Chromatographic clarification

In the past few years, process simplification and process intensification have emerged as key areas of focus when designing and implementing bioprocessing strategies for promoting a more efficient mAb production process. In the past couple of decades, the deployment of platform technologies such as using Chinese hamster ovary (CHO) cells as the expression platform have emerged as a standard practice in the industry to streamline the manufacturing process for higher efficiencies and flexibility.

Catalent employs a mix of different clarification technologies to meet the challenges of higher and higher cell densities to achieve desired clarification results. However, advancements in cell line engineering and development that have led to higher cell densities (>25 x 106 cells/ml) which has increased cell productivity and higher titer have also presented the unique challenge of presenting higher contaminants shifting the burden to downstream purification operations.

In this study, we explored a novel anion exchange (AEX) chromatographic approach designed for cell culture harvest in a single-use format. We explored the impact and savings that can be achieved by comparing multiple single use technology for clarification to understand the impact of using a novel single use technology to achieve chromatographic clarification.

Three different clarification trains were evaluated to explore the impact of chromatographic clarification (Figure 1). A traditional depth filter train employing a 3M Zeta Plus EXT series depth filter with 60SP02A grade media was used as a benchmark for evaluating depth filter performance. A second train exploring the effects of chromatographic clarification with the 3M Emphaze AEX hybrid purifier was used in conjunction with a primary depth filter. Lastly, the third train deployed the use of a single stage chromatographic clarification technology, 3M Harvest RC chromatographic clarifier.

In this study, we used three proprietary CHO cell lines representative of process-intensified cell line technology provided by Catalent to produce recombinant mo noclonal antibodies (mAb 1, mAb 2, and mAb 3). As shown in Table 1, two Catalent monoclonal GPEx CHO cell lines expressing an IgG antibody and one Catalent monoclonal GPEx Boost CHO cell line expressing an Fc-fusion protein were used for this study.

In addition, to understand the economic impact of using the novel chromatographic clarification technology, a cost of goods (CoGs) model was carried out using BioSolve to explore the impact on the manufacturing costs for a mAb product. The key parameters for the modelling are shown in Table 2.

In this study, we explored the concept of chromatographic clarification using a fibrous AEX media referred to as Harvest RC chromatographic clarifier. Current approaches for chromatographic clarification require multiple stages for the removal of large particles before subsequent steps of smaller and soluble impurities removal. This study evaluated the possibility of using the chromatographic clarifier as a stand-alone stage for chromatographic clarification. The technology is a bed of quaternary ammonium functionalised polypropylene fibres. Using scanning electron microscopy (SEM), cells were found adsorbed and captured onto the individual fibres of the technology (Figure 2). But in addition, soluble contaminants such as DNA/ chromatin/etc were also found to be removed as well to aid in subsequent downstream operations.

Figure 3A and 3B illustrates the results of comparing three different clarification trains.

Dynamic light scattering (DLS) revealed three distinct populations observed with depth filter filtrate (Figure 3A). One peak corresponding to 0.01 µm was observed which is consistent with where protein-based species would be. However, two additional peaks were also observed between 0.01 µm and 1 µm, which is an indication of the presence of DNA, chromatin, and cell debris.

The particle size distribution for both chromatographic clarification approaches resulted in a monodisperse particle size distribution centered around 0.01 µm consistent with the size of protein-based species, including mAbs. This suggests that the predominant species in the filtrate is now the product of interest utilizing the chromatographic approach to remove the DNA, chromatin, and cell debris.

The effect of having a cleaner clarified filtrate was evaluated on the performance of the Protein A capture step. Both chromatographic clarification approaches revealed a slight improvement in host cell protein reduction compared to traditional depth filtration during the clarification operation (Figure 3B). However, the biggestimpact was felt on the Protein A capture step further downstream where the aid in the removal of interferingDNA contaminants during the clarification ultimately enabled a cleaner purity with the Protein A eluate.

The improvement of using chromatographic clarified filtrate on Protein A was also revealed during the wash and acid strip steps of the Protein A operation. It was found that fewer contaminants were found bound to the ProteinA during the wash and strip steps of the Protein A when challenged with the two chromatographically clarified filtrate. In addition to the fact that Protein A eluate with chromatographic clarified filtrate resulted in lower impurities, it can also be implied there is less burden on the Protein A unit operation leading to higher cycle usage and enhanced performance.

We explored the financial ramifications of deploying the Harvest RC chromatographic clarifier as a single step chromatographic clarification. A BioSolve cost analysis was modeled using the experimental results. The relative contribution of capital, materials, consumables, labour, and other costs to the total cost of the chromatographic clarifier process compare to a traditional two stage clarification train. Using a single-stage chromatographic clarification approach, an improvement of 16% in CoGs was observed. The 16% improvement can be traced to the process compression to a single stage reducing in consumable waste generated and the yield improvement observed with a chromatographic clarification approach.

This is the first study to show the use of chromatography to capture whole cells, cell debris, and soluble impurities in a single clarification step for a variety of different cell cultures. Overall, the findings suggest that the Harvest RC is well suited for past, current, and upcoming challenging cell cultures. The results indicate the benefits of using chromatography for clarification in the form of the clarifier. These benefits include reduced DNA, higher recovery yield, robust scalability, and higher purity compared to traditional clarification technologies.

The findings from this case study were further supported using an economic model to show that the inclusion of the Harvest RC has a beneficial impact in terms of reducing mAb manufacturing cost and reducing environmental impact. These benefits are a strong function of product yield improvement but other factors such as simplified deployment, faster processing and process simplification also play a role as well.

For more information visit www.3m.com/bioprocessing

Images and text were adapted from Almeida, A., Chau, D., Coolidge, T., El Sabbahy, H., Hager, S., Jose, K., Nakamura, M., and Volos- hin, A. (2022). Chromatographic capture of cells to achieve single stage clarification in recombinant protein purification. Biotechnolo- gy Progress, 38(2), e3227.