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Manufacturing by Jetting, the future in Protein A affinity matrix design
Protein A affinity chromatography continues to be the preferred method for commercial purification of antibodies because of its high selectivity and robust resin performance over repeated purification cycles. Reports estimate that US$125 billion of yearly sales will be generated from monoclonal antibody (mAb) products by 2020 (1). Most of those will be purified by large-scale Protein A affinity chromatography. With the continued growth and commercial importance of mAb production, availability of high-quality resin material and options for secondary sourcing are growing concerns. As current commercial patents for therapeutic antibodies expire and biosimilars enter the market, the cost of manufacturing will be of increasing interest.
The workhorses of today’s commercial mAb purification processes still are porous resins based on styrenic, acrylic, or agarose chemistry and produced by the same batch emulsification methodology that has been used since the mid-1900s. This technology produces resins that have a wide particle-size distribution and requires extensive screening to achieve the column performance demanded in modern processes. By contrast, a scalable continuous emulsification technology termed ‘jetting’ can be used to produce beads with a narrow particle-size distribution, without sieving, and resulting in almost quantitative yield.
It is not uncommon to overlook some important aspects of Protein A affinity chromatography. The most important factors to consider during the evaluation of Protein A resins are summarized below. Some factors will be impossible to evaluate at scale during design of a clinical process but still should be considered before selecting a resin.
- Dynamic binding capacity (DBC):Typically this is the starting point of every Protein A resin evaluation, as it has a significant impact on both productivity and buffer consumption. A small bead will result in a relatively higher DBC, especially at short residence times. But small beads will also generate higher back pressures and will be more sensitive to fouling.
- Purification Performance: The most critical contaminant in many processes is host-cell proteins (HCP). Copurification of HCP depends on both binding to a target mAb and the design and material used in a resin base matrix. In general, highly hydrophilic materials such as agarose show the best performance with respect to unspecific binding (2). The type of Protein A used could affect the required elution pH needed to obtain quantitative recovery and the resulting product pool volume.
- Cleaning in place (CIP): Development of effective cleaning protocols after purification is instrumental, both to eliminate carry-over and to maximize resin lifetime. Because of their high costs, Protein A resins are reused over many cycles in commercial manufacturing.
- Fouling of chromatography resins: This could be the result of many factors. It is feed dependent, and proteins, lipids, lipoproteins and anti-foam agents all can cause fouling. However, in the case of purification of mAbs from Chinese hamster ovary (CHO) cell cultures, the most common cause of fouling is protein fouling by a target protein or target protein variants. The most effective agent for removing precipitated proteins is sodium hydroxide. With modern, alkaline-stable, protein A ligands, 0.1 – 0.5 M sodium hydroxide is the standard cleaning agent. For difficult feed streams, sodium hydroxide might be insufficient, and more sophisticated clean-in-place (CIP) protocols must be developed (3). Severe fouling might require further optimization of harvesting and clarification methods.
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- Process Economics: Clearly, the price of a resin significantly affects raw material costs. In many cases, Protein A resins are the most expensive raw material in mAb manufacturing. To accurately estimate the contribution of Protein A resin cost for a given process, you need to take into account DBC, anticipated numbers of resin reuse, and buffer cost and consumption. Many companies are pursuing different types of continuous chromatography as a technology to decrease resin cost and buffer volumes. But such technologies are not yet available.
- Functional Lifetime: At the commercial stage, the number of cycles in which a resin can be used is an important factor that significantly affects overall process economy. With “easy” cell culture supernatants, more than 200 cycles often can be achieved. However, every cell culture supernatant and mAb are unique, and true resin lifetime cannot be predicted from simple alkaline stability data. For example, Gilead presented cycling data (3) for the newest resin from GE Healthcare, MabSelect™* PrismA™, with and without sample load using 0.3 M NaOH for CIP (15 minutes contact time). The difference in DBC after 150 cycles was 17% when comparing with and without sample load.
- Packing and Unpacking: In commercial manufacturing, resins are expected to last for more than 50 cycles and in some cases, at least 200 cycles. Therefore, resin aspects such as ease of packing and unpacking are important. For some processes, standard operating procedures require repacking every 25 cycles. Long-term storage stability, resistance to shear forces (to determine whether a resin slurry can be pumped), and generation of fines (which could clog column filters and frits) are important considerations.
To read the full ebook, click here. Continued in this article, we’ll be discussing security of supply and Purolite Life Sciences’ patented ‘jetting’ process.
Reducing the Costs of Biopharmaceutical Separations and Purifications with Novel Resins in partnership with BioPharm International. Alongside Purolite, YMC America, Process Technologies Group have contributed in sharing their expertise, research and insights in relation to biopharmaceutical separations & purification with novel resins.