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).
Antibody manufacturing provides an excellent example of the application of process redesign and streamlining principles to
increase productivity, cut costs, and maintain product quality. Most manufacturers use three chromatography steps for antibody
purification, starting with a very expensive Protein A capture step that is placed immediately after clarification, followed
by anion exchange (AEX) chromatography in flow-through mode to extract negatively charged contaminants, such as host-cell
protein (HCP), endotoxins, host DNA, and leached Protein A. Then, either cation exchange (CEX) chromatography or hydrophobic
interaction chromatography (HIC) in retention mode is used to remove positively charged residual contaminants and product
related impurities, such as aggregates and degradation products (13). Modern platform processes also serve as orthogonal strategies
for virus removal.
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).