Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody–polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to <1 µg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody. Biotechnol. Bioeng. 2009;102: 1141–1151. © 2008 Wiley Periodicals, Inc.     

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.     

Salt tolerant membrane adsorbers for robust impurity clearance

Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50–150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage ?X174 (pI ～ 6.7) at pH 7.5 and phage PR772 (pI ～ 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host‐cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Caprylic acid precipitation method for impurity reduction: An alternative to conventional chromatography for monoclonal antibody purification

We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes. Biotechnol. Bioeng. 2012; 109: 2589–2598. © 2012 Wiley Periodicals, Inc.     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

Development of a novel adenovirus purification process utilizing selective precipitation of cellular DNA

The use of recombinant adenoviral vectors for vaccination and gene therapy requires the development of purification processes that are cost-effective, scalable, and capable of robust host cell DNA clearance. An adenovirus purification process was developed which incorporates selective precipitation of host cell DNA, enabling a reduction in the use of costly nucleases and chromatographic resins while substantially improving DNA and protein clearance capabilities. In this work, three cationic detergents were evaluated for their capacity to selectively precipitate DNA from adenovirus-containing cell lysate. Parameters including pH, sodium chloride concentration, nonionic surfactant concentration, and cell density were investigated during development of the precipitation step. In a novel application, the cationic detergent domiphen bromide was found to have superior selectivity for host cell DNA. In addition, domiphen bromide-induced precipitation of adenovirus was shown to be reversible, which reduces the importance of mixing. Precipitation of DNA in the cell lysate coupled with primary clarification resulted in 3 logs of DNA clearance and improved impurity clearance in the subsequent ultrafiltration step. As a result, nuclease treatment and/or anion exchange chromatography can be eliminated, or included exclusively to improve process robustness.     

Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.     

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow‐rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log₁₀ of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750–757, 2015     

Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein

An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.     

Integrated flow-through purification for therapeutic monoclonal antibodies processing

An integrated all flow-through technology platform for the purification of therapeutic monoclonal antibodies (mAb), consisting of activated carbon and flow-through cation and anion exchange chromatography steps, can replace a conventional chromatography platform. This new platform was observed to have excellent impurity clearance at high mAb loadings with overall mAb yield exceeding 80%. Robust removal of DNA and host cell protein was demonstrated by activated carbon and a new flow-through cation exchange resin exhibited excellent clearance of mAb aggregate with high monomer recoveries. A ten-fold improvement of mAb loading was achieved compared to a traditional cation exchange resin designed for bind and elute mode. High throughput 96-well plate screening was used for process optimization, focusing on mAb loading and solution conditions. Optimum operating windows for integrated flow-through purification are proposed based on performance characteristics. The combination of an all flow-through polishing process presents significant opportunities for improvements in facility utilization and process economics.     

正辛酸沉淀在单克隆抗体纯化中的工艺优化与应用

为应对治疗性抗体快速增长的市场需求,抗体上游细胞培养规模和表达量水平已显著提高,而下游纯化工艺的生产效率则相对落后,下游处理能力已成为限制抗体产能的瓶颈。本研究以单克隆抗体mab-X为实验材料,优化了细胞培养液、低pH病毒灭活收集液2种模式的正辛酸(caprylic acid,CA)沉淀工艺条件,并研究了CA处理去除聚体、CA处理灭活病毒等2种应用,在小试的基础上,采用低pH病毒灭活收集液CA沉淀的模式进行了500 L细胞培养规模生产放大研究,对沉淀前后的产品质量和收率进行了检测和对比。结果显示,两种模式的CA沉淀均可显著降低宿主细胞蛋白(host cell protein,HCP)残留和聚体含量,在聚体去除实验中CA沉淀可去除约15%的聚体,病毒灭活研究显示CA对逆转录模型病毒具有完全的病毒灭活能力。在放大生产规模中,下游依次进行了深层过滤收获、亲和层析、低pH病毒灭活、CA沉淀及深层过滤、阳离子交换层析,CA沉淀过程中混合时间和搅拌速度显著影响CA沉淀效果,CA沉淀处理后低pH病毒灭活液中的HCP残留量降低了895倍,沉淀后产品纯度和HCP残留均已控制在单克隆抗体质量要求范围内,CA沉淀可以减少传统纯化工艺中的一个精纯步骤。总之,下游工艺中采用CA沉淀,能够精简传统纯化工艺,并完全满足mab-X的纯化质量要求,而且能提高生产效率、降低生产成本。本研究结果将推动CA沉淀在单克隆抗体下游纯化生产中的应用,为解决目前传统纯化工艺的问题提供参考。     

Effects of solution environment on mammalian cell fermentation broth properties: Enhanced impurity removal and clarification performance

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. Biotechnol. Bioeng. 2011; 108:50–58. © 2010 Wiley Periodicals, Inc.     

猪圆环病毒2型病毒样颗粒的高效组装技术研究*

目的:探索猪圆环病毒2型(PCV2)病毒样颗粒(VLPs)的高效组装技术,提高VLPs的稳定性。方法:利用大肠杆菌表达PCV2 Cap蛋白自组装为VLPs,分析不同离子强度下VLPs的稳定性。利用切向流技术添加尿素,降低pH,可使VLPs解组装,利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。结果:PCV2 Cap蛋白自组装VLPs在150mmol/L NaCl下稳定性较差,而在500mmol/L NaCl下可提高VLPs的稳定性,但仍较易发生聚集,核酸含量均较高。在150mmol/L NaCl、300mmol/L尿素和pH 5.5的缓冲体系条件下,能够使VLPs解组装。经25%~50%饱和硫酸铵(V/V)分级沉淀粗纯,阴离子交换层析500mmol/L NaCl下洗脱获得精纯Cap蛋白,蛋白质纯度≥95%,并能够有效去除核酸。通过切向流技术去除体系中的尿素,并将NaCl浓度提高至1mol/L、pH提高至8.0,改变蛋白质表面静电荷分布,实现VLPs的高效、均一再组装,组装效率≥99%,回收率为65.85%,并明显提高VLPs的稳定性,能够稳定保存6个月以上。结论:利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part I. clearance of minute virus of mice

Membrane adsorbers may be a viable alternative to the packed‐bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18–22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands. Biotechnol. Bioeng. 2013; 110: 491–499. © 2012 Wiley Periodicals, Inc.     

Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions

Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Performance was comparable to that observed using conventional HIC conditions with high salt.     

Polishing approach with fully connected flow‐through purification for therapeutic monoclonal antibody

The biopharmaceutical industry is evolving toward process intensification that can offer increased productivity and improved economics without sacrificing process robustness. A semi‐continuous downstream process linking purification/polishing unit operations in series can reduce or eliminate intermediate holding tanks and reduce overall processing time. Accordingly, we have developed a therapeutic monoclonal antibody polishing template comprised of a connected flow‐through polishing technologies that include activated carbon, cation exchange, and anion‐exchange chromatography. In this report, we evaluated fully‐connected pool‐less polishing with three flow‐through technologies, operating as a single skid to streamline and improve an mAb purification platform. Laboratory‐scale pool‐less processing was achieved without utilizing in‐line pH adjustment and conductivity dilution based on the previously optimized single process parameter. Two connected flow‐through configurations of polishing steps were evaluated: a two‐step process using anion exchange and cation exchange and a three step process using activated carbon, anion exchange and cation exchange chromatography. Laboratory‐scale proof of concept studies showed comparable performance between the batch purification process and the pool‐less process configuration. Three step polishing highly intensified the processes and provided higher process loading and achieved bulk drug specification with higher impurity clearance (>95%) and high overall mAb yield (>95%).     

Chromatographic clarification overcomes chromatin-mediated hitch-hiking interactions on Protein A capture column

Protein A capture chromatography is a critical unit operation in the clearance of host cell protein (HCP) impurities in monoclonal antibody (mAb) purification processes. Though one of the most effective purification steps, variable levels of protein impurities are often observed in the eluate. Coelution of HCP impurities is suggested to be strongly affected by the presence of chromatin complexes (Gagnon et al., 2014; Koehler et al., 2019). We investigated the effect of removal of DNA complex and HCP reduction pre-Protein A on the HCP clearance performance of the Protein A capture step itself. We found that only reduction of DNA in the Protein A load consistently lowered HCP in the Protein A eluate. Reduction of HCP in the Protein A load stream did not produce a significant increase in the chromatography HCP clearance performance. These results are consistent across three different biosimilar therapeutic mAbs expressed by the same Chinese hamster ovary (CHO) cell line (i.e., CHO^BC® of Polpharma Biologics). This result demonstrates that optimization of the mAb purification process utilizing Protein A as the primary capture step depends primarily on being able to effectively clear DNA and associated complexes early in the process, rather than trying to incorporate HCP reduction at the harvest cell culture fluid.     

Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as “shoulder,” indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1−3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%.     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part II. Virus,host cell protein,DNA clearance,and antibody recovery

Anion exchange membrane adsorbers are used for contaminant removal in flow‐through polishing steps in the manufacture of biopharmaceuticals. This contribution describes the clearance of minute virus of mice, DNA, and host cell proteins by three commercially available anion‐exchange membranes: Sartobind Q, Mustang Q, and ChromaSorb. The Sartobind Q and Mustang Q products contain quaternary amine ligands; whereas, ChromaSorb contains primary amine based ligands. Performance was evaluated over a range of solution conditions: 0–200 mM NaCl, pH 6.0–9.0, and flow rates of 4–20 membrane volumes/min in the presence and absence of up to 50 mM phosphate and acetate. In addition contaminant clearance was determined in the presence and absence of 5 g/L monoclonal antibody. The quaternary amine based ligands depend mainly on Coulombic interactions for removal of negatively charged contaminants. Consequently, performance of Sartobind Q and Mustang Q was compromised at high ionic strength. Primary amine based ligands in ChromaSorb enable high capacities at high ionic strength due to the presence of secondary, hydrogen bonding interactions. However, the presence of hydrogen phosphate ions leads to reduced capacity. Monoclonal antibody recovery using primary amine based anion‐exchange ligands may be lower if significant binding occurs due to secondary interactions. The removal of a specific contaminant is affected by the level of removal of the other contaminants. The results of this study may be used to help guide selection of commercially available membrane absorbers for flow‐through polishing steps. Biotechnol. Bioeng. 2013; 110: 500–510. © 2012 Wiley Periodicals, Inc.     

Adsorptive filtration: A case study for early impurity reduction in an Escherichia coli production process

Primary recovery of intracellular products from Escherichia coli requires cell disruption which leads to a massive release of process-related impurities burdening subsequent downstream process (DSP) unit operations. Especially, DNA and endotoxins challenge purification operations due to their size and concentrations. Consequently, an early reduction in impurities will not only simplify the production process but also increase robustness while alleviating the workload afterward. In the present work, we studied the proof of concept whether a nonwoven anion exchange filter material decreases soluble impurities immediately at the clarification step of E. coli DSP. In a first attempt, endotoxin burden was reduced by 4.6-fold and the DNA concentration by 3.6-fold compared to conventional depth filtration. A design of experiment for the adsorptive filtration approach was carried out to analyze the influence of different critical process parameters (CPPs) on impurity reduction. We showed that depending on the CPPs chosen, a DNA lowering of more than 3 log values, an endotoxin decrease of approximately 7 logs, and a minor HCP clearance of at least 0.3 logs could be achieved. Thus, we further revealed a chromatography column protecting effect when using adsorptive filtration beforehand.