ALTERNATIVES TO PROTEIN A: Improved Downstream Process Design for Human Monoclonal Antibody Production - A two-step process—a cation exchange capture column followed by anion exchange flow-throu

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ALTERNATIVES TO PROTEIN A: Improved Downstream Process Design for Human Monoclonal Antibody Production
A two-step process—a cation exchange capture column followed by anion exchange flow-through membrane chromatography—resulted in >80% process yield, higher batch capacity, and shorter process time.


BioPharm International


ABSTRACT

Affinity purification schemes for antibody production have certain limitations keeping up with cell culture expression levels as they reach and exceed 10 g/L. New downstream purification processes are based on low cost, long lasting, and high binding (40–100 mg/mL) cation exchange resins.

Replacing column chromatography with membrane chromatography makes processes less validation-intensive and more operationally friendly without compromising product quality. These non–Protein A purification processes can be as simple as a cation exchange capture column followed by anion exchange flow-through membrane chromatography. These processes represent a significant process improvement in antibody purification schemes.

A purification process with these two separation mechanisms along with additional specific viral reduction steps meets the requirements for endogenous and adventitious agents and other process-related contaminant removal. These two-step purification schemes typically result in >80% process yield and significantly improved batch capacity and process time, leading to efficient facility usage.



Purification schemes for antibody production based on affinity chromatography are trying to keep pace with increases in cell culture expression levels. There have been reports of 10 g/L of antibody production from mammalian cell culture and predictions that 20 g/L will be reached in the next few years.1 This ramping up of productivity is challenging in downstream processing.2 Significant advances are needed to prevent purification from becoming a bottleneck.

Several innovative approaches are being implemented to streamline purification processes. These include using high-binding resins that can handle high flow rates,3 decreasing cycle time by eliminating a few process steps, and implementing disposable unit operations for chromatography.4

A majority of these advancements have been carried out on affinity purification schemes. Polishing steps have also been the subject of easy modifications. A simplified affinity purification scheme with a disposable polishing membrane chromatography is reported recently1 which is more productive than traditional affinity processes.

This article outlines a different approach for purifying human monoclonal antibodies (HuMAbs). We have developed a non-affinity purification platform with efficient ion-exchange capture chromatography.5 Optimizing such ion exchange capture met the demands of purity, contaminant removal, and recovery, and we were able accomplish this in two or three downstream processing steps.

Streamlining Capture Chromatography

Affinity chromatography became the traditional choice for antibody processes of large volumes of upstream cell culture harvest because of its specificity, ability to handle unconditioned feed straight from bioreactors, scale-up robustness, and protein purity level. However, Protein A chromatography has some inherent limitations because of its high cost and leachability. Second- and third-generation affinity resins offered several improved qualities such as alkali compatibility, low ligand leaching, better recyclability, and higher binding. Mixed-mode resins and affinity by mimetic ligands were used in some cases, but those have limited application. Thus, the most common antibody platform technology for mammalian cell culture is affinity capture followed by one or two polishing steps to clear the process- and product-related contaminants and viruses.

Normally, ion exchange separations are implemented as polishing steps, either as a flow-through mode or a bind-and-elute mode, to remove the last traces of contaminants. We took on the challenge of developing an efficient ion-exchange alternative to an affinity column to meet all the demands of capture chromatography.

Few examples of a purification platform approach based on a cation exchange (CEX) capture step appear in the literature. However, such a process can offer several advantages over affinity purification schemes, provided the purity (>95%) and yield (80–90%) are comparable to conventional designs. We will discuss the development of simplified downstream processes for HuMAbs, based on cation capture and one or more polishing steps. A wide variety of cation exchange resins are available at relatively low cost. By careful screening methods and with the ability to manipulate process conditions, binding capacities can be improved several fold over affinity resins. In addition, for high-titer feedstocks, larger columns are much more affordable and fewer cycles are needed.


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