Vaccine Scale-up and Manufacturing

Donald F. Gerson, Bhawani Mukherjee, and Rattan Banerjee

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Abstract Vaccine licensing and manufacturing are interdependent. e vaccine product and license depend on the process, equipment and facility. Vaccines typically are complex mixtures of epitopes, antigens, impurities, and adducts, making the reproducibility of the vaccine substance dependent on the manufacturing process. Regulators worldwide are aware of these issues, and operate on the precept that the process denes the vaccine. Creating a new robust, reproducible vaccine manufacturing process is key to successful long-term vaccine supply. Process development, equipment selection, and facility design dene the nal vaccine manufacturing process, and must embody the science behind the vaccine. e long lead-times in manufacturing process design, facility implementation, and start-up necessitate parallel investment in vaccine discovery and industrialization, simultaneously increasing nancial risk and ensuring expedient delivery of the vaccine product to the population upon licensure. Success in new vaccine development can only be dened by the sustained long-term provision of consistent vaccine to billions of people. Introduction ere are two major parallel pathways in vaccine development. Product development is the pathway from discovery research to clinical evaluation of putative protective antigens. Process development is pharmaceutics, process evaluation, production optimization, stabilization, assay validation, packaging and other practical activities required to turn an antigen into a vaccine. ere are three major stages in the development of a vaccine: research leading to antigen discovery, development leading to a small-scale manufacturing process for clinical materials, and manufacturing leading to large-scale mass production of the vaccine for worldwide use. During process development, exploratory research determines scaleable methods that will produce the same antigen made during discovery research. e techniques and methodologies are used for the production of phase I, II and III clinical materials, and ultimately dene the manufacturing process.

e establishment of manufacturing involves the simultaneous development of cGMP facilities and equipment, process engineering for scale-up, renement of QC methods, validation and proof the mass produced vaccine is bioequivalent (Gerson, 1998). Optimality criteria for the manufacturing process are based on the technology and the expected economics of the product. Manufacturing economics are very dierent for a high-priced vaccine for the developed world and for a low-priced vaccine for the developing world. With a high-prot vaccine, reduced process development reduces initial investment, speeds product introduction and gives fast nancial return, covering high capital and operational costs. With a low-priced vaccine, enhanced process development increases initial investment, reduces operational costs later on, and allows a low sales price. e total cost to society is least in the long run if operational costs are minimized. Balances between investments in development, capital cost and operating cost are made in all cases, but decisions depend on the price and volume expectations for the product. Most probably, an AIDS vaccine for the developing world will always be produced in large volumes and purchased at low cost with, essentially, donated funds. Low-cost manufacturing for the developing world raises concern for the balance of quality and need. Just because the need is great or urgent, just because the price is low, just because the location is the developing world, there is no reason to assume, encourage or allow low quality manufacturing. Well designed processes and facilities can produce very large quantities of low cost, high quality vaccines. e requirements for quality, purity, good manufacturing practices, and regulatory compliance no longer dier around the world. Manufacturing facility design and construction is converging on a worldwide standard of acceptability, and long-term planning for AIDS vaccine manufacturing must take this into account. Process development Once a vaccine candidate is selected by pre-clinical testing and phase I/II clinicals, process development should begin. e current regulations are that clinical materials must closely

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represent the intended nal product and be made in cGMP compliance. HIV vaccine development projects have not given clearcut results, making decisions on process development and manufacturing preparedness problematical. Vaxgen had the courage to proceed with process development and facility planning and construction on the basis of positive immunogenicity results. Unfortunately there was no protection: had there been protection, Vaxgen would have been ready to begin manufacturing upon licensure. Should process development and manufacturing preparedness be delayed until proof of ecacy, there will be about a 5-year gap before product is available for distribution. Progression from laboratory method to large-scale manufacturing Fig. 17.1 shows diagrammatically the progression from laboratory method to large-scale manufacturing. Following antigen discovery, systematic, iterative process development studies transform the discovery process into a practical, highly reproducible, robust, cost-eective large-scale manufacturing process that will make hundreds of millions of doses of vaccine. Manufacturing clinical grade vaccines using a well-dened process in an appropriate facility, under increasingly stringent good manufacturing practices (cGMP), is the basis for the success of phase III clinical trials. Licensure depends on successful proof of ecacy and documented proof of the optimization and reproducibility of the manufacturing process, and the compliance of the manufacturing facility. e transition from laboratory method to industrial process adds many layers of scientic, technical, regulatory and organizational knowledge and implementation to the vaccine production method. Knowledgeable and wise application of the multiple elds of endeavor involved in vaccine industrialization usually yields a successful product. However, numerous failures at this transition attest to the diculties. Process development occurs in incremental steps from discovery through clinical trials, and continues as process im-

provement after licensed manufacturing begins. is progression from discovery laboratory through process development laboratories to large-scale manufacturing facilities and product launch is shown diagrammatically in Fig. 17.2. Emphasis on process development is a major success factor in being rst to market and protable with new vaccines and biopharmaceuticals. Inadequate process development is associated with late-stage product development failure, or for high production costs for successful products (Pisano, 1997). Inadequate attention to process development details may result in failure or abandonment of a good product due to unforeseen manufacturing diculties or unnecessary high cost, with tragic consequences. Cost estimates made before process development can lead to the abandonment of a perfectly good product on the basis of incorrectly perceived high production costs. Costs can almost always be brought into line with expectations if the process is subjected to an appropriate level and duration of process development by those skilled in this aspect of new vaccine development. Manufacturing process development involves an iterative optimization of each sequential process step to dene methodologies appropriate to the scale of production. A properly implemented manufacturing process development program contributes signicantly to product success by providing engineering data, prospective process validation at a manufacturing scale, and accurate cost information for sound economic decisions. At the present time, global capabilities are less than adequate for process development, clinical materials manufacturing and eventual large-scale manufacture of a successful HIV vaccine. With the vaccine production technologies available today, it is possible to design and construct exible, generic production facilities that could make any of the HIV (or TB or malaria) vaccine products currently envisioned. Once the precise product has been decided upon, procedures to implement the required process in such a generic plant could be developed. Pre- construction of facilities for clinical materials manufacturing, and most importantly for large-scale manufacturing could be done by a contract manufacturing organization,

Figure 17.1 The progression of product and process development from laboratory method and phase I trials, to large-scale manufacturing process and phase III trials, leading to licensed product launch.

Figure 1. A diagram of the progression of product and process development Uncorrected proofs not for distribution from laboratory method and phase I trials, to large-scale manufacturing process

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would result in benecial use. A contract manufacturing organization independent from a vaccine development company, devoted to the eventual large-scale manufacturing of an AIDS vaccine, would greatly accelerate the introduction of all successful vaccines to the developing world. Large-scale manufacturing technologies Current status of AIDS vaccine manufacturing processes Most current AIDS vaccine manufacturing processes are in early stages of process development, and considerable eorts will be required for eective scale-up. e processes to be used for AIDS vaccines under development to date involve three major categories of production technology: microbial fermentation, cell culture or egg-based production. In each production technology, a given host organism may carry one of many possible HIV-specic gene constructs. is is an advantageous situation: unlike traditional vaccine manufacturing in which vaccines were made from the respective pathogens, each having unique and unusual physiologies and requiring highly specic, specialized processing approaches, the processes for AIDS vaccines fall into only 3 distinctly dierent categories. In addition, each methodology is of average nature within its type. is situation greatly reduces Vaccine Industrialization the risk involved in early decisions to proceed with manufacFigure 17.2 Interplay between product and process knowledge turing facility design and construction. A plant designed to development, appropriate facilities and the production of clinical meet the average needs and to have greater operational latitude materials in the development of a commercial vaccine product. could be accommodated to whichever product is successful.
Figure 2. Interplay between product and process knowledge development, appropriate facilities either aproduction of clinical materials basis, development CMO, on and the protable or not-for-prot in the and used of a commercial vaccine product vaccine to be licensed. In this way, the by the rst successful

risk of capital expenditure would be separated from any one individual product, greatly increasing the probability that it
D. Gerson 2

Large-scale manufacture of HIV vaccines e generalized processing steps involved in the manufacture of an AIDS vaccine are as follows (Fig. 17.3): e conceptual manufacturing steps are as follows:

Figure 17.3 The generalized processing steps involved in the manufacture of an AIDS vaccine: Seed Banks, Growth, Harvest, Purication, Bulk formulation, and lling and packaging. processing steps involved in the manufacture of an Figure 3. The generalized

AIDS vaccine: Seed Banks, Growth, Harvest, Purification, Bulk Formulation, and Filling and Packaging. Uncorrected proofs not for distribution

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Cell-culture based viral vector vaccine manufacturing (Fig. 17.4) Animal cells can be replicated in a bioreactor with appropriate media and controlled operating conditions. b All nutrients and supplements required for cell growth are mixed with highly puried, pharmaceutical grade water in Media Preparation Tanks before being sterilized and transferred to the bioreactors. e production of puried water and other process utilities is a major part of any manufacturing fac cility. Cell inoculum from the cell bank is placed into the rst seed fermentor to initiate the process. Growth of animal cells in bioreactors is a dicult and exd acting process, requiring equipment with precise operational variable control and absolute sterility. Equipment failure due e to inadequate design is the most common cause of manufacturing failures or interruptions. Once the number of cells has increased by 510 times, the cells are transferred to the next f stage leading to the production bioreactor, where the cells increase another 5- to 10-fold. Usually, there is a series of fermentors, each 510 times larger than the previous leading to the penultimate production volume. A 10-fold multiple balances the economics of production with the technical diculties of ree bulk vaccine intermediate manufacturing technoloscale-up; other multiplication factors have been used industrigies encompass all vaccine candidates under consideration at ally but if smaller, add capital cost, or if larger, add scale-up this time: microbial, cell and egg. While traditional vaccines risk, without signicant benet. By the time the cell growth have utilized multiple doses of the same antigen, it appears step ends, the total number of cells has increased thousands at this time that an AIDS vaccine immunization series may of times. involve multiple doses of dierent antigens and that the antiAt the nal growth step, the cells in the production biogens may be produced by dierent production technologies. It reactor are infected with genetically modied virus containing is foreseen that manufacturing technologies for chick embryo genes for the desired HIV antigen, and the virus is allowed to cells will become the same as the manufacturing technology multiply. for other animal cells, and that primary cells will be replaced At the Vaccine Industrialization end of the virus growth cycle, the contents of the by avian or other cell lines, further reducing the complexity of production bioreactor are centrifuged or ltered to separate planning. the cells from the supernatant. Depending on the process, eiIn the following section, each process technology is dether the cells or the supernatant will contain the virus. e scribed in greater detail. product is held in a harvest hold tank for further processing a Master and working seed banks: To ensure that the starting materials are identical for each batch, all biological processes begin with a vial from a certied seed bank system for microbial or animal cells, or virus. Growth: Fermentation in a series of increasingly large bioreactors develops the desired quantities of cells to produce the product. In viral processes, the cells are usually grown to maximum density, then infected with the virus and maintained to maximize virus production. Harvest: e active vaccine intermediate is separated from the growth media using cross-ow or dead-end ltration, or centrifugation. Purication: e active vaccine intermediate is puried by ltration and chromatography. Bulk lling: Puried vaccine intermediate is formulated to nal bulk, or API, specications and lled into appropriate sterile bulk containers. Final product lling and packaging: Final formulated vaccine is lled into syringes, vials or other nal delivery containers, lyophilized if necessary, labeled, packaged and prepared for shipment to the nal destination.

Figure 17.4 Cell culture-based viral vector vaccine manufacturing.

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and cross-ow ltration to separate the virus from most cellular contaminants. Virus purication proceeds by isolating the virus from most of the remaining contaminants by chromatographic separation or cross-ow ltration. Typically, large quantities of buer are required at this step, and one of the objectives of process development is to reduce processing requirements to achieve the desired level of purity at minimum total cost. Following virus purication, the active vaccine Intermediate is formulated to its nal composition and prepared for bulk lling into sterile transfer vessels. Bulk vaccine intermediate is then stored prior to nal lling and packaging. Equipment turn-around requires automated cleaning cycles and batch-to-batch maintenance.

chromatography column. Puried product is then formulated, lled into transfer vessels and stored prior to nal lling and packaging.

Egg-based viral vector vaccine manufacturing (Fig. 17.6) In this process, chicken embryo broblast (CEF) cells are utilized as host cells to grow MVA, or another virus that has been genetically modied to include genes for HIV antigens. MVA has been adapted to grow exclusively on chicken embryo cells and will not grow on human cells, making it a suitable live viral vaccine vector for use in populations with unsuspecting HIVpositive individuals. A major issue with virus production from eggs is sterility control. Eggs come from a non-sterile environment and are dicult to open without occasional contamination. MVA is a Microbial-based manufacturing (Fig. 17.5) Microbial production is very similar to, but slightly less strinvery large virus, and cannot be terminally lter sterilized, so gent than, cell culture. e seed fermentor containing sterile the only protection from contamination is aseptic technique media is inoculated with recombinant microbial cells containand the quality of the facility and environment. All MVA proing appropriate HIV constructs and is controlled to promote cesses require aseptic operations in clean rooms: processes that optimal microbial cell growth. An important dierence beare facilities, personnel and training intensive, hard to maintween animal cell cultivation and microbial cell growth is that tain and expensive to operate. microbial cells usually require an order-of-magnitude more is situation illustrates a classical quandary in producagitation and aeration, signicantly changing the nature of the tion: while eggs appear to be inexpensive and oer the promfermentation and utility supply equipment. e cells multiply ise of low operating costs, the required large and high-quality in a series of 10-fold steps leading to the production bioreacfacilities, the high operational expenses, the impossibility of tor. economies of scale, and the relatively high frequency and cost At the end of cultivation, the biomass is centrifuged to of batch failures combine to make it questionable whether the separate the product from media and other contaminants. If apparent savings can be realized. Fermentor and cell-culture the vaccine is the cells, they are recovered, washed and sent to based operations may appear to be more complex and expenbulk lling. In another possible process, the cells are processed sive at the lab level, but at full-scale they result in lower xed in a homogenizer to release the target plasmids or protein. and variable facilities costs, true economies of scale and much Lysed cell paste from the homogenizer is then resuspended lower batch Vaccine Industrialization failure rates, resulting in low unit manufacturing with a suitable buer solution in the lysate hold tank. Residual costs. cell debris is then removed from the lysate by cross-ow ltrais process begins with fertilized specic pathogention. e product, rDNA or protein vaccine intermediate is free eggs (SPF eggs) produced in a highly specialized chicken stored in the claried broth tank before being puried on a population and facility. e size of an SPF chicken and egg

Figure 17.5 Microbial-based vaccine manufacturing.

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Figure 17.6 Egg-based viral vector vaccine manufacturing.

production facility appropriate to the quantities of HIV vaccine required has yet to be estimated, however, approximately 20 million eggs/year could be required for a full scale facility. SPF eggs are shipped to the manufacturing facility, checked for viability and sanitized. Egg-Based Viral Vector Vaccine Figure 6. Chicken embryos are harvested from surface sanitized chicken eggs. e embryos are incubated with trypsin to separate the CED cells, which are recovered from the ltrate by centrifugation. All these operations are manual and are conducted in a biosafety hood in a class B environment. e recovered CED cells are infected with virus seed in roller bottles, and incubated in a warm room. e cells do not grow in this system, but the virus multiplies. At the end of the incubation period the cells and virus are harvested and centrifuged to begin the purication process. e recovered cells are broken to release the target virus particles, then claried and puried by centrifugation. Bulk formulated vaccine is prepared and lled into transfer vessels under aseptic conditions. All these operations are carried out under aseptic conditions in a class B environment.

of multi-dose and single-dose presentations of the vaccines. In terms of facility planning, this is an extremely important consideration: lling and packaging space is clean room space and is very expensive to build and operate. For many traditional vaccines for the developing world, multi-dose presentations Manufacturing have been optimal: cost per dose is minimized for manufacturing, shipping and storage. However, multi-dose vials require many syringes and must be entered many times. If a needle is used twice, the entire contents of the vial could be contaminated and disease could be spread between vaccinees. Given the known danger of HIV transmission by multiple uses of the same needle, greater social safety will be achieved by a single dose pre-lled syringe presentation. Use of a pre-lled plastic disposable syringe device such as the BD UnijectTM injection device (BD and Uniject are trademarks of Becton Dickinson and Company) will be highly desirable. is device has the additional advantage that it is designed to prevent secondary use, reducing the dangers associated with the unauthorized recycling and re-use often associated with traditional syringe devices (Gerson, 2005).

Filling and packaging Manufacturing facility Filling and packaging is always a costly, space- and labor-conCombining the characteristics described above with reasonVaccine Industrialization suming activity. Fig. 17.7 compares the benets and detriments able estimates of process yields and the manufacturing space
Dosage Presentation Single Dose Features More expensive Increased Safety Less Expensive Decreased Safety

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Multi-Dose

Figure 17.7 Risk and cost table for single dose and multi-dose nal product presentations of an AIDS vaccine.
Figure 7. Risk and cost table for single dose and multi-dose final product presentations of an AIDS Vaccine.

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requirements for dierent basic types of manufacturing activities, it is possible to derive a basic estimate of the facilities requirements for an AIDS vaccine manufacturing facility. e usual estimate of the capacity of a vaccine facility assumes the need to immunize approximately 5% of the population each year, this is 50 million immunization schedules per billion persons. Early in the lifecycle of a new vaccine, this is low, as the population is unimmunized, and considerably more catch-up doses are required to immunize a wider age range than would be needed in a steady-state situation. Once only by those entering the target age-class require immunization, capacity may appear to be high, providing the opportunity to export vaccine to other areas or to make other vaccines. Later in the product and plant life cycle, the population will have increased, reducing the excess capacity. Any manufacturing facility for the developing world must meet the same quality standards as a vaccine facility in the developed world. To meet standard vaccine facility design criteria at minimum cost and with maximum exibility, a facility should be composed of production, quality, service and administrative modules along a central spine, facilitating construction, process ow, personnel ow and potential expansion (Fig. 17.8). e overall size of an HIV vaccine facility, with a capacity of 50 million immunizations per year, and thus servicing a billion people, is approximately 425,000 sq ft (42,500 m2). e Vaccine Industrialization expected number of employees, based on stang standards in the USA, is approximately 500. e capital cost would be expected to be approximately US$350 million, if constructed in the USA (Mukherjee and Gerson, 2004). e impact of dierent locations on the cost is subject to analysis based on factors for construction, equipment, utilities and operational costs for pharmaceutical facilities around the world. Preliminary estimates indicate that costs are less sensi-

tive to location than may have been imagined. is is primarily due to the lack of tax considerations, the insensitivity of equipment costs to location, and the need for well-educated workers. Clearly, this topic requires full economic location analysis at the appropriate time. Summary and conclusions Vaccine development involves multiple parallel pathways. Product development, from laboratory to clinical proof of ecacy is one pathway, and process development from laboratory method to pilot plant studies to manufacturing facility design and start-up is another. Regulatory development leading from IND or equivalent to licensure, and economic and social development leading to a social desire and a nancial ability to purchase and administer the vaccine are also parallel elements of vaccine development. e only useful measure of vaccine development success is delivery to and protection of millions or billions of people. All these, and many other parallel eorts comprise the multi-dimensional scientic, manufacturing, and social eort required to eectively develop a vaccine product that will benet mankind. Acknowledgments e authors would like to thank Luke Gong, Nancy BordaDiaz, and Mark Chmielewski for their skill and patience in making the gures. References
Gerson, D.F., et al. (1998). Transfer of processes from development to manufacturing. Drug Information J. 32, 1926. Gerson, D.F. (2005). Safety Guides New Syringe Designs. Pharma Manufacturing, April 2005, 255. Mukherjee, B. and Gerson, D.F. (2004). Manufacturing Vaccines for the Developing World. BioPharm Int. 17(10), 2430. Pisano, G.P. (1997). e Development Factory. Harvard Business School Press, Boston, MA.

Figure 17.8 Design for an economical AIDS vaccine production facility, composed of production, quality, service and administrative modules along a central spine with linear product, process, and personnel ows.

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