Chromatographic capture of cells to achieve single stage clarification in recombinant protein purification

Abstract Recent advancements in cell culture engineering have allowed drug manufacturers to achieve higher productivity by driving higher product titers through cell line engineering and high‐cell densities. However, these advancements have shifted the burden to clarification and downstream processing where the difficulties now revolve around removing higher levels of process‐ and product‐related impurities. As a result, a lot of research efforts have turned to developing new approaches and technologies or process optimization to still deliver high quality biological products while controlling cost of goods. Here, we explored the impact of a novel single use technology employing chromatographic principle‐based clarification for a process‐intensified cell line technology. In this study, a 16% economic benefit ($/g) was observed using a single‐use chromatographic clarification compared to traditional single‐use clarification technology by improving the overall product cost through decreased operational complexity, higher loading capacity, increased product recovery, and higher impurity clearance. In the end, the described novel chromatographic approach significantly simplified and enhanced the cell culture fluid harvest unit operation by combining the reduction of insoluble and key soluble contaminants of the harvest fluid into a single stage.


| INTRODUCTION
Monoclonal antibody (mAb) therapeutics is one of the biggest product segments in the pharmaceutical industry. Six out of the top 10 pharmaceuticals sold in 2018 were mAbs and a staggering 18 antibodies were approved the following year by the US FDA. 1 The global mAb market has been valued at USD $143.5 billion and expected to grow at a compound annual growth rate of 15% from 2020 to 2026. To meet the market demand, there has been an increased expenditure in research and development in the industry to accelerate the speed from discovery to clinic. In the last 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. Thus, advancements in novel technologies that promote these areas will play a major role in supporting the continued growth of the mAb therapeutic market space.
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. In the last 5 years, over 80% of approved recombinant therapeutic mAbs were expressed in mammalian cell culture systems and predominately in CHO cell lines. 2,3 Further advancements in the understanding of cell biology and cell culture have led to higher cell densities (>25 Â 10 6 cells/ml), increased cell productivity and longer cell culture durations which result in a higher titer and ultimately higher productivity. However, the increased cell mass and corresponding increase in soluble and insoluble contaminant loading have challenged the traditional approaches for separation and purification operations shifting the burden further downstream.
Traditionally, the cell culture harvest unit operation is the first step for clarification and responsible for the removal of cells, cell debris, and other large insoluble aggregates. Legacy clarification strategies use a combination of centrifugation, depth filtration, and membrane filtration for clarifying cell culture fluid (CCCF). Together, these approaches are still widely regarded as the benchmark for clarification performance. Centrifugation is a popular approach using centrifugal force to separate large components in a mixture according to their density and particle size properties. 4,5 Depth filtration relies on a complex porous media containing a mixture of naturally derived filter aids, cellulose, and a resin binder to retain large particulates while letting the soluble product (e.g., mAb) through based on a size exclusion principle. Depth filtration have been the most prevalent single-use technology deployed for clarification in many biopharmaceutical settings. Additionally, novel filter media designs with a wider range of pore sizes and different filter media morphology have been explored to improve filter media utilization. 6 However, the fundamental principle for separation has largely been limited to using size and density as the basic principle of separation. In recent years, newer concepts such as acoustic wave capture, precipitation, and flocculation, have been explored to replace and/or complement legacy clarification strategies to overcome capacity limitations, but still primarily rely on traditional size and sedimentation clarification principles.
As a result, each of the legacy and emerging clarification technologies have encountered similar technical and economic challenges that have been described more extensively in other reviews and articles. [7][8][9] In this study, we explored a novel anion exchange (AEX) chromatographic approach designed for cell culture harvest in a single use format. The adoption and innovation in single use technology have proven to further the transformation of the biomanufacturing industry to be more efficient and flexible from traditional stainless-steel technology. Single use technology can be used and disposed of without having the drawbacks of capital investments and cost associated with cleaning and validation along with shorter turnaround time which would all ultimately lead to higher productivity. 10,11 However, as bioprocesses have evolved to yield higher cell densities, the ability to clarify current and future cell cultures with higher levels of insoluble and soluble impurities with traditional single use clarification technologies have proven to be difficult. Current single use technology for cell culture harvest are primarily constructed out of fibers from naturally derived ingredients to provide the structure for mechanical sieving, and recent studies have reported the incorporation of adsorptive properties to aid for a better separation. 12,13 Khanal et al. specifically goes into the effect of using highly charged resin binders combined with a porous depth filter media to enhance the adsorption of soluble contaminants. 12 Thus, charge-based separation that was typically reserved for downstream processing are now being explored in the clarification stages of cell culture harvest. The objective of this study was to evaluate the potential of using a novel single use technology resembling chromatography for the clarification of raw CCF referred to as 3M™ Harvest RC Chromatographic clarifier in this study. This single stage would utilize fibrous AEX technology engineered to capture the entire range of particles found in raw cell culture broth from cells to DNA in a single-step approach for clarification. Current chromatography technologies for mAb production exist in the form of columns, resins, or functional membranes. Chromatography generally exploits the physical and chemical differences between biomolecules to achieve a higher degree of separation compared to upstream filtration. However, the increase in cell density, cellular debris and other impurities have now caused new challenges for both clarification and downstream chromatography. The primary limitation of all the current chromatographic media types is that they are not well suited to handle the initial level of large particulates present in cell culture. Large quantities of solids can easily foul chromatographic devices that will reduce overall performance and effectiveness. 14 In the last decade, we saw the attempt of using chromatography for clarification in the form of expanded bed adsorption (EBA). However, the original concept of EBA was to use chromatography to not only achieve high separation efficiency but also capture the product. 15,16 In certain applications, EBA had success where the levels of contaminants were low and the titer was moderate enough to where the EBA could be deployed to replace several traditional unit operations, namely centrifugation, filtration, and capture chromatography. However, like other column and bead-based technology, EBA was susceptible to fouling and clogging once the impurity loading specifically with cells and cell debris got too high. There were other drawbacks associated with deploying EBA at manufacturing scale which involved the complexity to clean and validate the EBA column that needed to be regenerated and reequilibrated before each use. 15 In addition, the flow rate of the column can be very restricted in order to keep the bead-based bed suspended thus slowing down the process. 17  HCPs with chromatographic clarification compared to conventional clarification strategy alleviating the burden on downstream purification. 21 We have previously reported that this approach can be effectively used across multiple molecules and is platformable. 22 It has been further reported that the advantages of deploying chromatographic clarification in this manner have led to improved viral clearance performance and reduced impurity challenges to downstream polishing columns and filters by generating a cleaner filtrate prior to the capture step. 23,24 Similar to conventional chromatographic modalities, current chromatographic clarification can only be achieved by combining adsorptive hybrid filters with other conventional clarification techniques to remove larger insoluble contaminants.
To our knowledge, this is the first reported study to utilize chromatography during clarification without any additional conventional clarification techniques needed. In this study, 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 ( Figure 1).This article primarily addresses the use of a single stage fiber chromatographic clarification technology for high cell density cultures as compared to traditional clarification techniques. The GPEx ® and GPEx ® Boost cell lines studied within this article were chosen as good clarification challenge cases, representative of process intensified mAb production conditions expected for modern pharmaceutical production processes. 25 Clarification is a critical unit operation in the production of mAbs because it directly affects yield, product consistency and performance of downstream unit operations.
By implementing the novel strategies presented in this study, we have shown the ability to demonstrate process intensification and compression to drive improved yields and lower operating costs.

| Cell culture
Three proprietary CHO cell lines were used to produce harvests contaminant recombinant monoclonal antibodies (mAb 1, mAb 2, and mAb 3). Two Catalent monoclonal GPEx CHO cell line expressing an IgG antibody and one Catalent monoclonal GPEx Boost CHO cell line expressing an Fc-fusion protein were used for this study. These three cell lines were grown in commercially available cell culture medium and passaged every 3-4 days in shaker flasks in a cell incubator until a suitable cell number was attained to inoculate the production vessel.
The production phase was performed in single-use bioreactors incorporating a fed-batch process. The basal medium used was a commercially available cell culture medium supplemented with matching commercially available concentrated feeds at regular intervals to maintain a healthy cell culture environment. The production vessel was monitored daily, and process parameters maintained within the platform limits.

| Cell culture conditions
The cultures were harvested when protein production leveled off, reaching a protein concentration of 1.00 g/L for the 3 M cell line, 1.86 g/L for the GPEx cell line, and 6.2 g/L for the GPEx Boost line.
The detailed information for each cell culture used in this study can be found in Table 1 below.

| Clarification
The 3M™ Zeta Plus™ 60SP02A depth filters were first prepared by

| Host cell DNA quantification
Detection of CHO DNA by quantitative polymerase chain reaction (qPCR) was carried out using the resDNASEQ preparation kit from F I G U R E 1 Process flow diagram of different single use clarification trains combined with Protein A Thermo Fischer as part of an integrative system that includes sample preparation, TaqMan ® assay and master mix, standard DNA, instruments, and software (AccuSEQ™) and has a limit of quantitation of 6 pg DNA/ml. Results are generated from a 7500FEST real-time PCR system and reported as pg DNA/mg of protein.

| HCP quantification
Detection of HCP by enzyme-linked immunoassay (ELISA) using Cygnus CHO HCL ELISA Kit F550-1 kit. The antibodies used in this procedure are polyclonal and designed to broadly react with most HCP's that might co-purify with the desired product.

| Protein quantification
Protein quantification in media was carried out using a 0.1 ml (2.1 Â 30 mm) POROS A 20 μm column with a six point internal calibration standard in sample matrix using a bind/elute buffer system. A standard curve was generated off the area of the peaks, and sample areas were evaluated against the standard curve.

| Turbidity and particle size analysis
Turbidity of the CCCF was measured using an ORION™ AQ4500 turbidity meter (Thermo Fisher Scientific, Waltham, MA) in NTU units.
Dynamic light scattering (DLS) particle size distribution in CCCF was measured using a Nanotrac Flex DLS Analyzer (Microtrac, Montgomeryville, PA). Particle size distribution from 1 to 6 μm was measured in intensity mode.

| BioSolve modeling
Cost of goods (CoGs) modeling was conducted using BioSolve Version  Table 2.
In order to understand the impact of 3M™ Harvest RC on the manufacturing costs for a mAb product, two main processes were modeled: a state of the art baseline process, which contained a two-stage clarification train including a primary depth filter followed by chromatographic clarification using the 3M™ Emphaze™ AEX Hybrid Purifier; and a next generation process with a single clarification step, 3M™ Harvest RC solution. All other unit operations were kept the same between the two processes. The key parameters for each of the unit operations including loading, unit operation size and yield are shown in Table 3.
To understand the impact of particular features for a single stage clarification stage using 3M™ Harvest RC, incremental changes were made to the baseline process to understand the impact 3M™ Harvest RC has on mAb manufacturing costs. To understand where the cost savings could be realized deploying this new technology, two hypothetical situations showing the impact of having higher product recovery and condensing of multiple stages to one were modeled using Biosolve according to Table 3 to understand the impact of the these two features that were realized when deploying 3M™ Harvest RC. The key parameters for each of the unit operations in these hypothetical processes including loading, unit operation size and yield are also shown in Table 3. In the first hypothetical process, the yield for the primary clarification depth filter step was set at 100% so that the overall clarification stages would mimic the 98% overall yield from the 3M™ Harvest RC. This would help us understand the impact of having a higher yield. The second hypothetical process was modeled by combining two clarification stages from the baseline process into a single hypothetical step. This single hypothetical clarification step was modeled such that it had no overall impact on the number or cost of consumables or hardware used in clarification.

| Chromatographic clarification with 3M™ Harvest RC
In this study, we explored the concept of chromatographic clarification using a fibrous AEX media referred to as 3M™ Harvest RC. Current approaches for chromatographic clarification require multiple stages for the removal of large particles followed by smaller and Harvest RC as a stand-alone stage for chromatographic clarification. As shown in Figure   contribute to any filter caking that is typically observed with traditional clarification strategies using depth or membrane filtration. 26,27 Currently the most widely applied single-use clarification technology is depth filtration. However, conventional technologies such as depth or membrane filters can foul and cake with higher biomass leading to lower throughputs. The higher contaminant profile can also make depth filter performance more sensitive to batch variations due to naturally derived component variations in depth filters. 8 Because of these effects, some report up to a 50% safety margin can be deployed during scaling of depth filtration clarification systems from laboratory trials to clinical and commercial deployment. 28 Because the 3M™ Harvest RC technology is fully defined and utilizes AEX chromatography rather than a combination of surface caking and bed loading, the scaling across different cell culture volumes is much more predictable. To confirm this, we evaluated the performance using a high cell density culture (mAb 3) across three different scales. The differential pressure during filtration followed the same pressure profile across all three scales of bench, pilot, and production scale devices (Figure 4). A standard parameter for assessment of clarification efficiency is turbidity.

| Depth filter clarification
A single stage depth filter was selected as the base line performance for its prevalence and simplicity. A 3M™ Zeta Plus™ 60SP02A depth filter was chosen because of its dual layer configuration which allows it to capture large particles such as cells as well as smaller particles such as cell debris due to the nominal pore size rating between 0.2 and 10 um.
Per the manufacturer's recommendation of usage, a pressure criterion of 15 psid was used as the endpoint for the depth filtration. Using mAb 2 in Table 3, a throughput capacity of 88 L/m 2 was achieved after

| Improved clarified fluid quality using chromatographic clarification
In the first clarification train, two peaks were observed centered around 0.01-1 μm suggesting the presence of DNA, chromatin, and cell debris that were not captured during depth filter clarification ( Figure 6a). As a result, with the chromatographic clarification method, a monodisperse particle size distribution was observed around 0.01 μm corresponding to the mAb where the peaks corresponding to the presence of DNA, chromatin, and cell debris were not found.  The removal of DNA-protein complexes using chromatographic clarification by 3M Harvest RC was supported through multiple measurements of impurity removal, DLS, and acidified turbidity. Figure 7 shows that turbidity ratio was measured to be below one throughout   Figure 10 3M Harvest RC had $98% product recovery with the same concentration before and after clarification. This higher product recovery has large ramifications in terms of process simplicity and process economics.  The reduction in other costs observed includes insurance; waste management; maintenance and utilities. The reduction in these costs are: 11.6%; 6.6%; 12.1 and 12.5%, respectively. Since all these reductions, except for the waste management costs, are around 11%, they are thought to be driven primarily by the 11% increase in the capacity of the process due to the 11% increase in DSP yield. With respect to the waste management costs, which are reduced by less than 11%, it is thought that the increased yield, as shown from Figure 10, will give rise to more product to be handled by the downstream process which therefore requires more consumables downstream to deal with the increased amount of product. Consequently, some of the reduction in cost, resulting from the increased yield, is balanced out by an increase in the cost of disposing of these extra consumables. Figure 11b shows the contribution of the various features of Harvest RC to the overall 16% reduction in mAb manufacturing costs. By far the largest contributor, 10 out of the 16%, to the overall reduction in mAb manufacturing costs, is the downstream process yield increase that results from the use of 3M™ Harvest RC and gives rise to an increase in mAb production capacity of approximately 11% F I G U R E 1 1 Biosolve model results. (a) shows the percentage contribution of capital (blue), materials (red), consumables (yellow), labour (light blue) and other costs(green) to the CoGs for the baseline process and the 3M ™ Harvest RC process. All values were normalized to the mAb manufacturing cost per gram for the baseline process. (b) shows the relative contribution to the reduction in the mAb manufacturing cost of the increased downstream processing (DSP) yield (blue), combining the two clarification steps into one (yellow), the removal of the peruse flush (red) and other factors (green) such as the reduced number of capsules, reduced waste and differences in capsule cost. The percentages presented are relative to the mAb manufacturing cost for the baseline process. (c) shows impact of implementation of 3M™ Harvest RC on the process mass intensity (black), capacity (white) that is, the quantity of product than can be produced using the process) and the DSP recovery (gray). (d) shows the reduction in the process mass intensity (PMI) due to increased DSP yield (gray), combining the two clarification steps into one (diagonal hash), the removal of the peruse flush (horizontal hatch) and 3M™ Harvest RC (white) compared to the baseline process (black) (Figure 11c). This is because of the high value of the mAb product when compared to the materials used to produce and purify it and is intuitive (i.e., if the mAb did not have that relatively high value then the process would be economically unviable). Just under 1% point of the reduction in manufacturing costs is due to moving from multiple clarification steps to a single clarification step. The remaining cost savings, labeled as other, covers the reduced number of consumables and differences in consumable cost.
In addition to the cost savings resulting from the use of Harvest RC, a reduction in the process mass intensity (PMI) of $26% is also observed-as shown in Figure 11c. PMI is a measure of the quantity of process water, materials and consumables required. A lower PMI indicates that less of these things are required to manufacture a gram of product and therefore indicates that the process has a reduced environmental impact. This reduced PMI is observed for a number of reasons which include: a reduction in the quantity of process water required for flushing; lower clarification consumable requirements, that is, 10 capsules used during the Harvest RC clarification compared to 24 capsules for the baseline process as shown in Table 2; and the increased yield which results in a more efficient process overall.

| CONCLUSION
To our knowledge, this is the first study to use chromatography to capture whole cells, cell debris, and soluble impurities in a single clarification step for a variety of cell cultures. Overall, the findings suggest that the 3M™ Harvest RC is well suited for past, current, and upcoming challenging cell cultures. The results indicate the benefits of utilizing chromatography in the form of the 3M™ Harvest RC such as reduced DNA, higher recovery yield, robust scalability, and higher purity compared to traditional clarification technologies. These findings were further supported using an economic model to show that the inclusion of 3M 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.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.