It is important to note that a platform process for purifying antibodies must accommodate both charge and hydrophobicity variation between the molecules themselves. The constant regions of most immunoglobulins is consistent in physical and chemical behavior, but single amino acid changes in variable regions can drastically shift either the isoelectric point (pI) of the protein or the relative regional hydrophobicity. So the process must be able to remove a wide range of charge variants as well as various hydrophobic species (e.g., aggregates.)

Moesta (Bristol-Myers): mAbs, Fc-fusion proteins and adnectins developed by Bristol-Myers Squibb have large conserved regions, resulting in physical properties that allow one to achieve the vast majority of purification using platform technology. Charge heterogeneity in the variable region and post-translational modifications then require individually optimized polishing steps. For antibodies, this is usually a combination of an anion exchange step (either in flow-through or bind and elute mode) coupled with a second chromatography step. One must evaluate the remaining purification objective, select the best method, and optimize it. Viral inactivation and filtration steps, as well as diafiltration and concentration steps can be standardized and made to fit with a drug-product formulation platform if available.

PharmTech: Is it possible to develop a purification platform for various classes of products (e.g., mAbs and enzyme products)?

PharmTech: Can purification platforms accommodate the rising titers that upstream processes are yielding?

Atkinson (Cook): The rising titers are both a blessing and a curse for downstream unit productivity. The capacity of most chromatography resins is basically sufficient for the increased titers, but the buffer consumption and the throughput become a challenge with very high titers. In this case, limiting the number of unit operations, for example, moving from a three-column antibody process to a two-column process becomes much more attractive. Engineering solutions, such as buffer blending and even possibly simulated-moving bed chromatography can also be considered to manage the increased productivity.

Most downstream unit operations, with the exception of viral filtration, are inherently scalable to an industrial scale. I believe that the biggest downstream bottleneck for high-titer processes has become viral filtration. Most chromatography unit operations can scale effectively, but the need to use an expensive, low-throughput filter can create an inefficient bottleneck in the overall purification process for mammalian cell-derived products.

Scale-up challenges include the high upfront cost for consumables (e.g. resins, bags, filters) as well as the challenges with liquid handling. Bulk volumes of liquid and intermediate holds are inherently inefficient. Engineering solutions for liquid transfer, mixing, and minimizing storage of liquids should be explored.

Moesta (Bristol-Myers): The technology available today can accomplish the manufacture of proteins up to the metric-ton scale. However, few products to date require this large scale of manufacture.

Increasing demand for proteins, combined with higher titers in fermentation, can enable implementation of alternative technologies, such as protein precipitation and crystallization. These technologies provide a means to improve purification throughput while significantly reducing cost. Some examples include blood fractionation products and recombinant insulin.

A key scale-up challenge is chromatography because there are physical limits based on resin-flow characteristics (e.g., back pressure and compression). The next step for industry is the use of simulated moving-bed technology, which can increase throughput.

Powell (Asahi): Process engineers can accommodate rising titers using a combination of liquid handling systems and modern virus-removal filters. Because the ability to rapidly and reproducibly create accurate buffers in a minimal footprint is a common bottleneck during downstream processing, Asahi Kasei Bioprocess offers IBD inline buffer dilution systems to generate on-demand diluted buffers for capture, polishing, and virus-removal. Customized skids are easily integrated with existing equipment to improve purification efficiency.

Additionally, next-generation virus-removal filters facilitate reliable processing at concentrations of up to 50 g/L. Before processing high titers, basic physics of production must be considered. Such factors include the viscosity of the feed material, mechanism of mass transfer, and filter efficiency. Purification becomes easier as the ratio of contaminants to product decreases yet caution must be used as high product levels often reduce cell viability.

PharmTech: With regard to scale up, how do downstream process platforms perform? Are there limitations?

Moesta (Bristol-Myers): Large plants, such as Bristol-Myers' Devens plant in Massachusetts, can have long piping runs between pieces of equipment with significant hold-up volumes. If appropriately designed, process piping either drains by gravity or can be blown out with compressed air, minimizing losses. Filter housings often require water flushes for adequate yield recovery. If properly optimized, large-scale process performance can meet or exceed that observed at smaller scale.

Powell (Asahi): Scaling out, as opposed to up, is the preferred approach. During scale up, a facility transitions to larger diameter columns and filter housings before launching trains of production units in parallel. Besides reagent disposal, additional challenges include space as well as the use of water and buffer.

Holding tanks may be required for byproducts that cannot be released directly into the environment. However, properly designed chromatography systems from Asahi Kasei Bioprocess can reduce the ratio between the hold-up volume and the filter or liquid chromatography (LC) column volume to minimize the waste burden and improve operational efficiency.

PharmTech: Do these scale-up problems require customized solutions?

Powell (Asahi): Obstacles created by process scale up require customizes solutions to a certain extent, especially with respect to automation. When a company moves forward with commercial production, a plant-wide distributed control systems have historically been the preferred method to control and gather data from each step in the process. But for smaller scale production. such as orphan drugs, "islands of automation" are still preferable.

Finer, more accurate monitoring of this nature streamlines operations and enables tanks to open to skids at the proper time. Distributed control systems provide greater access to information in a manufacturing plant, thereby allowing euipment-related problems to be identified and addressed prior to impacting production.