Improving Bioprocessing Productivity Through the Application of Single-Use Protein A Columns

Transcription

1 Improving Bioprocessing Productivity Through the Application of Single-Use Protein A Columns By K. John Morrow, Jr. Ph.D. In the last two decades, the bioprocessing industry has confronted the demands of more rapid and efficient antibody purification as the number of antibody therapeutics in development continues to increase. The ever-increasing antibody titer concentrations creates a manufacturing bottleneck if downstream technologies are unable to advance rapidly enough to meet these demands. The increases on the upstream side are constantly pressuring those working in downstream to eliminate these bottlenecks and improve productivity and manufacturing efficiencies. As such, the appeal of pretested, disposable technologies holds great promise for the simplification of the production process. Single-use technology has gained popularity for a variety of reasons, including flexibility, cost savings, and reliability. With many early-stage clinical trials underway, pharma and biotech companies are outsourcing development to CMOs in order to delay capital investment. This spurs a demand for development of multiple drug products produced at a single site. Even at large scale, disposable purification technologies that can handle larger volumes are becoming viable alternatives. Additionally, the trend toward smaller, high potency drugs to treat specific diseases continues to drive the demand for a range of disposable technologies. The current state of the industry has been summarized by Langer 1 : Although much of the discussion surrounding downstream purification concerns resolving or avoiding short-term capacity constraints, the long-term implications

2 to the industry are more crucial and significant in terms of improving overall bioprocessing, increasing the number of world-class facilities, and lowering overall costs. Langer s surveys have determined that downstream processing is one of the top areas in which both industry suppliers and end users are developing and evaluating new technologies and other options for dealing with major challenges. These include: (1) cost of chromatography materials, (2) lack of disposable options, (3) cost of membranes, (4) cleaning and validation costs, and (5) improving efficiency of operations. Properties of Antibody Resins The mainstay of antibody purification is protein A, which binds antibodies of the immunoglobulin G class (IgG) with high affinity. This 56 kda surface protein, originally found in the cell wall of the bacterium Staphylococcus aureus, has proven to be the best all-round choice despite some drawbacks. Three principal types of Protein A resins are referred to by Liu et al 2, including glass or silica-based, such as the Prosep va and Prosep va Ultra from Millipore; the agarose-based Protein A Sepharose Fast Flow, MabSelect from GE Healthcare; and organic polymer-based, such as polystyrene-divinylbenzene Poros A and MabCapture, from Applied Biosystems. The primary drawback of Protein A is its high cost. In one study obtained, costs of $5K to $10K per kg of monoclonal antibody purified (depending on the resin type 3 ) and it may represent 70% of the cost of a downstream purification process. Various factors influence these numbers, including product amount, batch size, column size, and the process time demanded by the particular protocol. Despite the cost, Protein A is still favored, as it can yield a highly purified product in a single step (see Figure 1). Combining low-cost protein A resins with prepacked disposable cartridges can be an extremely effective strategy to reduce overall downstream operations costs. Prepacked Columns According to Langer 4, there is growing interest in prepacked column technology, given the dynamics of the industry s growing push to improve downstream performance. Although designed for single use, because of their considerable cost, prepacked columns are frequently reused, and this trend will continue until an inevitable price adjustment occurs. Langer mentions a number of products that are now available, and Table 1 summarizes those currently available in the marketplace. Available Prepacked Columns for Single Use Company Brand Features EMD Millipore Chromabolt ion exchange resins columns Protein A and other affinity resins Atoll MaxiChrom AC Process-Scale Columns (single or campaign-use) Repligen OPUS Custom packing services. Prepacked disposable columns and a 45 cm ID prepacked column. Thermo Fisher/Life Technologies GoPure column range of POROS resins Grace Provance Silica based protein A Table 1. Various Prepacked Column Products.

3 Figure 1. Purification time chart. Note substantial savings with Provance. Figure 2. Column cost chart. Provance columns yielded a 64% reduction when compared to agarose. Prepacked columns are gaining popularity with CMOs because they offer convenience and flexibility with a wide range of formats for different batch sizes. They are more environmentally friendly, considering they avoid the use of large volumes of purified water for cleaning. Moreover, they remove the labor-intensive tasks of packing columns before operation and cleaning them afterward. Grace has recently developed Protein A prepacked silica columns which go by the trade name of Provance. These columns provide a number of key advantages over other options. They eliminate packing and cleaning steps, and they shorten purification time compared to agarose (Figure 1). There is a striking column cost reduction of 64% (Figure 2) with a resulting 57% total savings in cost of operations. In addition, there is high dynamic binding capacity (Figure 3). Dr. Kiran Chodavarapu is a research scientist and specialist in singleuse technology at Grace. He discussed the company s products and their advantages. ProVance columns combine disposable column technology with cost-effective silica resin for optimal value and performance. The columns are constructed of high-quality polypropylene with no leachable or extractable components. This makes the columns effective and viable for single-use manufacturing. Steel or glass components, on the other hand, are cost prohibitive for single use. Silica has traditionally not been considered due to the fact that it cannot withstand clean-in-place (CIP) requirement of traditional purification processes. But ProVance columns use silica resin, as it is ideal for disposable manufacturing where CIP is not needed. ProVance offers an inexpensive platform with high-performance features not available from other products. Additionally, silica is an incompressible resin, which means it will not collapse during high pressure runs as will softer resins such as agarose. Binding of the Protein A ligand to the silica substrate is carried out using proprietary linking techniques. Figure 3

4 The Choice of Protein A According to Chodavarapu, the current trend Harvest Removal of cells and cell debris is toward smaller bioreactor batches, reflecting the interest of biopharma companies in specialized treatments. Such trends consist of Centrifugation/filtration prior to chromatography targeted therapies, personalized medicines, Protein A Yields highly purified Chromatography product in a single step drugs for orphan diseases, and in-country/ for-country manufacturing. Previously in Low ph hold Inactivates endogenous/ the blockbuster era, the industry focused on for viral inactivation adventitious viruses large-scale projects in which so much product was required that the operations ran 24/7 for a year or more, he explained. Now there are Additional polishing Removal of product/process related many specialty therapies under investigation. chromatography impurities and viruses This means the industry is turning to smaller bioreactors and higher titers, and a typical Additional polishing Removal of product/process related campaign may be only two to three months. While there are some alternatives, there is no option that possesses the high affinity chromatography impurities and viruses Removes endogenous/ and stability of Protein A for IgG molecules. Viral filtration adventitious viruses In addition, other alternatives such as Ion exchange simply lack the specificity to reach a high level of purity in a single step. Ion Ultrafiltration/ Final, formulated bulk exchange is 1/3 the cost of Protein A, a big savings, but due to the lack of specificity, the cost of operations is actually higher than diafiltration drug substance Figure 4. Typical purification protocol. Protein A, Chodavarapu stated. While the industry will continue to pursue alternatives, it appears that the dominant status of protein A will not be overthrown in the foreseeable future. There simply is no alternative that possesses this level of specificity. The downstream bottleneck will be a growing challenge for antibody purification. In some cases extremely high titers of 15 to 30 grams/liter of antibody have been achieved. However, extremely high titers may actually be too much of a good thing, as such high concentrations can cause aggregation and precipitation problems. Currently, most protocols work with upstream production levels in the 0.2 to 5 g/l range, which is optimal for the ProVance columns. A flow chart of a typical purification protocol is shown in Figure 4. Looking Forward Langer has characterized the bioprocessing industry as risk-averse, not surprising for an industry that is heavily regulated and focuses on the cost of goods and maximizing the bottom line. This mind-set discourages quick adoption of radical technologies which could potentially fail due to unforeseen shortcomings, thus causing changes within the industry to be more incremental rather than truly disruptive. We don t expect rapid changes in technologies, but rather ongoing gradual improvements in the coming years, Chodavarapu stated. The industry will step carefully as it negotiates the requirements of a global post blockbuster biomanufacturing environment, adjusting according to regional needs and the regulatory requirements for a range of new antibodies in development.

5 References 1 Langer ES Trends in Downstream Bioprocessing: Special Coverage, Purification and Separation. Biopharm International. September 1, Liu HF, Ma J, Winter C and Bayer R Recovery and purification process development for monoclonal antibody production. MAbs Sep-Oct; 2(5): Kobayashi S and Ueda Y Comparing protein A resins for monoclonal antibody purification. Biopharm International. December. 4 Langer ES Disposable Chromatography: Options Are Increasing. Genetic Engineering and Biotechnology News Dec 10, 2013.