Enhancing Protein A performance in mAb processing: A method to reduce and rapidly evaluate host cell DNA levels during primary clarification

Abstract The use and impact of 3M™ Emphaze™ AEX Hybrid Purifier, a single‐use, fully synthetic chromatographic product, was explored to reduce host cell DNA (HC‐DNA) concentration during the primary clarification of a monoclonal antibody (mAb). An approximately 5‐log reduction in HC‐DNA was achieved at an Emphaze AEX Hybrid Purifier throughput of 200 L/m2. The appreciable reduction in HC‐DNA achieved during primary clarification enhanced Protein A chromatography performance, resulting in a sharper and narrower elution profile. In addition, a 24× improvement in host cell protein (HCP) removal and fewer impurities nonspecifically bound to the Protein A column were observed compared to those resulting from the use of depth filtration for clarification. The use of a rapid, qualitative acidification assay to facilitate HC‐DNA monitoring was also investigated. This assay involves the acidification‐induced precipitation of HC‐DNA, enabling the easy and rapid detection of DNA breakthrough across purification media such as Emphaze AEX Hybrid Purifier by means of turbidimetric and particle size measurements.


| INTRODUCTION
The modern production of monoclonal antibodies is a complex, multistep process requiring the interplay between many different unit operations to purify the target product with good yield and high purity. [1][2][3][4][5][6] Traditionally, monoclonal antibody (mAb) production has been divided into two distinct stages, upstream processing and downstream purification, separated by a product capture step, typically Protein A chromatography. 3,4 Upstream processing generally entails cell line development, cell culture cultivation, and the clarification of harvested cell culture fluid (HCCF). During HCCF clarification, large insoluble debris, such as host cells, is removed.
Depending upon HCCF characteristics, a single clarification step may suffice, or multiple unit operations in series, such as centrifugation followed by depth filtration, may be required to achieve the desired level of clarity. The clarified cell culture fluid (CCCF) produced during upstream processing is typically passed through a sterilizing grade membrane to remove any residual smaller debris and provide bioburden control before capture chromatography and downstream processing.
Protein A affinity chromatography commonly serves as the capture step in mAb production. The Fc region of a mAb exhibits a strong affinity and high selectivity toward Protein A, binding the target protein to the chromatography ligand or resin. 4,7,8 During loading, the mAb is bound to the chromatography resin, whereas other impurities such as host cell proteins (HCPs) generally interact to a lesser extent with Protein A ligands and flow through the column to be discarded.
After loading onto the Protein A column, the bound mAb is washed to remove residual HCPs and other contaminants. After washing, a change in the mobile phase pH results in elution of the target mAb molecule. Processing of the Protein A eluate then continues in the downstream unit operations to remove residual contaminants and further increase the target mAb purity.
This sequential mAb purification scheme establishes a cascade where the removal of impurities at one stage of the process directly impacts the performance of subsequent unit operations. Therefore, achieving a significant reduction in contaminants early in the process enables simpler, more efficient mAb production. Upstream clarification unit operations readily illustrate this concept by effectively removing macroscopic, insoluble debris such as host cells. However, soluble impurities such as chromatin and its HCP, DNA, and RNA constituents are not significantly reduced during primary clarification and are left for removal during downstream unit operations. As a result, downstream unit operations are often scaled to accommodate a high level and range of impurities, reducing their effectiveness. For instance, additional unit operations, such as the inclusion of ancillary depth filters and chromatography columns, must often be incorporated into downstream unit operations to achieve the target throughput and purity. 8 One soluble contaminant in HCCF typically not cleared during primary clarification is chromatin, a complex of HC-DNA coiled around histone proteins as well as RNA and other proteins. [9][10][11][12] The presence of chromatin has been documented to directly impact mAb purification and recovery, presenting a challenge to the affinity chromatography and downstream processing steps. 10 In the case of Protein A chromatography, chromatin and HCPs have been found to nonspecifically bind to Protein A ligands, reducing the number of binding sites for the target mAb and effectively lowering the dynamic binding capacity of the affinity column. 10,13 In addition, chromatin has been shown to enable the coelution of HCPs with the target mAb from the Protein A column.
Through interactions with chromatin, HCPs are not completely removed during the wash step and can "hitchhike" across the Protein A column and co-elute with the desired mAb. 10,11 When HCPs and DNA are not completely removed during Protein A chromatography, the performance of downstream unit operations is also impacted. Failure to remove DNA can potentially lead to increased turbidity during neutralization after a low pH hold viral inactivation procedure, thus requiring the addition of a filtration step. 8,14 A number of chromatin-directed clarification strategies have been proposed to address the presence of chromatin/DNA and the challenges it creates during and after Protein A chromatography. These approaches generally aim to significantly reduce chromatin levels during primary clarification or alter the operating conditions used during Protein A chromatography. Recent efforts have focused on selective precipitation to isolate and remove chromatin during primary clarification. In this case, chromatin precipitation is typically achieved through the addition of fatty acids, such as caproic or octanoic acid, allantoin or ethacrine, to CCCF. [9][10][11][12]15 Rather than inducing the precipitation of soluble contaminants such as chromatin and DNA, other methods have incorporated the precipitation of the target mAb or target protein. A peptide or biopolymer featuring a Protein A epitope is introduced to the cell culture and binds the desired mAb, subsequently inducing precipitation. Upon isolation and washing of the precipitate, the mAb can be released into solution by pH modulation, allowing further purification. 16,17 Other methods to overcome the challenges chromatin/DNA presents to Protein A chromatography involve modifying the operating conditions for Protein A chromatography itself. One strategy used chromatofocusing using a pH gradient through the Protein A column to reduce HCP coelution with the target mAb. 18 An alternative approach made use of two distinct flow rates, high and low, to influence residence time and minimize nonspecific binding and HCP hitchhiking as well as to improve overall column utilization. 19 Changes to the column washing strategy have also been considered to improve HCP and other contaminant clearance across Protein A chromatography. Washes introducing additives that disrupt protein-protein interactions, such as urea, isopropanol, elevated pH, and increased salt concentration, have shown promise in reducing HCP levels in Protein A eluate. 14,20 Rather than modifying HCCF through the addition of extraneous material or altering the Protein A elution conditions, this study targeted the removal of HC-DNA, a common component of chromatin, with a single-use, advanced anion exchange media: Emphaze AEX Hybrid Purifier. Comprising solely synthetic materials, Emphaze AEX Hybrid Purifier provides Q-functional chemistry on a polypropylene nonwoven scaffold followed by a 0.2 μm asymmetric polyamide membrane. 21 The high positive charge of Emphaze AEX Hybrid Purifier enables the removal of negatively charged soluble impurities, including HC-DNA. In this investigation, Emphaze AEX Hybrid Purifier was incorporated upstream of Protein A following depth filtration to remove HC-DNA during primary clarification.
To assess the performance of techniques for the removal of soluble impurities such as HC-DNA, HCP, and chromatin, the levels of these contaminants in the cell culture fluid must be monitored. Quantitative assays such as Picogreen, qPCR, and/or ELISA are typically conducted on aliquots collected over the course of purification to measure their concentration. These assays provide a means to accurately and quantitatively determine the effectiveness of HC-DNA removal for a given solution; however, they are often costly and time consuming. Furthermore, multiple fractions may be collected and submitted for analysis but are typically not processed or analyzed until after the clarification process has been completed. With the challenge posed by current analytical techniques, the ability to rapidly identify the presence of significant soluble contaminant levels, such as HC-DNA, during purification will aid in the development and design of primary clarification approaches that provide higher-quality, standardized material to downstream unit operations. A rapid qualitative assay for HC-DNA using turbidimetric readings and particle size analysis was explored during this investigation to facilitate the estimation of HC-DNA reduction during primary clarification and potential breakthrough across purification media such as Emphaze AEX Hybrid Purifier.

| Turbidity and particle size analysis
Turbidity measurements were performed on sample aliquots of approximately 12 mL each using an ORION™ AQ4500 turbidity meter (Thermo Fisher Scientific). Dynamic light scattering (DLS) measurements were performed with a Microtrac Nanotrac Wave Flex (Microtrac) to characterize the particle size distributions in the samples collected.

| Rapid qualitative assessment of HC-DNA levels through acidification
An assay was developed to rapidly and qualitatively monitor HC-DNA breakthrough across Emphaze AEX Hybrid Purifier during primary mAb clarification and may be used as a proxy for chromatin. pH adjustments, especially acidification, during mAb production are known to impact solution stability and potentially result in precipitation. The acidification of HCCF has been explored as a purification technique, in which a decreased pH precipitates HC-DNA and other high-molecular-weight biological contaminants, thereby facilitating their removal. 22 The assay considered during this study provides a means to obtain an estimate of HC-DNA levels in real time during clarification without the delay often associated with more rigorous To assess the utility of the turbidimetric assay, HC-DNA levels were quantified via qPCR and correlated to the turbidimetric values before and after acidification. A qualitative indication of the HC-DNA breakthrough was estimated by considering the ratio of turbidity measurements before and after acidification: Turbidimetric ratio = post acidification turbidity initial sample turbidity: When trace HC-DNA levels were present, little to no precipitation occurred upon acidification, and the sample turbidity remained relatively unchanged: the turbidity of an aliquot containing 60 ppb HC-DNA was 0.92 FNU before acidification and 0.76 FNU after, resulting in a turbidity ratio slightly less than 1. In contrast, when higher levels of HC-DNA were present, a discernable rise in turbidity was observed. At a HC-DNA concentration of 6.2 × 10 5 ppb, for example, turbidity increased from 5.9 FNU to 18.4 FNU upon acidification, yielding a turbidity ratio of approximately 3.
As presented in Figure 2B, higher turbidity ratios were observed as the HC-DNA concentration increased, which provided a means to qualitatively assess HC-DNA levels during clarification. A turbidity ratio near or less than 1.0 corresponded to a low HC-DNA concentration measuring less than 100 ppb. As the FNU turbidity ratio reached and exceeded 1.5, the HC-DNA concentration approached the >1 × 10 6 ppb concentration present in unclarified material.
In practice, there are several methods available to assess turbidity.
Formazin and nephelometric measurements are commonly used in biopharmaceutical applications during upstream processing to monitor clarification. Both nephelometric and formazin turbidity measurements make use of light scattering to assess and quantify clarification; however, the wavelength used differs between the two. Nephelometric turbidity measurements are commonly conducted using a broadband light source, whereas formazin turbidity relies on infrared wavelengths. 23 Both turbidity measurement techniques provided a qualitative assessment of HC-DNA levels, but with different levels of sensitivity.
As evidenced in Figure 3, the use of formazin turbidity measurements in the qualitative assay provided higher sensitivity to HC-DNA  DLS can also provide information on the quality of CCCF. Owing to a diverse mixture of components such as host cells, HCPs, HC-DNA, and the target protein, HCCF typically presents a wide and polydisperse particle size distribution. As shown in Figure 4A, the depth filtered feed evaluated during this study revealed particles ranging in size from 0.1 to 1 μm. Upon acidification, the DLS spectra shifted to contain a population of larger particles consistent with aggregation and precipitation. Following the clarification of HCCF via depth filtration and Emphaze AEX Hybrid Purifier, the particle size distribution became more defined, resulting in monodisperse populations at low throughputs. As illustrated in Figure 4, samples containing trace levels of HC-DNA exhibited a monodisperse particle size distribution centered at approximately 0.01 μm before and after acidification, consistent with the size of protein-based species, including mAbs. Purifier throughput of 200 L/m 2 , the turbidity ratio was 0.87, and a monodisperse particle size distribution was observed. When the throughput increased to 300 L/m 2 , the turbidity ratio increased to 1.0, and a second particle size population consistent with HC-DNA began to develop in the DLS spectra. Through the removal and reduction of HC-DNA, Emphaze AEX Hybrid Purifier also provided a more consistent and standardized filtrate pool, as clearly demonstrated by the particle size distributions presented in Figure 4. Without the removal of HC-DNA during primary clarification, CCCF typically presents a polydisperse particle size distribution, as shown in Figure 3A. When DNA began to break through Emphaze AEX Hybrid Purifier at throughputs above 200 L/m 2 , a secondary particle size population began to appear. Consistent with the quantitative measurements, at throughputs of 400 L/m 2 or less, there was no apparent shift in particle size distribution upon acidification, and the smaller particle size population remaining dominant in the DLS spectra indicated that appreciable HC-DNA breakthrough did not occur. At higher throughputs, the larger F I G U R E 5 DNA reduction with depth filtration and at different Emphaze AEX Hybrid Purifier throughputs. Log reduction values (LRV) above Emphaze AEX Hybrid Purifier throughputs denote DNA reduction relative to the initial depth filter pool. The highlighted Emphaze AEX Hybrid Purifier samples at throughputs of 500 and 600 L/m 2 had significantly higher DNA concentrations than the earlier fractions and were consistent with the findings of the turbidimetric assay: the turbidity ratios were found to be greater than 1.5 particle size population associated with HC-DNA continued to evolve, approaching a distribution similar to that of the starting depth filter pool.

| Lower HC-DNA levels facilitate improved Protein A performance
An improvement in Protein A performance was noted in the quality of the absorbance profiles during elution and the acid strip ( Figure 6).
When high levels of HC-DNA were present, a broader absorbance peak was observed during the elution of tociluzimab, as evidenced in the elution profile of material from the depth filter pool. A reduction in HC-DNA levels during upstream clarification provided a benefit in terms of Protein A column regeneration. 25 As presented in Figure 7, the elution of HCPs during the acid stripping of Protein A was quantified as a function of DNA concentration in the feed. Without a reduction in the HC-DNA levels in the feed, a marked increase in the amount of nonspecifically bound HCPs was observed F I G U R E 6 UV absorbance spectra at 280 nm acquired during Protein A chromatography: (A) UV absorbance over the course of the entire Protein A chromatography step, (B) profile during elution, and (C) UV absorbance of material during the column acid stripping during Protein A regeneration. Following purification of a feed clarified by conventional depth filtration, acid washing of Protein A resulted in the elution of 500 to 1,000-fold more HCPs relative to clarification where HC-DNA was significantly reduced. Less HCP build up on the Protein A resin was observed when a 5-log reduction in HC-DNA concentration was achieved using Emphaze AEX Hybrid Purifier. After clarification with Emphaze AEX Hybrid Purifier, low HCP concentrations ranging from 40 to 400 ng/mL eluted during the acid stripping of the Protein A column.
Lower HC-DNA levels were also found to reduce turbidity formation during pH adjustment of the Protein A eluate following viral inactivation. As illustrated in Figure 8

| CONCLUSION
When included in upstream purification, Emphaze AEX Hybrid Purifier provided a significant reduction in HC-DNA levels compared to standard depth filtration. Emphaze AEX Hybrid Purifier reduced HC-DNA concentration by approximately 5 logs. Therefore, Emphaze AEX Hybrid Purifier provides a cleaner, more standardized clarified fluid to enable improved HCP clearance during Protein A chromatography.
For example, when a 5-log HC-DNA reduction was accomplished during primary clarification, Protein A was over 20× more effective at HCP removal. Furthermore, only an order of magnitude reduction in HC-DNA using Emphaze AEX Hybrid Purifier was found to improve Protein A performance, providing 5× greater HCP clearance relative to that achieved by traditional primary clarification approaches. The dashed line at the top of the plot denotes the detection limit of the turbidity meter. Trendlines have been included for clarity and do not represent a fit to the data regeneration will allow improved cycle times and help minimize the exposure of the Protein A column to caustic conditions that are detrimental to the affinity ligands. 26 A rapid qualitative acidification assay was developed as part of this study to facilitate the determination of HC-DNA breakthrough across Emphaze AEX Hybrid Purifier and to provide a method to estimate HC-DNA levels. A turbidimetric assay relying on measurements before and after acidification was developed to quickly screen for the presence of HC-DNA and determine the capacity of Emphaze AEX Hybrid Purifier.
Ratios of formazin turbidity measurements before and after acidification that were below 1.0 were found to indicate low HC-DNA concentrations or HC-DNA-free samples. When the formazin turbidity ratios increased above 1, high levels of HC-DNA were present, and these ratios were associated with breakthrough across Emphaze AEX Hybrid Purifier.
Supporting the results from the acidification assay, particle size measurements provided a means to identify significant quantities of DNA. In samples containing little or no HC-DNA and chromatin-related species, a monodisperse particle size distribution was observed cen-