Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography.

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Safety risk management for low molecular weight process-related impurities in monoclonal antibody therapeutics: Categorization, risk assessment, testing strategy, and process development with leveraging clearance potential.

Process-related impurities (PRIs) derived from manufacturing process should be minimized in final drug product. ICH Q3A provides a regulatory road map for PRIs but excludes biologic drugs like monoclonal antibodies (mAbs) that contain biological PRIs (e.g. host cell proteins and DNA) and low molecular weight (LMW) PRIs (e.g., fermentation media components and downstream chemical reagents). Risks from the former PRIs are typically addressed by routine tests to meet regulatory expectations, while a similar routine-testing strategy is unrealistic and unnecessary for LMW PRIs, and thus a risk-assessment-guided testing strategy is often utilized. In this report, we discuss a safety risk management strategy including categorization, risk assessment, testing strategy, and its integrations with other CMC development activities, as well as downstream clearance potentials. The clearance data from 28 mAbs successfully addressed safety concerns but did not fully reveal the process clearance potentials. Therefore, we carried out studies with 13 commonly seen LMW PRIs in a typical downstream process for mAbs. Generally, Protein A chromatography and cation exchange chromatography operating in bind-and-elute mode showed excellent clearances with greater than 1,000- and 100-fold clearance, respectively. The diafiltration step had better clearance (greater than 100-fold) for the positively and neutrally charged LMW PRIs than for the negatively charged or hydrophobic PRIs. We propose that a typical mAb downstream process provides an overall clearance of 5,000-fold. Additionally, the determined sieving coefficients will facilitate diafiltration process development. This report helps establish effective safety risk management and downstream process design with robust clearance for LMW PRIs.

Process optimization and protein engineering mitigated manufacturing challenges of a monoclonal antibody with liquid-liquid phase separation issue by disrupting inter-molecule electrostatic interactions.

We report a case study in which liquid-liquid phase separation (LLPS) negatively impacted the downstream manufacturability of a therapeutic mAb. Process parameter optimization partially mitigated the LLPS, but limitations remained for large-scale manufacturing. Electrostatic interaction driven self-associations and the resulting formation of high-order complexes are established critical properties that led to LLPS. Through chain swapping substitutions with a well-behaved antibody and subsequent study of their solution behaviors, we found the self-association interactions between the light chains (LCs) of this mAb are responsible for the LLPS behavior. With the aid of in silico homology modeling and charged-patch analysis, seven charged residues in the LC complementarity-determining regions (CDRs) were selected for mutagenesis, then evaluated for self-association and LLPS properties. Two charged residues in the light chain (K30 and D50) were identified as the most significant to the LLPS behaviors and to the antigen-binding affinity. Four adjacent charged residues in the light chain (E49, K52, R53, and R92) also contributed to self-association, and thus to LLPS. Molecular engineering substitution of these charged residues with a neutral or oppositely-charged residue disrupted the electrostatic interactions. A double-mutation in CDR2 and CDR3 resulted in a variant that retained antigen-binding affinity and eliminated LLPS. This study demonstrates the critical nature of surface charged resides on LLPS, and highlights the applied power of in silico protein design when applied to improving physiochemical characteristics of therapeutic antibodies. Our study indicates that in silico design and effective protein engineering may be useful in the development of mAbs that encounter similar LLPS issues.

Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage: Identification, Characterization, and Mitigation.

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019.

Evaluation of continuous nonaffinity capture chromatography for a recombinant enzyme-optimization for a changing perfusion feedstream and comparison to batch processing.

This work presents the optimization and critical evaluation of continuous capture chromatography in the downstream process of a recombinant enzyme. For the upstream manufacturing of this molecule, a perfusion process was implemented due to benefits for product quality and productivity. This process is, however, characterized by low titer and significant changes over the course of the harvest duration in terms of active enzyme concentration and impurity content. We evaluated the feasibility and benefits of a continuous capture operation. This case study illustrates the design approach that can be utilized to address challenges presented by a changing feedstream, and the statistical measures that can be employed to characterize and optimize the operating space under material and time constraints. Process economic modeling in conjunction with Monte Carlo simulations indicate that even for a nonaffinity capture step utilizing a relatively cheap ion-exchange resin, the smaller column volume used in a continuous set-up results in cost savings compared to the batch process. We compare this option to the scenario of repeated processing using a small capture column in batch mode. Our analysis establishes that continuous processing becomes economically attractive for processes where only a small portion of the potential column lifetime can be utilized or for column steps with slow mass transport and shallow breakthrough curves. In cases where column breakthrough is sharp and resin lifetime is relatively short, continuous processing may offer an improvement over traditional batch processing, but much of the productivity and cost savings can be realized through repeated column cycling. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1195-1204, 2018.

Impact of Magnetic Stirring on Stainless Steel Integrity: Effect on Biopharmaceutical Processing.

Stainless steel containers are widely used in the pharmaceutical and biopharmaceutical industry for the storage of buffers, process intermediates, and purified drug substance. They are generally held to be corrosion resistant, biocompatible, and nonreactive, although it is well established that trace amounts of metal ions can leach from stainless steel equipment into biopharmaceutical products. We report here that the use of stainless steel containers in conjunction with magnetic stirring bars leads to significantly aggravated metal contamination, consisting of both metal particles and significantly elevated metal ions in solution, the degree of which is several orders of magnitude higher than described for static conditions. Metal particles are analyzed by scanning electron microscopy with electron-dispersive X-ray spectroscopy, and metal content in solution is quantitated at different time points by inductively coupled plasma-mass spectrometry. The concentration of iron, chromium, nickel, and manganese increases with increasing stirring time and speed. We describe the impact of buffer components on the extent of metal particles and ions in solution and illustrate the effect on model proteins.

Liquid-liquid phase separation causes high turbidity and pressure during low pH elution process in Protein A chromatography.

Turbid elution pools and high column back pressure are common during elution of monoclonal antibodies (mAbs) by acidic pH in Protein A chromatography. This phenomenon has been historically attributed to acid-induced precipitation of incorrectly folded or pH-sensitive mAbs and host cell proteins (HCPs). In this work, we propose a new mechanism that may account for some observations of elution turbidity in Protein A chromatography. We report several examples of turbidity and high column back pressure occurring transiently under a short course of neutral conditions during Protein A elution. A systematic study of three mAbs displaying this behavior revealed phase separation characterized by liquid drops under certain conditions including neutral pH, low ionic strength, and high protein concentration. These liquid droplets caused solution turbidity and exhibited extremely high viscosity, resulting in high column back pressure. We found out that the droplets were formed through liquid-liquid phase separation (LLPS) as a result of protein self-association. We also found multiple factors, including pH, temperature, ionic strength, and protein concentration can affect LLPS behaviors. Careful selection of process parameters during protein A elution, including temperature, flow rate, buffer, and salt can inhibit formation of a dense liquid phase, reducing both turbidity (by 90%) and column back pressure (below 20 pounds per square inch). These findings provide both mechanistic insight and practical mitigation strategies for Protein A chromatography induced LLPS.

Double-peak elution profile of a monoclonal antibody in cation exchange chromatography is caused by histidine-protonation-based charge variants.

We have systemically investigated unusual elution behaviors of an IgG4 (mAb A) in cation exchange chromatography (CEX). This mAb A exhibited two elution peaks under certain conditions when being purified by several strong CEX columns. When either of the two peaks was isolated and re-injected on the same column, the similar pattern was observed again during elution. The protein distribution between the two peaks could be altered by NaCl concentration in the feed, or NaCl concentration in wash buffer, or elution pH, suggesting two pH-associated strong-and-weak binding configurations. The protein distributions under different pH values showed good correlation with protonated/un-protonated fractions of a histidine residue. These results suggest that the double-peak elution profile associates with histidine-protonation-based charge variants. By conducting pepsin digestion, amino-acid specific chemical modifications, peptide mapping, and measuring the effects of elution residence time, a histidine in the variable fragment (Fab) was identified to be the root cause. Besides double-peak pattern, mAb A can also exhibit peak-shouldering or single elution peak on different CEX resins, reflecting different resins' resolving capability on protonated/un-protonated forms. This work characterizes a novel cause for unusual elution behaviors in CEX and also provides alternative avenues of purification development for mAbs with similar behaviors.

Engineering of novel Staphylococcal Protein A ligands to enable milder elution pH and high dynamic binding capacity.

We describe novel Staphylococcal Protein A ligands that enable milder elution pH for use in affinity chromatography. The change in elution pH is the result of point mutations to the protein sequence. Two novel ligands are investigated in this study. The first, designated Z(H18S)4, represents a histidine to serine substitution single mutation. The second, designated Z(H18S, N28A)4, is a double mutant comprising histidine to serine and asparagine to alanine mutations. Both are compared against the unmutated sequence, designated Z4, which is currently utilized in a commercially available Protein A stationary phase for the purification of molecules containing Fc domains. The ligands are coupled to a chromatography support matrix and tested against a panel of antibodies and an Fc fusion protein for elution pH, dynamic binding capacity, step-wise elution, and capture from clarified culture media. Results demonstrate that the novel ligands result in milder elution pH, on average >0.5 pH units, when tested in a pH gradient. For step-wise elution at pH 4.0, the Z(H18S, N28A)4 ligand showed on average a greater than 30% increase in yield compared to Z4. Importantly, for the antibodies tested the mutations did not result in a decrease in dynamic binding capacity or other desirable attributes such as selectivity. A potential application of the novel ligands is shown with a pH sensitive molecule prone to aggregation under acidic conditions.

Development and scale-up of a commercial fed batch refolding process for an anti-CD22 two chain immunotoxin.

We describe the development and scale-up of a novel two chain immunotoxin refolding process. This work provides a case study comparing a clinical manufacturing process and the commercial process developed to replace it. While the clinical process produced high quality material, it suffered from low yield and high yield variability. A systematic approach to process development and understanding led to a number of improvements that were implemented in the commercial process. These include a shorter inclusion body recovery process, limiting the formation of an undesired deamidated species and the implementation of fed batch dilution refolding for increased refold titers. The use of a combination of urea, arginine and DTT for capture column cleaning restored the binding capacity of the capture step column and resulted in consistent capture step yields compared to the clinical process. Scalability is shown with data from 250 L and 950 L scale refolding processes. Compared to the clinical process it replaces, the commercial process demonstrated a greater than fivefold improvement in volumetric productivity at the 950 L refolding scale.

Process scale separation of an anti-CD22 immunotoxin charge variant.

We describe the analytical characterization and process scale separation of a deamidated variant of an immunotoxin. The different charge variants of the immunotoxin were separated using analytical ion-exchange HPLC. These charge variants were analyzed by peptide mapping and LC-MS/MS to identify the site of modification, which was determined to reside in the toxin portion of the molecule. Using a cell-based bioassay it was also determined that deamidation led to reduced biological activity, requiring it be controlled during manufacturing. This was accomplished using process scale anion-exchange chromatography. The process was capable of reducing the deamidated form to a level low enough for the resulting product to maintain acceptable biological activity. Keys to the successful control of this impurity at process scale were a good understanding of structure-function relationship and the availability of an analytical HPLC assay to provide a surrogate for the cell-based bioassay.