William K. Wang

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.

Monoclonal antibody fragment removal mediated by mixed mode resins

Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities are required.

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.

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.

Cathepsin L Causes Proteolytic Cleavage of CHO 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.