STREAMLINING AND REDESIGNING EXISTING MANUFACTURING PROCESSES

Many processes for biopharmaceutical manufacturing were designed at a time where process efficiency was considered unimportant (3). More recently, manufacturers have sought to increase the efficiency of each unit operation, but they are only now starting to consider redesigning the entire process train to see whether cost savings can be made. The trend toward process streamlining owes a lot to FDA's quality-by-design (QbD) principles, which are derived from the design-of-experiments (DOE) concept. The design space of a manufacturing process is littered with efficiency peaks and troughs, but there is not always a simple path leading to the most efficient process. Therefore, process design incorporating efficiency and quality from first principles involves going back to the drawing board and evaluating the critical attributes that contribute to an efficient process.

Most companies are applying these principles and actively streamlining their processing strategies wherever possible. Antibodies take center stage because they represent more than half of all biopharmaceutical products in development, and their common properties make it possible for companies to share process efficiency data that are applicable across platforms (10, 11). It is for this reason that antibody manufacturing has benefitted from the development of so-called generic platform processes, which are broadly similar for all antibodies but can be tweaked to match the specific properties of individual products (12).

Realizing that no further cost savings could be gained by scaling up the above process, Pfizer explored the design space around the standard process and found that certain modifications could reduce costs considerably without affecting the quality of the antibody (14). The company introduced two types of process modifications, one in which the order of the polishing steps was reversed and another in which different separation technologies were used to increase process capacity (i.e., using membrane absorbers for the flow-through chromatography step and replacing the depth filtration step with continuous centrifugation) (15). These changes increased the efficiency of purification to such an extent that, for some antibody products, the cation-exchange step became unnecessary, reducing the process from three columns to two columns or even a single column. Not only did this save the direct costs of column resin and buffers, but also reduced the process time by > 45% which doubled the productivity in terms of batch processing (14).

EYEING SIMPLER TECHNOLOGIES

The capacity crunch in downstream processing has been avoided or overcome in other industries by adopting simple and inexpensive technologies (16). In bulk-chemical, conventional pharmaceutical, and food and detergent industries, expensive processing solutions, such as chromatography, would never be considered because the costs of implementation would not be sustainable in these high-volume, low-margin processes. This simple approach could be applied to biopharmaceutical manufacturing.

Several recent developments suggest that simpler technologies could find a niche in biopharmaceutical manufacturing, particularly in the early processing steps where the complex mixture of particulates and solutes have the most potential to foul expensive membranes and resins (16, 17). Tangential flow microfiltration, depth filtration, and (continuous) centrifugation are the current methods of choice for the clarification of the feed stream, and one or more of these processes may be employed in series to remove larger particulates until finally a polishing depth filter or dead-end filter can be used to remove fines and thus reduce feed stream turbidity (18). Efficient and inexpensive clarification becomes more challenging with higher-titer cell culture processes because these are characterized by a greater cell density and often a longer process time, resulting in a higher solids content, more particle diversity (size and physical properties), and—most challenging of all—a greater proportion of fine particles that escape coarse filtration. A technology that is widely used in the beverage industry and in wastewater processing is the use of flocculants to link small particles together and create aggregates that are easier to remove. Flocculation is achieved using polymers that bind simultaneously to the surfaces of several particles through electrostatic interactions, creating larger particles that may sink under gravity, or may be removed more easily by centrifugation or filtration.

In the bioprocessing industry, flocculation has been used to help remove whole cells from fermentation broth, but more recently it has also been used to remove fine cell debris and proteins. A simple and inexpensive strategy recently applied in antibody manufacturing is the creation of a calcium phosphate precipitate by adding calcium chloride to a final concentration of 30 mM and then potassium phosphate to a final concentration of 20 mM. Precipitation traps cell debris in larger particles, allowing removal by centrifugation for 10 min at 340 X g and yields a clear supernatant with the recovery of ~95% of the antibody (19). Interestingly, this strategy also removes some soluble host cell proteins and nucleic acids. Flocculation does not introduce any additional impurities to the feed stream because the flocculant is removed along with the aggregated particles.

Precipitation is widely used as a purification approach in the bulk-chemical industry, and given that precipitation can be induced by simple changes in the environment, such as varying the temperature or pH, increasing the salt concentration (i.e., salting out), or adding organic solvents, it should be easy to apply the same principles in bioprocessing (20). Precipitation has therefore been used to remove soluble impurities from the feed stream during antibody manufacturing, and these solids can then be trapped by filtration or pelleted by centrifugation leaving a clear feed stream relatively enriched for the target protein (20). In an innovative adaptation of this approach, the antibody can be precipitated under mild conditions and recovered from a collected pellet, thus removing many contaminants in a single step (21). This is possible because the mild precipitation conditions allow the protein to be redissolved without loss of activity. Several groups have developed methods to precipitate antibodies in large-scale processes, and this method could replace Protein A chromatography in the long term (22, 23). Precipitation methods using n-octanoic acid are used for the removal of contaminants in at least two industrial antibody manufacturing processes (24, 25).

In the final purification steps, another traditional technology that is being considered for use in biopharmaceutical manufacturing is crystallization. Crystallization involves the separation of a solute from a supersaturated solution by encouraging the growth of crystals. The crystallization process involves the formation of a regularly structured solid phase, which impedes the incorporation of contaminants or solvent molecules, and therefore yields products of exceptional purity (26). It is this purity which makes crystallization particularly suitable for the preparation of pharmaceutical proteins, coupled with the realization that protein crystals enhance protein stability and provide a useful vehicle for drug delivery (27). Protein crystallization has been developed into a commercial technology for drug stabilization and delivery, and several current manufacturing processes involve crystallization including the production of recombinant insulin, aprotinin, and Apo2L (28).