Streamlining cation exchange chromatography process development for therapeutic monoclonal antibody purification.

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

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

One-step purification of monoclonal IgG antibodies from mouse ascites: An evaluation of different adsorption techniques using high performance liquid chromatography

Recent advances in computational power, scientific understanding, and analytical capabilities are enabling more mechanistic modeling in biotechnology. This chapter describes the development and application of a mechanistic model for industrially relevant monoclonal antibody–cation exchange (CEX) chromatography processes. Typical monoclonal antibody purification processes utilize a protein-A chromatography capture step followed by two polishing chromatography steps. CEX chromatography is being used extensively in monoclonal antibody purification as one of the polishing steps and has been shown to be effective at removing high molecular weight species and providing some removal of host cell proteins (HCPs) and DNAs. The CEX chromatography process is operated in bind and elute mode with the following process steps: equilibrate column with solution at low salt concentration, load column to a target product concentration, wash with equilibration solution, and elute product with a linearly increasing salt gradient. Truly leveraging a mechanistic modeling approach requires changing the way process development is performed.

A simple and rapid method for the purification of murine and human monoclonal antibodies from ascites fluids and cell culture supernatants is described. The method, based on the use of hydroxylapatite (HAP) column chromatography, is applicable on both analytical and preparative scales. In our work on purification of monoclonal antibodies, we have found that the combination of a single step elution of impurities followed by linear gradient elution of antibody provides an excellent purification of the antibody from cell culture and ascites fluids. The procedure provides very good resolution at high flow rates. The cell culture supernatant can be pumped on the preparative column at the rate of 2-3 ml/min without any measureable back pressure. The binding is independent of the flow rate. This method has been successfully used to purify several monoclonal antibodies of different subtypes from cell culture supernatants.

ABSTRACTAn 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.

Screening of six cation exchange resins for high binding capacity, monomer purity and step yield: A case study

Purification of antibodies by precipitating impurities using Polyethylene Glycol to enable a two chromatography step process

Ensuring the quality and safety of biopharmaceutical products requires the effective separation of monoclonal antibodies (mAbs) from host cell proteins (HCPs). A major challenge in this field is the enzymatic hydrolysis of polysorbates (PS) in drug products. This study addresses this issue by investigating the removal of polysorbate-degrading HCPs during the polishing steps of downstream purification, an area where knowledge about individual HCP behavior is still limited. We investigated the separation of different mAb formats from four individual polysorbate degrading hydrolases (CES1F, CES2C, LPLA2, and PAF-AH) using cation exchange (CEX) and mixed-mode chromatography (MMC) polishing steps. Our research identified a key challenge: The similar elution behavior of mAbs and HCPs during chromatographic separation. To investigate this phenomenon, we performed high-throughput binding screenings for recombinant polysorbate degrading hydrolases and representative mAb candidates on CEX and MMC chromatography resins. We then employed a three-step strategy that also served as a scale-up process, optimizing separation conditions and leading to the successful removal of specific HCPs while maintaining high mAb recovery rates (>96%). This strategy involved the use of surface response models and miniature columns for screening, followed by validation on larger columns using a chromatography system. Our results highlight the critical role of the inherent properties of mAbs for successful separation from HCPs. These results underscore the need to tailor the purification process to leverage the slight differences in binding behavior and elution profiles between mAbs and specific HCPs. This approach lays the foundation for developing more effective strategies for overcoming the challenge of enzymatic polysorbate degradation, paving the way for improved quality and safety in biopharmaceutical products.

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

Purification of monoclonal antibodies from tissue culture medium depleted of IgG

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data: Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification

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.

An improved method for the purification of IgG monoclonal antibodies from culture supernatants

Influence of ligand density variations on the two peak elution behavior of a monoclonal antibody in cation exchange chromatography