption, which can contribute variously to impurity clearance and to reduction in product yield. The adsorption may occur on the different components of the depth filter, that is, filter aid, binder, and cellulose filter. We measured adsorption of several model proteins and therapeutic proteins onto filter aids, cellulose, and commercial depth filters at pH 5-8 and ionic strengths <50 mM and correlated the adsorption data to bulk measured properties such as surface area, morphology, surface charge density, and composition. We also explored the role of each depth filter component in the adsorption of proteins with different net charges, using confocal microscopy. Our findings show that a complete depth filter's maximum adsorptive capacity for proteins can be estimated by its protein monolayer coverage values, which are of order mg/m2 , depending on the protein size. Furthermore, the extent of adsorption of different proteins appears to depend on the nature of the resin binder and its extent of coating over the depth filter surface, particularly in masking the cation-exchanger-like capacity of the siliceous filter aids. In addition to guiding improved depth filter selection, the findings can be leveraged in inspiring a more intentional selection of components and design of depth filter construction for particular impurity removal targets.

The removal of host cell proteins (HCPs) is crucial in biopharmaceutical production, as residual impurities can impact product safety and efficacy. While a number of studies have demonstrated that depth filtration can provide significant HCP removal, there is little information on its effectiveness in removing specific HCPs. This study examines the application of liquid chromatography‐mass spectrometry (LC‐MS) to track HCP removal during depth filtration, providing a detailed analysis of HCP behavior with two commercial depth filters. Our findings reveal significant variability in HCP breakthrough behavior, with transmission patterns showing minimal correlation with either the protein isoelectric point or hydrophobicity, highlighting the unique behavior of individual HCPs. Both the X0SP and X0HC depth filters achieved almost complete removal of Lipoprotein Lipase, and the X0SP filter also effectively removed Lysosomal Acid Lipase (LAL), both known to degrade polysorbate in monoclonal antibody formulations. However, neither filter provided significant removal of Alpha‐enolase, Carboxypeptidase D, Glutathione S‐transferase, or Phospholipase B‐like 2. The X0SP filter showed equal or better removal for 18 out of 20 problematic HCPs, with greater HCP removal seen at lower conductivity. This work provides a detailed framework for understanding and optimizing depth filtration processes, offering insights into the effectiveness of depth filters for removal of problematic HCPs.

Depth filtration can be very attractive for initial clarification because of low capital costs and ease of operation. However, there is currently no fundamental understanding of the effects of the filter pore size and morphology on the overall capacity and filtrate quality. The objective of this study was to examine the flux, capacity, and filtrate turbidity of a series of depth filters with different pore size ratings and multilayer structures for the filtration of yeast cell suspensions. Data were analyzed using available fouling models to obtain insights into the flux decline mechanisms. Filters with small pore size provide high filtrate quality at low capacity, with the reverse being true for the larger pore sizes. The multilayer structure of commercial depth filters leads to improved performance, although the choice of layer properties is critical. The highest capacity was achieved using a multilayer filter in which the upper layer allows significant yeast cell penetration into the filter matrix but still protects the retentive layer that is needed for a high quality filtrate.

Flow and residence time distribution in small-scale dual-layer depth filter capsules

Quantitative interpretation of protein breakthrough curves in small-scale depth filter modules for bioprocessing

Depth filtration is an attractive method for initial clarification of broth for removing cells and cell debris. Electrospun nanofibers with their large specific surface area and a porous structure are known as attractive materials in filtration processes. However, dead-end filtration of cells through nanofiber mats (NFM) always leads to cake formation and increasing resistance. In this study, for the first time, a nanofiber sponge (NFS) or nanofiber aerogel was synthetized from polyamide 6 (PA6) building blocks. The NFS was flexible, highly porous and mechanically stable. The pore size of the NFS was tuned between 8 and 26 µm during the cryogenic processing step. Volumetric flux and filtration efficiency of the NFS depended on the pore size and the results were compared with those for NFM from the same PA6 nanofiber material. Dead-end filtration of Saccharomyces cerevisiae was feasible at a low differential pressure of 3.5 kPa and cell filtration efficiency was > 99 %. Modelling of the filtration process revealed that cake formation is prevented by NFS filters since cells are able to penetrate into the filter and to adsorb on their internal surface. The filtration characteristics were also compared with commercial depth filters and revealed the high flux of NFS filters along with the possibility to avoid filter aids and a low environmental impact. PA6-NFS filters may become a new and cost effective generation of filters for removing different cells or cell debris from broth and other applications.

DNA retention on depth filters

Sonically enhanced depth filtration of aerosols

Experimental and theoretical analysis of loading characteristics of different electret media with various properties toward the design of ideal depth filtration for nanoparticles and fine particles

Impact of virus filter pore size / morphology on virus retention behavior

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product-related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell-based ultra scale-down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non-hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (~10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand.

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.

A single-stage clarification was developed using a single-use chromatographic clarification device (CCD) to recover a recombinant protein from Chinese Hamster Ovary (CHO) harvest cell culture fluid (HCCF). Clarification of a CHO HCCF is a complex and costly process, involving multiple stages of centrifugation and/or depth filtration to remove cells and debris and to reduce process-related impurities such as host cell protein (HCP), nucleic acids, and lipids. When using depth filtration, the filter train consists of multiple filters of varying ratios, layers, pore sizes, and adsorptive properties. The depth filters, in combination with a 0.2-micron membrane filter, clarify the HCCF based on size-exclusion, adsorptive, and charge-based mechanisms, and provide robust bioburden control. Each stage of the clarification process requires time, labor, and utilities, with product loss at each step. Here, use of the 3M™ Harvest RC Chromatographic Clarifier, a single-stage CCD, is identified as an alternative strategy to a three-stage filtration train. The CCD results in less overall filter area, less volume for flushing, and higher yield. Using bioprocess cost modeling, the single-stage clarification process was compared to a three-stage filtration process. By compressing the CHO HCCF clarification to a single chromatographic stage, the overall cost of the clarification process was reduced by 17%-30%, depending on bioreactor scale. The main drivers for the cost reduction were reduced total filtration area, labor, time, and utilities. The benefits of the single-stage harvest process extended throughout the downstream process, resulting in a 25% relative increase in cumulative yield with comparable impurity clearance.

Background Recombinant therapeutic proteins are usually produced by cell culture technology using genetically modified host cell lines. During the manufacturing process, a mixture of the protein of interest and host cell derived impurities, including host cell proteins (HCPs) and other process related impurities are produced. Those process related impurities will be cleared or minimized though the process by optimization of process purification. Residual HCPs in the final drug substance may affect the quality, safety and efficacy and may result in clinical adverse effects. HCPs are typically quantified using immunoassays such as enzyme-linked immunosorbent assay (ELISA). The development and the validation of those assays are really challenging mainly due to the wide variety of possible HCPs in products. Although generic ELISA kits are commercially available to quantify HCPs from different recombinant systems, a process specific assay is required before drug registration and commercialization for biologics. The reagents, polyclonal antibodies and HCP standards, need to be carefully prepared and characterized to ensure a correct quantitation of the HCPs in the final product. Here we describe the first step of the development of a production platformspecific HCP-ELISA assay for UCB’s biopharmaceuticals produced in a Chinese Hamster Ovary (CHO) cell line, i.e. the production of mock material containing the HCPs using a null cell line. Materials and methods 2L and 80L stirred tank bioreactors (Sartorius) were run for 14 days in a fed-batch mode in a chemically defined medium. Feed was added daily from day 3 onwards. If required, antifoam was added to the bioreactor by manual injections. Dissolved Oxygen (DO), pH, and temperature were controlled at set points. DO was controlled using a multi-stage aeration cascade via a ring sparger. Viable cell concentration and cell viability were measured using a ViCell cell counter (Beckman Coulter). The osmolality was measured using an osmometer (Advanced Instruments).The off-line pH was measured using a BioProfilepHOx (Nova Biomedical). The glucose, lactate, glutamine and ammonia concentrations were measured with a BioProfile Analyzer 400 (Nova Biomedical). On the day of harvest, the clarification was performed by centrifugation, depth filtration and sterile filtration. Part of the material was further clarified by tangential flow filtration (TFF) on the Uniflux 10 system (GE Life Sciences) using low molecular weight cut-off membranes and concentrated before being diafiltered into an appropriate buffer. The antigens from the mock run were assessed for their total HCP content by 2D DIGE and analyzed by the DeCyderTM 2D 7.2 software.