Challenges in the purification of monoclonal antibodies (MAbs) include reducing production cost, developing robust processes for both product purity and viral clearance, and integrating upstream and downstream processes. Increases in titers for MAb feed streams and clinical doses have led to the need to develop new products and technologies or adapt existing ones to meet these challenges. This paper will discuss the status of some of these new technologies and products and their potential for improving platform processes, as well as issues related to their implementation.
Purifying proteins for therapeutic use requires highly selective and robust technologies to achieve the very high purity required for biopharmaceuticals. Processing such valuable product streams at large-scale while manufacturing to such high standards requires careful technology design and execution. This is because feed stocks containing the target protein most often are obtained from the culture of live cells and are variable in both product content and composition. Since 2003, 26 of the 31 newly approved protein therapeutics are produced in either E. coli microbial biosynthesis or mammalian cell culture.1 Because proteins degrade easily as a result of extremes of pH or temperature, or contact with common solvents, controling the variables during processing is especially important to avoid having to remove impurities formed during processing, because these impurities affect manufacturing yields and the activity profile of the therapeutic protein.2
The lowest production cost is achieved not by increased process complexity but by the opposite: Operating faster and with the fewest and most efficient downstream steps reduces investments in validation and subsequent batch failures.3 Some high-dose monoclonal antibody (MAb) products require an annual production of metric tons of material to supply the market. Demand for quantities this large exceed current manufacturing capabilities requiring companies to invest in additional capability to meet demand. This delay can contribute to missed revenue. Current platforms for the large-scale production of MAbs, for example, show that it is essential to design and operate an integrated series of purification steps that are both high yielding and product molecule–specific.4
Emerging strategies to improve process efficiency include high-throughput technologies, using disposable equipment, and using new filters and adsorbents that combine inexpensive substrates with greater functionalities, such as charge or lipid binding. In addition, increasing volumetric titers into downstream processing also has created technological bottlenecks that must be solved. How to take advantage of the multitude of emerging technologies while streamlining and improving a platform downstream process for MAbs remains one of the major challenges faced by practitioners of the art today.
Although some types of disposables such as media and buffer bags have been in use for years, the use of purification-related disposables is relatively new. The advantages in their implementation include reductions in preparation and product changeover, ease of implementation and validation, elimination of cleaning procedures, and a reduction in capital outlay.5 New and improved disposable technologies include single-use chromatography membranes and ready-to-use columns, ultrafiltration cassettes, and chromatography and ultrafiltration flow paths containing single-use monitors and transducers. However, it is not always easy to incorporate into existing processes as a one-for-one swap without additional development work. In addition, manufacturing costs must be considered, taking into account the advantages of disposables discussed above compared to disadvantages such as increased material demand and validation of leachables.6
The use of disposable membranes in MAb purification has shown potential to replace traditional resins, especially for anion exchange (AEX) membranes that are used in flow-through mode, in which the impurities bind to the membrane while the antibody flows through. The binding capacity of the target protein has previously been a problem for membranes because of their lower surface-to-bed volume ratio as compared to resins,7 so flow-through operations present an ideal situation for membrane use. The advantages of these membranes include improved mass flow properties because of the porous structure of the membrane hierarchy, and ease of preparation before use.8 The initial preparation time is significantly reduced because column preparation operations such as packing and qualifying, and post-use cleaning and storage are eliminated.
Figure 1 shows data comparing the DNA removal properties of two commercially available membranes, Sartobind Q (Sartorius Stedim Biotech, Germany) and Mustang Q (Pall Corporation, New York), as compared to various AEX resins. The starting material was affinity-purified broth from a Chinese hamster ovary (CHO) cell line expressing a MAb, and the flow was comparable based on a linear flow rate of 200 cm/h. The data are shown as DNA fold reduction, which is the starting value in the load material divided by the final value in the flow-through pool. The graph shows that both membranes were better at removing DNA than the resins tested, possibly because of faster, and therefore, more efficient charge interactions between DNA and the functional groups on the nonporous surface of the membrane. Additional experimentation also has shown a six-fold difference in the total amount of DNA that can be removed by a membrane as compared to resin.
However, host cell protein (HCP) removal by membranes can be a challenge. Side-by-side comparison of the Sartobind Q membrane to Q Sepharose Fast Flow resin has shown the membrane to be significantly less capable of removing HCP for some antibodies (Figure 2). However, new advances in salt tolerant membranes, such as the STIC from Sartorius Stedim and the Chromasorb from Millipore (Bedford, MA), which includes changes in the ligand type as well as the support and attachment chemistry, have shown significant improvements in HCP removal capability as compared with traditional membranes (Figure 2).
The question that remains for these disposable membranes relates to the cost of goods. Given the higher cost of membranes, their use makes sense during early-phase manufacturing when the number of batches is small, but how do they stack up in commercial manufacturing? The answer may lie in the ability of the membrane to be used multiple times. We have shown that reusing the Sartobind Q membrane up to 10 times does not affect its ability to remove DNA, but validation for reuse is an issue because it has yet to be proven.
Another advance in disposable technology is the single-use ultrafiltration cassette, such as the SIUS from Novasep (Pompey, France). In a comparison with standard multi-use cassettes, the SIUS showed a significant increase in flux capacity and reduced processing time for both concentration and diafiltration unit operations (Figure 3). The starting material in this case was a purified MAb, concentrated and diafiltered on small (100 cm2 or equivalent) 50 kD cassettes using an AKTA Crossflow system from GE Healthcare (Uppsala, Sweden). Although Figure 3 shows the data for only a single antibody, the same trend was seen with other therapeutic antibodies as well. These single-use cassettes can be paired with new disposable flowpath technologies to produce a true single-use unit operation that eliminates post-use cleaning and storage. Again, we must determine the cost of goods for commercial manufacturing as opposed to early-phase use.