ALTERNATIVES TO PROTEIN A: Improved Downstream Process Design for Human Monoclonal Antibody Production

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.

Early process schemes

For several HuMAbs, with expression levels as high as 500 mg/L, a cation capture step with 15 to 20 mg/mL capacity was developed, and then the process was scaled up successfully 10,000 fold with step recovery of 85 to 95%. These early process schemes consisted of three columns and an in-process tangential flow filtration (TFF) step. As HuMAb expression levels approached 4 g/L, we needed to integrate our upstream and downstream platform technology development to reduce the constraints posed by existing purification schemes. The next generation process was a result of a carefully crafted cation capture step with a high binding resin (40 to 100 mg/mL resin) and well-designed orthogonal polishing step(s). This setup was comparable to the affinity processes both in terms of yield and purity (Figure 1).

Figure 1. Ion exchange purification schemes for human monoclonal antibody production processes

Product quality and step yield with cation capture–based purification schemes for various HuMAbs are presented in Table 1. Process conditions were optimized for various process schemes with different HuMAbs. In the interest of brevity, we do not list details of resin type, column size, or other aspects. Cation capture resulted in >97% purity and 82% recovery for various HuMAbs.

Table 1. Product purity and step yield for the cation exchange capture step

Eliminating the In-Process Diafiltration Step

Although column chromatography accounts for the lion's share of costs in a purification process, intermediate diafiltration step(s) add substantial costs and time. In certain cases, optimizing process conditions on the columns before and after the in-process tangential flow filtration (TFF) step can be accomplished by selecting appropriate buffer species that are compatible with both columns. Dual buffer systems can be developed for a transition between ion exchange columns, and doing so may eliminate a TFF step. We have found that membrane chromatography is a viable alternative for processing large volumes if dilution is necessary to decrease conductivity of the load to a polishing step.

Updating the Polishing Steps

In addition to modifying the capture step, we took a multi-pronged approach to modifying the other chromatography steps with the goal of improving process time, economics, and validation. Innovative approaches for reducing or substituting existing polishing steps of generic purification processes are attempted by using state-of-the-art technologies, including disposable chromatography. Smooth transition of chromatography steps without prior conditioning of column feeds by a diafiltration step was accomplished by modifying buffer conditions, which made simple dilution possible. Under these conditions, flow-through membrane chromatography was handy because of its high flow rates.

These membranes not only offered fast processing time but also retained a contaminant clearance capability equivalent to column chromatography. Table 2 shows the comparison between Q resin and Q membrane chromatography. Each antibody has optimized process conditions. We show that the resin- and membrane-based methods are comparable in the performance of recovery and that Q membrane chromatography is a good substitute for resin chromatography with the same ligand, and can enable faster processing and easier validation.

Table 2. Performance comparison of anion exchange (AEX) chromatography showing that Q column and Q membrane chromatography are approximately equal

Designing a Simple Purification Scheme

Cation capture chromatography removes process-related contaminants to such a low level that a single polishing step is enough to clean the residuals. An additional advantage of cation capture is that it can reduce or remove the high molecular weight product-related species and easily find optimized conditions. Protein A capture does not have this ability.

Table 3. Viral clearance comparison for non-affinity purification schemes

Membrane chromatography also has been shown to reduce adventitious agents by significant levels. We set up cation capture and anion membrane units along with specific steps for viral inactivation and removal; these downstream process designs achieved log reduction values (LRV) >20 and provide a >10 LRV safety factor for therapeutic grade antibodies (Table 3). Clearance capability for process-related contaminants by ion exchange purification schemes for various HuMAbs are presented in Tables 4 and 5. The progressive stages of non-affinity downstream process development are illustrated in Figure 1.

Table 4. CHO host cell protein removal by non-affinity purification schemes

A shortened purification scheme was designed for therapeutic grade HuMAbs by combining the desirable characteristics of cation exchange capture and efficiency and the simplicity of membrane chromatography. An additional advantage of this process was that it minimized validation requirements. Including a clarification and a recovery step, overall process recovery was more than 85%.

Table 5. CHO DNA removal by non-affinity purification schemes

Scaling up the ion exchange schemes

Our ion exchange purification platform has been proven to be easy to scale up for commercial-scale manufacturing and has allowed multiple campaigns per year for different HuMAbs. These processes, originally designed for early clinical phase trials, needed minor changes to fit the process in large manufacturing and commercialization, but they paid off in large gains in time and resources during product development.

These new simplified process designs resulted in up to a 10-fold improvement in the processing time over the original ion exchange processes in addition to reducing the equipment, resin, buffer, and validation costs (Figure 2). Thus, the higher batch capacity of this improved downstream platform technology significantly reduces the cost of developing and manufacturing antibodies.^6,7

Figure 2. Comparison of process technology designs and their impact on the facility output

ALAHARI ARUNAKUMARI, PhD, is director of process development at Medarex Inc, 908.479.2451, aarunakumari@medarex.com JUE (MICHELLE) WANG, PhD, is senior manager of purification process development at Medarex, and GISELA FERREIRA, PhD, is a scientist in purification process development at Medarex.

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