Host Cell Protein Reduction Research Articles

Optimization for Simultaneous Removal of Product/Process-Related Impurities of Peptide Fc-Fusion Protein Using Cation Exchange Chromatography

Fc fusion proteins are used as therapeutic agents with unique structures by combining the Fc domain of an antibody with other active proteins, cytokines, and enzymes. Peptide Fc-fusion proteins are complex fusion molecules that possess a structure different from that of monoclonal antibodies (mAbs) and are difficult to express, thereby affecting their quality. Many product/process-related impurities generated during the production of peptide Fc-fusion proteins pose a risk to the robustness of pre-existing three-column platforms for the purification of mAbs. Thus, we first evaluated the effect of pH, conductivity, and dynamic binding capacity (DBC; g of product per liter of resin) on the separation of host cell protein (HCP) and high molecular weight (HMW) and low molecular weight (LMW) proteins in strong cation exchange chromatography and then established an operating range using the design of experiments (DoE). Based on our studies, the optimal removal rates of HCP and HMW were achieved under the following conditions: 8 CV of wash buffer, 20–23 g/L of resin DBC, and an elution buffer conductivity of 63–66 mS/cm. The conductivity of the wash buffer used to remove the LMW was 50 mS/cm. In addition, reproducibility was confirmed by scaling up two batches using the Fractogel® EMD SO3− (M) resin. As a result of confirming with a validated test method in all batches, >55% yield, >98.2% purity, and >27% HCP reduction rate were satisfied. The cation exchanger exhibited an acceptable step yield and effectively reduced product/process-related impurities within the established range.

Impact of freeze-thaw processes on monoclonal antibody platform process development.

Freezing of cell culture supernatant (CCS) is a standard procedure in process development of monoclonal antibody (mAb) platform processes as up- and downstream development are usually separated. In the manufacturing process of mAb, however, freezing is avoided, which poses the question of comparability and transferability from process development to manufacturing. In this case study, mAb CCS from Chinese hamster ovary (CHO) cells is frozen and thawed in a novel active freezing device and subsequently captured by protein A chromatography. Critical quality attributes such as host cell protein (HCP) concentration and soluble mAb dimer shares have been monitored throughout the case study. Furthermore, cryo-concentration of individual proteins was investigated. The main factors that drive cryo-concentration are diffusion and natural convection. Natural convection in freezing processes was found to increase at warmer freezing temperatures and thus slower freezing, leading to higher concentration gradients from top to bottom of a freezing chamber. The freeze concentration was dependent on protein size and correlated to diffusivity, where smaller proteins are exposed to higher cryo-concentration. Our results suggest that as a result of freezing processes, large particles based on mAb and specific host cell proteins (HCPs) expressing a certain affinity to mAbs are formed that have to be removed before purification. This leads to a significant improvement in HCP reduction by the protein A step, when compared with reference samples, where twice as much HCP remained in the eluate. Furthermore, HCP and mAb dimer concentrations in protein A eluate were dependent on the freezing temperature. As a conclusion, CCS should be frozen as rapidly as possible during process development to minimize issues of transferability from process development to manufacturing.

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., CHOBC ® 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.

Process characterization strategy for a precipitation step for host cell protein reduction.

Process characterization using QbD approaches has rarely been described for precipitation steps used for impurity removal in biopharmaceutical processes. We propose a two‐step approach for process characterization in which the first step focuses on product quality and the second focuses on process performance. This approach provides an efficient, streamlined strategy for the characterization of precipitation steps under the Quality by Design paradigm. This strategy is demonstrated by a case study for the characterization of a precipitation using sodium caprylate to reduce host cell proteins (HCP) during a monoclonal antibody purification process. Process parameters were methodically selected through a risk assessment based on prior development data and scientific knowledge described in the literature. The characterization studies used two multivariate blocks to decouple and distinguish the impact of product quality (e.g., measured HCP of the recovered product from the precipitation) and process performance (e.g., step yield). Robustness of the precipitation step was further demonstrated through linkage studies across the overall purification process. HCP levels could be robustly reduced to ≤100 ppm in the drug substance when the precipitation step operated within an operation space of ≤1% (m/v) sodium caprylate, pH 5.0–6.0, and filter flux ≤300 L/m2‐hr for a load HCP concentration up to 19,000 ppm. This two‐step approach for characterization of precipitation steps has several advantages, including tailoring of the experimental design and scale‐down model to the intended purpose for each step, use of a manageable number of experiments without compromising scientific understanding, and limited time and material consumption.

Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single-chain variable fragment.

Single-chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L-based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed-mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel™), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere™). The DoE designs of the polishing step included both binding and flow-through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere™ in binding-mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9>98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere™ below its theoretical pI, while the majority of HCPs were in the flow-through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.

One-step affinity capture and precipitation for improved purification of an industrial monoclonal antibody using Z-ELP functionalized nanocages.

Protein A chromatography has been identified as a potential bottleneck in the monoclonal antibody production platform, leading to increased interest in non-chromatographic capture technologies. Affinity precipitation using environmentally responsive, Z-domain-elastin-like polypeptide (Z-ELP) fusion proteins has been shown to be a promising alternative. However, elevated temperature and salt concentrations necessary for precipitation resulted in decreased antibody monomer content and reduced purification capacity. To improve upon the existing technology, we reported an enhanced affinity precipitation of antibodies by conjugating Z-ELP to a 25 nm diameter, self-assembled E2 protein nanocage (Z-ELP-E2). The enlarged scale of aggregate formation and IgG-triggered crosslinking through multi-valent binding significantly outperformed traditional Z-ELP-based methods. In the current work, we sought to develop an affinity precipitation process capable of purifying industrial monoclonal antibodies (mAbs) at ambient temperature with minimal added salt. We discovered that the mAb-nanocage complex aggregated within 10 min at room temperature without the addition of salt due to the enhanced multi-valent cross-linking. After precipitating out of solution, the complex remained insoluble under all wash buffers tested, and only resolubilized after a low pH elution. Through optimization of key process steps, the affinity precipitation yield and impurity clearance met or exceeded protein A chromatography performance with 95% yield, 3.7 logs host cell protein reduction, and >5 logs of DNA reduction from mAb cell culture. Because of the operational flexibility afforded by this one-step affinity capture and precipitation process, the Z-ELP-E2 based approach has the potential to be a viable alternative to platform mAb purification.

Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor.

We successfully transferred a two-stage batch precipitation-based antibody capture step to continuous mode using continuous tubular reactors. The precipitation process solely employs a cheap mineral salt (CaCl2 ) and an organic solvent (ethanol) and could replace the costly protein A capture step in the purification of recombinant antibodies from cell culture supernatant. The time from startup untill attaining steady state conditions was reached in less than 15 minutes and both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield of > 90 percent, with a host cell protein reduction from 42 777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm, could be achieved for the antibody investigated. The precipitated antibody can be dissolved at very high concentrations (> 40 g/L) in numerous buffer systems of various pH and high and low ionic strength, thereby rendering a subsequent concentration or buffer exchange step redundant. This system enables cell culture supernatants with low or high antibody titer to be processed with constant reactor size and without changing any parameters or increasing precipitant consumption. Aggregate levels were below 1% under all conditions tested. Purification by precipitation did not affect binding to CD16a or the isoform distribution of the antibody.

High productivity purification of immunoglobulin G monoclonal antibodies on starch-coated magnetic nanoparticles by steric exclusion of polyethylene glycol

We achieved exceptionally high capacity capture of monoclonal IgG by adding 200nm starch-coated magnetic particles as nucleation centers, adding polyethylene glycol (PEG), then collecting the particle-associated antibody in a magnetic field. Experimental data suggest that accretion of IgG begins on particle surfaces then continues with fusion of particle-centric accretions up to about 1mm in a process that closely parallels PEG precipitation. An embedded nanoparticle mass of 1.3% of the IgG mass is adequate to enable efficient magnetic collection of the associated IgG. Recovery of purified IgG averaged 98% up to loads of 78mg of IgG per mg of particles. Converted to an equivalent volume of settled particles, this represents about 58g IgG per mL of nanoparticles, which is roughly 1000 times higher than the average capacity of commercial protein A porous particles packed in columns. When applied to cell culture harvest clarified by centrifugation and microfiltration, performing the nanoparticle technique under physiological conditions permitted only a 10-fold reduction of host cell protein (HCP) contamination and IgG recovery less than 50%. Application of a more capable clarification method and operating the nanoparticle method at 0.5–1.0M NaCl supported more than 99% HCP reduction and 87% IgG recovery. The high salt concentration also dramatically diminished the influence of operating pH on selectivity. The nanoparticle step was followed by sample application without buffer exchange to a column packed with multimodal electropositive-hydrophobic particles that reduced HCP to 2ppm. Aggregate content was reduced from 4.9 to 3.6% at the nanoparticle step, then to less than 0.05% at the multimodal step. The multimodal step also removed residual PEG. Overall IgG recovery was 69%. The ability of the system to achieve purity similar to protein A, but dramatically higher productivity than packed columns, suggests that the technique could evolve as a credible option for industrial purification of monoclonal antibodies.

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.

Screening of peptide ligands that bind to the Fc region of IgG using peptide array and its application to affinity purification of antibody

Small peptides have attracted great interest for affinity purification since they are more stable, less immunogenic, and less expensive compared to protein ligands. In this study, screening of peptides that has affinity to Fc region of IgG was investigated from amino acid sequence of the IgG Fcγ receptor, which is well known as one of the IgG-Fc binding proteins, using a spot-synthesized peptide array. High affinity octamer peptides, NKFRGKYK and NARKFYKG, for mouse IgG were obtained by amino acid substitution assays. Both peptides also recognized human IgG-Fc. The association constants for human IgG-Fc were 8.9×106 (NKFRGKYK) and 6.5×106M−1 (NARKFYKG). Purity of the human IgG-Fc from cell culture medium using the peptide immobilized resins was 83% for NKFRGKYK and 68% for NARKFYKG. These peptide ligands were used for the purification of monoclonal antibody (MAb) from cell culture supernatants. The yields obtained for the antibody were found to be 69% and 80% for NKFRGKYK and NARKFYKG, respectively. The residual DNA and host cell protein reduction obtained by these peptides resin were in the range of 4.2 and 1.3 log reduction value (LRV) (NKFRGKYK), 3.9 and 1.5LRV (NARKFYKG), respectively, comparable to those reported for Protein A. Therefore, it was indicated that the screened NKFRGKYK and NARKFYKG peptide would be useful as affinity purification ligands for IgG.

Fast and scalable purification of a therapeutic full‐length antibody based on process crystallization

The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2 M guanidine–HCl or a combination of 0.85% phosphoric acid followed by 2 M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20 mg/mL.