Host Cell Protein Clearance Research Articles

Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product.

Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs with mAbs can sometimes result in co-purification of trace levels of 'hitchhiker' HCPs during the manufacturing process. Purified mAb-1 product produced in early stages of process optimization had high HCP levels. In addition, these lots formed delayed-onset particles containing mAb-1 and its heavy chain C-terminal fragments. Studies were performed to determine the cause of the observed particle formation and to optimize the purification for improved HCP clearance. Protease activity and inhibitor stability studies confirmed that an aspartyl protease was responsible for fragmentation of mAb-1 resulting in particle formation. An affinity resin was used to selectively capture aspartyl proteases from the mAb-1 product. Mass spectrometry identified the captured aspartyl protease as CHO cathepsin D. A wash step at high pH with salt and caprylate was implemented during the protein A affinity step to disrupt the HCP-mAb interactions and improve HCP clearance. The product at the end of purification using the optimized process had very low HCP levels, did not contain detectable protease activity, and did not form particles. Spiking of CHO cathepsin D back into mAb-1 product from the optimized process confirmed that it was the cause of the particle formation. This work demonstrated that process optimization focused on removal of HCPs was successful in eliminating particle formation in the final mAb-1 product.

Development of advanced host cell protein enrichment and detection strategies to enable process relevant spike challenge studies.

An orthogonal chromatography methodology for the enrichment of host cell protein (HCP) species relative to monoclonal antibody (mAb) products was developed and applied for the successful enrichment of HCP from post-Protein A process pools for seven different mAb products. An advanced two-dimensional liquid chromatography/mass spectrometry platform (2D-LC/MS(E) ) was utilized to demonstrate that the HCP enriched material was representative, in terms of species content, to pre-enriched process pools. The HCP enrichment methodology was scaled up for two different mAb products, and this process relevant enriched HCP material was used to conduct advanced spike challenge studies to demonstrate the utility of the approach for the understanding of (1) quantitative HCP clearance, (2) individual species clearance, and (3) species clearance redundancy across polishing chromatography steps. The combined ability to enrich process relevant HCP, detect individual HCP species with 2D-LC/MS(E) technology, and conduct advanced challenge studies with process relevant material surmounts prior limitations to high integrity process challenge study implementation, and facilitates significant process understanding for development of risk-based control strategies and strategic process design. This also demonstrates implementation of a foundational strategy for conducting spike-challenge studies using process-relevant impurities isolated from processes of interest using orthogonal approaches.

Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies.

The performance of amino acids in Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography for monoclonal antibody purification was investigated. Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies. Improved clearance of impurity, high molecular weight species (HMW) and host cell proteins (HCP) was observed in the purification processes when using the amino acids as base-buffer constituents, additives or eluents compared with that of buffers without these amino acids. In addition, we designed a buffer system in which the mobile phases were composed of only a single amino acid, histidine, and applied it to the above three chromatographies. Effective HMW and HCP clearance was also obtained in this manner. These results suggest that amino acids may enhance impurity clearance during the purification of monoclonal antibodies.

Two-stage chromatographic separation of aggregates for monoclonal antibody therapeutics

Aggregates of monoclonal antibody (mAb) therapeutics, due to their perceived impact on immunogenicity, are recognized as a critical quality attribute by the regulatory authorities as well as the industry. Hence, removal of aggregates is a key objective of bioprocessing. At present, this is achieved by a combination of two or more orthogonal chromatographic steps with possible modalities of ion exchange, hydrophobic interaction and mixed mode. A two-stage chromatographic purification process consisting of ion-exchange and hydrophobic interaction modes is proposed in this paper for effective and efficient control of aggregates for a mAb therapeutic. The proposed scheme does not require any intermediate processing of the process stream. Further, baseline separation is achieved for monomer and aggregates resulting in robust performance. This was possible because the proposed operational scheme allowed for an addition of selectivities of the two chromatography modes vs. the traditional two column scheme where part of the separation of aggregates achieved by the first column is lost upon pooling. The proposed process scheme yielded improved separation of aggregates (0% vs. 1–2%) at >95% recovery and reduced overall process time (6h vs. 14h) for a typical application. Further, clearance of host cell proteins was also shown to have improved with the suggested process scheme. Successful implementation of the proposed scheme has been demonstrated for two different monoclonal antibody therapeutic products.

A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies.

Co-purification of a subset of host cell proteins (HCPs) with monoclonal antibodies (mAbs) during the capture of mAbs on Protein A affinity chromatography is primarily caused by interactions of HCPs with the mAbs. To date, there is limited information about the identity of those HCPs due to the difficulty in detecting low abundance HCPs in the presence of a large amount of the mAb. Here, an approach is presented that allows identification of HCPs that specifically associate with the mAb, while avoiding interference from the mAb itself. This approach involves immobilization of purified mAb onto chromatography resin via cross-linking, followed by incubation with HCPs obtained from supernatant of non-mAb producer cells that are representative of the expression systems used in mAb manufacturing. The HCPs that bind to the mAb are recovered and identified using mass spectrometry. This approach has not only allowed a comprehensive comparison of HCP subpopulations that associate with different mAbs, but also enabled monitoring of the effects of a variety of wash modifiers on the dissociation of individual HCP–mAb interactions. The dissociation of HCPs that associated with the mAb was monitored by enzyme-linked immunosorbent assay and mass spectrometry. This approach can be utilized as a screening tool to assist the development of effective and targeted wash steps in Protein A chromatography that ensures not only reduction of HCP levels copurified with the mAb but also removal of specific HCPs that may have a potential impact on mAb structural stability and patient safety. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1114–1124, 2014

Affinity precipitation of a monoclonal antibody from an industrial harvest feedstock using an ELP‐Z stimuli responsive biopolymer

In this work, a proof of concept elastin-like polypeptide-Z domain fusion (ELP-Z) based affinity precipitation process is developed for monoclonal antibody (mAb) purification from industrial harvest feeds. Greater than 99% mAb recoveries are obtained during the initial binding step of the process for both pure mAb and the mAb harvest feeds. Great than 90% overall mAb yields are also obtained for the subsequent elution step of the process with no measurable mAb aggregation. The process is shown to result in more than 2 logs of host cell protein (HCP) and more than 4 logs of DNA clearance from the harvest feed. While the overall mAb yield and HCP clearance for the affinity precipitation process was comparable to Protein A chromatography the DNA clearance was clearly superior. Performance is maintained for mAb final elution concentrations up to 20 g/L, demonstrating the ability of the process to both concentrate and purify the mAb. Effective ELP-Z regeneration is also demonstrated using 0.1 M NaOH with no adverse effect on subsequent capture efficiency. Finally, the reusability of the ELP-Z construct and robustness of the process is demonstrated for up to three purification-regeneration cycles with minimal product and impurity carryover and high yields and purity. This work demonstrates that the ELP-Z based precipitation approach can be successfully employed as an affinity capture step for industrial mAbs.

Evaluation of high‐capacity cation exchange chromatography for direct capture of monoclonal antibodies from high‐titer cell culture processes

Advances in molecular biology and cell culture technology have led to monoclonal antibody titers in excess of 10 g/L. Such an increase can pose concern to traditional antibody purification processes due to limitations in column hardware and binding capacity of Protein A resins. Recent development of high capacity cation exchangers can make cation exchange chromatography (CEX) a promising and economic alternative to Protein A capture. This work investigates the feasibility of using CEX for direct capture of monoclonal antibodies from high titer cell culture fluids. Two resin candidates were selected from seven newer generation cation exchangers for their higher binding capacity and selectivity. Two monoclonal antibodies with widely differing pI values were used to evaluate the capability of CEX as a platform capture step. Screening of loading pH and conductivity showed both resins to be capable of directly capturing both antibodies from undiluted cell culture fluid. At appropriate acidic pH range, product loading of over 65 g/L resin was achieved for both antibodies. A systematic design of experiment (DOE) approach was used to optimize the elution conditions for the CEX step. Elution pH showed the most significant impact on clearance of host cell proteins (HCPs). Under optimal conditions, HCP reduction factors in the range of 9-44 were achieved on the CEX step based on the pI of the antibody. Apart from comparing CEX directly to Protein A as the capture method, material from either modality was also processed through the subsequent polishing steps to compare product quality at the drug substance level. Process performance and product quality was found to be acceptable using the non-affinity based process scheme. The results shown here present a cheaper and higher capacity generic capture method for high-titer antibody processes.

Feasibility study of semi‐selective protein precipitation with salt‐tolerant copolymers for industrial purification of therapeutic antibodies

We present a feasibility study for an antibody capturing process from clarified cell culture fluid using semi-selective protein precipitation with salt-tolerant copolymers. Protein precipitation is mediated by hydrophobic and electrostatic interactions with the copolymer that can be customized for the respective target. Precipitation yield with different copolymers at ionic strength of 2-22.5 mS cm⁻¹ and pH 5.0-pH 5.7 was evaluated using pure monoclonal antibody solutions. Optimized parameters were used to elucidate yield and purity of various antibodies precipitated at physiological conditions from cell culture fluid of CHO, NS0, and SP2/0 cell culture fluid. Precipitated protein was easily redissolved in small volume, enabling concentrating monoclonal antibodies (mAb) more than 40-fold and up to 100-fold, while residual polymer was removed to >98% using cationic polymer attached to silica flakes. mAb recovery of >90% and host cell protein clearance of >80% were achieved, not requiring any pre-dilution of cell culture fluid. Precipitation showed no impact on mAb binding affinity when compared to non-precipitated mAb. The obtained yield and purity were lower compared to a protein A based purification and loss of mAb was factor 1.5-3.0 higher. Yet, for high titer mAb purification processes being implemented in the future, precipitation is an attractive option due to its ease of scalability and cost-effectiveness.

The Comparison of Chemiluminescent- and Colorimetric-detection Based ELISA for Chinese Hamster Ovary Host Cell Proteins Quantification in Biotherapeutics

Biologics manufacturing requires the clearance of Host Cell Proteins (HCPs) from recombinant therapeutic protein to acceptable low levels to ensure product purity and patient safety. To ensure adequate removal, a highly sensitive method, commonly in the form of Enzyme-Linked Immunosorbent Assay (ELISA), is necessary to quantify the HCPs amount in process intermediates and drug substance. We report the development of a chemiluminescent detection based ELISA (luminescent ELISA) in lieu of previously used colorimetric method (colorimetric ELISA) to improve assay sensitivity for the quantification of Chinese Hamster Ovary (CHO) HCPs in a monoclonal antibody product (mAb-A). For luminescent ELISA, Pierce Supersignal ELISA Femto was chosen as the substrate to replace colorimetric substrate TMB. The assay performance of luminescent and colorimetric ELISA was directly compared side-by-side. Our data show that luminescent ELISA has better signal/background ratio, broader linear range over logarithmic scales, and better linearity within the same linear range than colorimetric ELISA. Luminescent ELISA also demonstrates better lowend linearity, greater accuracy and precision. In addition, the Limit of Detection (LOD) and Limit of Quantification (LOQ) are significantly improved with luminescent ELISA as compared to colorimetric ELISA. In summary, luminescent ELISA is a more sensitive method and demonstrates superiority over colorimetric method for CHO HCP quantification.

Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography

Protein A chromatography is typically used as the initial capture step in the purification of monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Although exploiting an affinity interaction for purification, the level of host cell proteins in the protein A eluent varies significantly with different feedstocks. Using a batch binding chromatography method, we performed a controlled study to assess host cell protein clearance across both MabSelect Sure and Prosep vA resins. We individually spiked 21 purified antibodies into null cell culture fluid generated with a non-producing cell line, creating mock cell culture fluids for each antibody with an identical composition of host cell proteins and antibody concentration. We demonstrated that antibody-host cell protein interactions are primarily responsible for the variable levels of host cell proteins in the protein A eluent for both resins when antibody is present. Using the additives guanidine HCl and sodium chloride, we demonstrated that antibody-host cell protein interactions may be disrupted, reducing the level of host cell proteins present after purification on both resins. The reduction in the level of host cell proteins differed between antibodies suggesting that the interaction likely varies between individual antibodies but encompasses both an electrostatic and hydrophobic component.

Effects of resin ligand density on yield and impurity clearance in preparative cation exchange chromatography. I. Mechanistic evaluation

The effects of resin ligand density on cation exchange chromatography performance in preparative monoclonal antibody purification processes were evaluated. A set of agarose-based cation exchange resins spanning a relatively wide range of ligand densities was tested using three different monoclonal antibodies with unique impurity profiles. Experiments were performed in bind-and-elute mode using gradient elution at both intermediate protein loadings and near saturation capacity. Ligand density did not affect clearance of high molecular weight variants under any of the conditions tested; however, ligand density did affect resolution of a basic charge variant in one case and changed host cell protein clearance in another instance. In general, the results indicate that the relationships between ligand density, retention, and resolution are affected by both characteristic charge and protein surface charge distribution.

Overloading ion-exchange membranes as a purification step for monoclonal antibodies.

The present study examined the overloading of ion-exchange membrane adsorbers, a form of frontal chromatography, as the final purification step in the production of mAbs (monoclonal antibodies) produced from CHO (Chinese-hamster ovary) cells. Preferential binding of impurities over antibody product was exploited using commercially available cation- and anion-exchange membranes. Three different antibody feedstreams previously purified over Protein A and ion-exchange column chromatography were tested. Feedstream conductivity and pH were adjusted to induce product and impurity adsorption. Membranes were then overloaded in a normal flow mode, resulting in retention of impurities and breakthrough of purified antibody. Although some amount of the product also binds to the membranes (usually ≤30 g mAb/l membrane), yields of ≥99% were achieved by marginalizing the losses, typically by loading more than 3 kg mAb/l membrane. Analyses of the purified pools show consistent removal of impurities despite strong mAb–ligand interactions and high membrane loadings. The clearance of host cell proteins was affected by pH and conductivity, but was unaffected by flow rate, membrane properties or scale. The importance of the present study lies in our demonstration of an alternative use of ion-exchange membranes for fast, effective and high yielding purification of mAbs.

Monoclonal antibody purification using cationic polyelectrolytes: An alternative to column chromatography

The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.

Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli

Apolipoprotein A 1 Milano (ApoA-1M), the protein component of a high-density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co-purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA-1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing 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 phiX174 (pI approximately 6.7) at pH 7.5 and phage PR772 (pI approximately 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.

Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A‐IMilano in an industrial HIC unit operation

We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A-I(Milano) (apo A-I(M)) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale-up of biopharmaceutical manufacturing processes. Product association of the HCPs is confirmed using co-immunoprecipitation and Western blotting techniques. Two-dimensional gel electrophoresis and mass spectrometry techniques are used to confirm the identity of OppA and DppA. In this example, clearance of these difficult to separate HCPs decreased significantly when the process was scaled to a 1.4 m diameter column. Laboratory-scale experimentation and trouble shooting identified several key parameters that could be further optimized to improve HCP clearance. The key parameters included resin loading, peak cut point on the ascending side, wash volume, and wash salt concentration. By implementing all of the process improvements that were identified, it was possible to obtain adequate HCP clearance so as to meet the final specification. Although it remains speculative, it is believed that viscosity effects may have contributed to the lower HCP clearance observed early in the manufacturing campaign.

Viral clearance using monoliths

Clearance of biological impurities is an essential part of the manufacture of biotechnology-derived products such as monoclonal antibodies (mAbs). Salt is required during manufacture to solubilize the mAb product and stabilize it against aggregation, but salt can be a problem later during impurity clearance operations. In this work, the use of a traditional quaternary amine (Q) monolith, and a new salt-tolerant monolith were evaluated for the clearance of pathogenic impurities including viruses, DNA, and host-cell protein (HCP). The impact of flow rate, salt concentration, and presence of mixtures of impurities in the feed stream were evaluated. Both monoliths cleared DNA to the limit of detection at all salt concentrations, and both cleared virus and HCP equally well at no salt. At intermediate salt, clearance of HCP was greater for the salt-tolerant monolith, and only the salt-tolerant monolith cleared virus at elevated salt. In conclusion, monoliths successfully trapped impurities such as DNA, host-cell protein, and viruses, and at flow rates far greater than traditional chromatography columns packed with beads.

Host cell protein clearance during protein a chromatography: Development of an improved column wash step

Host cell protein (HCP) contaminant clearance is a significant concern during downstream process development for biopharmaceuticals. Protein A chromatography as a capture step for monoclonal antibodies and Fc fusion proteins can clear a large proportion of these impurities from cell culture harvest. Nevertheless, remaining levels of this process-related impurity class do present significant constraints on the rapid development of effective and robust polishing steps. Conventionally, an intermediate pH wash is employed between column loading and elution to minimize HCP levels after Protein A chromatography. A significant mechanistic finding presented in this work is that HCP contaminants that persist following Protein A capture predominantly comprise species that associate with the product in preference to direct interaction with the chromatographic resin. This suggests that the development of improved column wash techniques to maximize HCP clearance ought to focus on disrupting protein-HCP interactions rather than Protein A-HCP interactions. A higher wash pH to preserve product--Protein A binding along with the use of additive combinations to disrupt interactions between HCPs and the product are investigated. This strategy was successfully applied to develop a broadly applicable wash condition that has the potential for eliminating the need for product specific optimization of wash conditions. A combination of 1 M urea and 10% isopropanol in the wash buffer were successfully applied as a platform wash condition for Protein A chromatography. Use of this (and other similar) wash conditions are anticipated to aid the rapid development of effective downstream processes for monoclonal antibodies and Fc fusion proteins resulting in their rapid introduction into clinical trials.

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.

Demonstration of Robust Host Cell Protein Clearance in Biopharmaceutical Downstream Processes

Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.