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BioPharm International, July 2011 Issue (PDF)

Increased globalization, outsourcing, and expansion into nontraditional, or emerging, markets for product research, development, manufacturing, and distribution has put a spotlight on an extended network of supply-chain participants. A growing list of so-called unsafe products, including certain foods, toys, and more recently, active pharmaceutical ingredients and raw materials, originating in China and India has emphasized the need for increased supplier and material qualification and auditing. The focus is on quality control and regulatory compliance, an area of increasing concern for life-sciences companies as well as for the agencies responsible for ensuring the safety and efficacy of medications consumed by a global population.  

There is growing discussion regarding the use of cGMP as companies become more global and supply chains become more complex. To ensure compliance, the focus on cGMP processes and procedures needs to take into account all activities and participants engaged in the transformation and distribution of materials as they move from origin to point of final consumption. 

BE AWARE OF INCREASED REGULATORY OVERSIGHT

US agencies, including FDA, are not alone in their growing concern and awareness of the issues related to product safety and efficacy. Agencies in emerging markets, such as China's State Food and Drug Administration, are also increasing their oversight and requiring cGMP practices for the manufacture of all products that are destined for human consumption. Chinese officials are taking advantage of FDA's technical assistance, provided by the FDA offices established there in 2008, to ensure that their export-related regulations are in compliance with US and European standards, compiling facility audits where necessary. 

Despite these remedial actions, there is still much room for improvement, in particular in the chain of custody as materials and components move through an increasing number of contract research organization and contract manufacturing organization facilities. Recognition that the marketing authorization holder, or brand owner, of the product retains responsibility for its products has provided the need for a more collaborative environment across these operations, with increased oversight of activities at outsourced locations. In many cases, however, the logistics component of this supply-chain network is not included in the development of Service Level Agreements between all supply-chain participants, leading to, at times, variations in interpretation of standard operating procedures (SOPs).  

Unlike traditional markets, with mature storage and distribution infrastructure in place between key origin and destination points, many emerging markets are challenged with providing logistics resources, in some cases relying on nontraditional equipment and modes of transportation, such as bicycles or horses. Global networks of integrated carriers do not always extend to the point of manufacture and must rely on transportation partners, thereby incurring a high level of variation with regard to material handling and storage facilities. This Achilles heel of the global supply chain, so to speak, should not be ignored. Unless there is stringent specification and compliance in place for product storage, handling, and transportation, the risk of product contamination and cross-contamination is unacceptably high. There are additional risks tied to variations in temperature, humidity, and vibration that must be monitored and controlled.   

There is increased concern from FDA and other global regulatory bodies related to supplier qualification and monitoring as well as cold-chain and cool-chain compliance. There are also instances where FDA can issue a Warning Letter to a manufacturer for lack of monitoring and control of suppliers, especially when there is evidence of nonconformance to required procedures and it is shown that the company took no action. Such regulatory oversight is now being enforced across a broader spectrum of the supply chain. 

If these factors are ignored, the end user of the product—the patient—can be put at risk. In addition to the impact of compliance violations (i.e., regulatory citations, observations) having a high level of risk across an extended global network increases the likelihood of a supply-chain disruption. The ability to thoroughly investigate a product quality issue is severely compromised if the chain of custody is too variable or not understood by the sponsor company, or manufacturer.  

For instance, a stockout can be caused if there is a disruption in the flow of goods to the point of demand. Carriers and forwarders are crucial in ensuring that global trade compliance is adhered to and that there are no delays caused by misinterpretation of documentation or lack of sufficient information. The impact of a stockout due to inability to supply—for either logistics or regulatory compliance reasons—should not be underestimated. In addition to the financial considerations (which can be considerable), reputational risk and market perception have a high price. In certain cases, it is impossible to recover and some brands have suffered as a result.  

The product life cycle for most life-sciences products is long, thereby providing adequate time to develop a detailed strategy for product introduction, manufacturing, and distribution. The ideal time to think about an end-to-end product supply chain is during product research and development (R&D). The ability to ensure quality product delivery to the end user is as much a part of the product as any aspect of development. 

Evaluating and selecting suppliers of ingredients and components should be done holistically. Questions to ask include,  Is this supplier able to support our needs for increased supply? If not, what alternatives should be considered to ensure a sustainable product—at the detailed bill-of-material level? 

Including a supply-chain component in the company's market and commercialization strategy makes it possible to identify areas of risk and concern up front and facilitate any needed remedial action. This process should involve a team approach to ensure that all appropriate participants are included in the planning and implementation stages of development. In addition to scientists and product development specialists who understand the go-to-market  strategy,  the  team  should  include:									

Sourcing personnel to identify alternative suppliers for single or sole source materials, while working closely with strategic suppliers to assist them in developing capabilities to meet projected demand.

Quality and regulatory compliance personnel to incorporate a quality-by-design approach. Quality progams need to take into account product level quality not only for the components and finished product, but also for material handling equipment, packaging, and cross-contamination concerns. Inclusion of quality assurance and compliance personnel at the R&D stage will enable development of guidelines that can be used throughout the product life cycle.

Packaging experts for product protection. They can ensure that storage, handling, and packaging requirements are formalized and incorporated into  the product master file and supplier master file. Special handling requirements should be included in SOPs as well as in all product labeling and documentation. Language and literacy requirements across the extended supply network should be taken into account as well.

Risk-management, legal and finance experts to identify, qualify, and quantify risk factors. Participation by legal counsel and financial personnel can facilitate the development of a strategy that takes into account global trade legislation, including but not limited to rules of origin, denied parties, the Sarbanes–Oxley Act of 2002, the Foreign Corrupt Practices Act, and foreign currency rates of exchange. Legislation should be incorporated into contractual terms and conditions for suppliers, distributors, and end users.

Logistics and trade compliance personnel to consider modes of transportation across origin and destination pairs. Logistics includes many players—be sure to understand the weakest link in the chain and plan for potential problems. 

There are additional packaging and distribution considerations to keep in mind when considering the product life cycle and its supply-chain. For example: 					

Consider and provide protective packaging and monitoring devices for products that move through highly variable environments.

Define a cold-chain compliance program that takes into account seasonal variations across the possible trade lane configurations.

Consider locations where product could be exposed to hazards (e.g.,  the runway at an airport)  during trans-shipment points across the network and implement mitigation strategies and controls. 

Become informed about product-level literature required for global customs and trade compliance. Product definition and classification has a large impact on rates of duty and tariffs, as well as on  foreign customs inspections. Unforseen delays could take weeks or months to resolve, resulting in product degradation and, in  some cases, total loss.

Understand the impact of shipping and transportation prior to a weekend or during a holiday period and build these factors into guidelines and SOPs. 

Consider weather, including potential natural disasters, and have contingency plans in place to provide direction to logistics providers to enable them to make alternative arrangements. Cost should not be a reason to put product at risk.

A plan is only as the good as its execution. When a product is approved for manufacturing and commercialization, planning documents should be reviewed to identify any changes and be adjusted accordingly. Detailed SOPs should be communicated to all participants, from suppliers of raw materials and components (and their supply-chain partners) to providers of packaging, storage, and distribution services. 

A simple set of dashboard metrics should be developed and agreed upon, providing a framework for ongoing monitoring and control. Regular communication and adjustments will assist in fine-tuning the initial product-level planning and execution framework, thereby facilitating an environment of continuous improvement. The stakes—that is, patient safety—are high and so product planning must be equally thorough and stringent. 

Compliance Notes

Incorporating regulatory requirements into the product life cycle is crucial.

Impact of cGMP on Supply Chain

A fermentation process for the manufacture of virus like particles (VLPs) in 

) was transferred from an external collaborator and rapidly implemented in Pfizer's cGMP pilot plant. Challenges faced in the transfer were meeting the high oxygen demand of the original process, and attempting high density cultivation of 

 in a bioreactor system primarily designed as a seed tank for larger-scale mammalian and microbial culture. These concerns were overcome by an approach that combined process and equipment characterization, allowing suitable adaptation of the process to fit the pilot facility. 

Technology transfer of manufacturing processes for recombinant proteins frequently involves the reassessment of equipment capabilities and corresponding process requirements. In this context, this article describes the transfer of a fermentation process for the manufacture of Q-beta virus like particles (VLPs) in 

) from an external collaborator to Pfizer's cGMP pilot plant. In preparation for the technology transfer, establishment of appropriately organized teams as well as effective communication at all levels were key (see Figure 1). These early teams included both process development and analytical development scientists, since analytical assay support for an atypical product such as the Q-beta VLP was considered important for the success of this endeavor.  

Figure 1: Technology transfer team structure. (ALL FIGURES ARE COURTESY OF THE AUTHORS) 

The Q-beta VLP is an antigen delivery platform designed to serve as a key component of therapeutic and prophylactic vaccines (1–4). Each Q-beta VLP consists of 180 copies of a single coat protein from the 

 Q-beta, which together form an icosahedrally symmetric particle stabilized by disulfide bonds (5). The first step of Q-beta VLP production involves cytoplasmic expression of the coat protein (Q-beta monomer) in 

 Subsequent processing and purification steps ensure the formation and recovery of mature VLPs. Technology transfer challenges related to the Q-beta VLP manufacturing fermentation process included: a change in scale, significant equipment-related differences, and the high oxygen demand of the original process. 

Initially, the VLP fermentation process was transferred and implemented at the laboratory scale, which allowed further process characterization data to be collected. Following this, equipment characterization was undertaken to assess the capabilities of the cGMP pilot plant bioreactor system and compare it with the laboratory-scale system. Based on this information, required process adaptations were reviewed with our collaborators, and the process was successfully implemented in the cGMP pilot plant.  

CELL LINE, CULTURE MEDIUM, AND ANALYTICAL METHODS

The strain used for production of the Q-beta VLP was an 

 K-12 derivative that constitutively overexpresses the 

 repressor.  The target protein, Q-beta monomer, was expressed under the control of the hybrid 

 promoter induced by lactose or isopropyl beta-D-1-thiogalactopyranoside (IPTG) in a high copy-number plasmid. Plasmid selection pressure was maintained by a kanamycin resistance gene (6). The 

 production strain was transferred to Pfizer as vials from a cGMP working cell bank (WCB) containing a low-density cell suspension in culture medium with cryoprotectant.  

The fermentation process consisted of a seed culture stage and a fed-batch fermentation stage. The same yeast extract-enriched medium containing glycerol as the primary carbon source was used for cell bank preparation, seed culture preparation, and the fermentation batch phase (see Table I) (6, 7). For the fermentation fed-batch phase, two different fermentation feed media were used; both were similar in composition to the batch medium, but contained either a high concentration of glycerol alone or glycerol and lactose (see Table I) (6). Lactose was used as a carbon source and as the inducer.  

Samples were collected throughout the fermentation to assess culture growth and determine Q-beta monomer titer. Cell growth was examined by measuring absorbance at 600 nm (OD

), as well as determining dry cell weight with a 1 mL cell broth sample. Titer was determined by quantifying the amount of Q-beta monomer present in the soluble fraction of a lysed cell pellet using reverse phase-high performance liquid chromatography (RP-HPLC). Any fully or partially formed VLP present in the extract was first reduced to its monomer form by incubation with a reducing agent.  Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was also used to ascertain monomer identity based on comparison with a standard. Cell-free spent fermentation media samples were used to measure glycerol and lactose concentrations. Glycerol content was estimated using a Nova Bioprofile 300 Analyzer (Nova Biomedical, Waltham, MA) and lactose was measured using a YSI 2700 Biochemistry Analyzer (YSI Inc., Dayton, OH). Off-gas analysis was carried out during the fed-batch fermentation runs using Tandem off-gas analyzers by Magellan Instruments (Limpenhoe, UK) to obtain an estimate of the oxygen uptake rate (OUR). After subsequent processing and purification, the quality of the intact Q-beta VLP was assessed by several methods, including analytical size exclusion chromatography (SEC). The titer and product quality assays were transferred from our collaborator or developed in house. 

PROCESS TRANSFER TO LABORATORY-SCALE FERMENTATION SYSTEM

The objective of first transferring this process into the laboratory was to enable characterization of the fermentation protocol and allow the production of Q-beta VLP test batches for analytical characterization or development of the VLP purification process. It was understood that the VLP fermentation process implemented in the laboratory might require further adaptation to fit into Pfizer's cGMP pilot plant, as determined by the available equipment. 

Starting from a single cGMP WCB vial, a high-density research and development cell bank was quickly created for laboratory use (see Figure 2).  Frozen cell suspension from the cGMP WCB vial was used to inoculate a small volume of medium in a 250 mL shake flask; this culture was then used to inoculate a 3-L shake flask. When the culture reached the desired OD600, the cells were harvested by centrifugation and resuspended at a high density in fresh medium containing glycerol (20% v/v) as a cryoprotectant. Aliquots were frozen at –80 °C for long term storage. High density cell banks with cell concentrations of the order of 1 x 10

  colony forming units were used to minimize seed vial requirements per run and ensure consistency with respect to seed culture growth.  

Figure 2: Preparation of laboratory (non-cGMP) and cGMP cell banks for Q-beta virus-like particle (VLP) manufacture.  Lab cell bank was tested for bacteriophage and purity only; this cell bank was used at risk until test results were available.  The cGMP cell bank preparation did not include the Shake Flask 1 stage. 

The Q-beta VLP fermentation process to be transferred from our collaborator was a high-density fed-batch cultivation process that was inoculated using a shake flask seed culture. The fermentation itself consisted of three phases: a batch phase, an exponential fed-batch phase, and an induction fed-batch phase (see Figure 3). The batch phase lasted until glycerol in the batch medium was consumed, and was immediately followed by the exponential fed-batch phase, that involved the introduction of glycerol feed at a preset, exponentially increasing rate. Exponential feeding was continued for a set duration to achieve a target biomass concentration, at which time the glycerol feed medium was replaced with induction medium containing glycerol as well as lactose, and the induction fed-batch phase commenced. The induction medium was fed at a constant rate, equal to the feed rate at the end of the exponential feeding phase. The induction phase lasted a predetermined duration, after which the culture was harvested. This protocol was repeatedly shown to achieve a high titer of the Q-beta monomer along with a high biomass concentration (6). Significant biomass accumulation occurred during the induction phase despite the initiation of recombinant protein production. An oxygen-enriched air stream was used to satisfy the high oxygen demand of the culture during fermentation.  

Figure 3: Feed profiles for Q-beta virus-like particle (VLP) fermentation.  The solid line shows original feeding protocol. Feed I was the glycerol feed added at an exponential rate.  Feed II was the glycerol/lactose feed added at a constant rate for induction.  Dotted line shows the manner in which exponential feeding was extended for process characterization. 

Laboratory scale fermentations were carried out in a 15-L working volume, computer-controlled Sartorius Biostat C DCU3 fermentation system. Operating conditions for the 15-L scale fermentations were derived from the original process conditions, taking equipment limitations and prior fermentation experience into account (6). For all laboratory fermentations, the dissolved oxygen (DO) set point was 20%, with cascade control by agitation (500–1200 rpm) and airflow (10–15 Lpm). The temperature and pH were controlled at 30 °C and 6.8 respectively. Operating pressure was increased with respect to the original process to increase oxygen solubility, because a pure oxygen stream to enrich the process air was unavailable. 

A staged approach was followed for the technology transfer, which was intended to help understand the fermentation process requirements, such as oxygen transfer, and assess the laboratory fermentation system's ability to meet these requirements. This enabled adaptation of the original process for implementation in the laboratory without the use of oxygen, while maintaining productivity as close to the original process as possible. In practice, the staged approach was implemented by first performing uninduced batch fermentations, followed by uninduced fed-batch fermentations and finally induced fed-batch fermentations. Data from these runs drove discussions on process adaptation. 

Batch fermentations were conducted to determine the biomass concentration after all the glycerol present in the batch medium was consumed. This data was required for calculation of the feeding profile. The batch fermentation medium contained 5 g/L glycerol as the main substrate for growth; however, additional substrate was also available due to the presence of yeast extract (6). At the 15-L laboratory scale, a starting batch volume of 10 L was used, and the batch was inoculated with 2.4% v/v shake flask preculture broth. During the batch fermentations, all glycerol in the medium was consumed in 7–8 h (data not shown). 

Fed-batch fermentation with exponential feeding

Exponential fed-batch fermentations were designed to assess culture oxygen requirements and determine the maximum oxygen uptake rate that could be supported by the laboratory fermentation system. This would be observed on the DO profile as a sustained decrease in the DO value below the 20% set point. These fermentation runs included a batch phase as described earlier, followed by a fed-batch phase that involved the addition of glycerol feed medium at an exponentially increasing rate.  The equation used to define the exponential feeding profile has been well documented in literature (6–9):

 is volumetric flow rate of feed medium at time t (L/h), 

 is culture volume at feeding start (L), 

 is biomass (cell dry weight, g/L) at feed start (determined from batch fermentation), 

 is process time at feed start (7–8 h), 

 is concentration of glycerol in the feed medium (g/L), and 

 is glycerol yield coefficient determined from batch fermentations (g dry cell weight / g of glycerol at the batch phase end). 

It was observed that after about 8.5 h of feeding, to maintain the dissolved oxygen set point at 20%, the agitation and airflow reached maximum values of 1200 rpm and 15 Lpm respectively, and that beyond this time the set point could not be maintained (see Figure 4A). Taking this into account, the duration of the exponential feed phase was adjusted to 8 h for future laboratory runs, which was shorter than described in the original process but deemed satisfactory to allow sufficient biomass accumulation for induction. The OUR at this time was measured at approximately 230 mM/L–h.  

Figure 4: Q-beta virus-like particle (VLP) fermentation at the lab scale. (A) Characterization experiments to observe process oxygen requirements - dissolved oxygen and oxygen uptake rate profiles for fermentations conducted with the extended exponential feeding.  (B)  Dissolved oxygen and oxygen uptake rate profiles for modified fermentation process with shortened exponential feed phase to decrease peak oxygen demand. Colors show dissolved oxygen profiles for different batches. 

These fermentation runs were conducted to complete the laboratory-scale fermentation process transfer and assess final Q-beta VLP production with a shorter (8 h) exponential feeding phase prior to induction. The batch and fed-batch phases were conducted as described earlier. At the end of the exponential feed stage, glycerol feed medium was replaced with the induction medium containing glycerol and lactose. The induction medium was added at a constant feed rate equal to the feed rate reached at the end of the exponential feed phase. The induction phase duration was maintained at 5 h as described for the original process. The culture density increased significantly during the induction phase, and the cell density at harvest was more than twice that at induction. The OUR decreased after induction and remained at approximately 150 mM/L–h (see Figure 4B). Some lactose accumulated during the early part of the induction phase as the culture adapted to this new carbon source (data not shown). As expected, the harvest cell density and final titer were approximately 80% and 85% of the corresponding values for the original process, respectively (see Figure 5A). The specific productivity, calculated as grams Q-beta monomer per grams dry cell weight, corresponded very well with the original process, and product quality after purification was also satisfactory (see Figure 5B and 6A).  

Figure 5: Culture growth and productivity data at the laboratory scale. (A) Growth for the adapted fermentation and original process.  Original process is in red. (B) Productivity of the adapted process was comparable to the original process. 

Fermentation process transfer to the laboratory was completed in approximately three weeks, including preparation of the development cell bank.  A well-characterized small-scale fermentation process that fully utilized the capabilities of the 15-L laboratory fermentation system was now available to produce test batches of Q-beta VLP for development.  

Figure 6: (A) Adapted process showed comparable product quality at the lab scale.  Analytical size exclusion chromatography (SEC) was one of several methods used to test VLP quality.  SDS-PAGE was used to observe the monomer (inset). (B) Adapted process showed comparable product quality at the pilot scale.  Analytical SEC and SDS-PAGE were among the assays used to used to check VLP quality (inset). 

PROCESS TRANSFER TO PILOT-SCALE FERMENTATION SYSTEM

The cGMP pilot plant fermentation system had a 100 L maximum working volume and had previously been used as a seed tank to support larger scale mammalian and microbial cultivation. Thus, it was expected that this system would have certain operating constraints that might require further adjustment of the laboratory-scale process prior to implementation. To prepare for the transfer, the pilot plant bioreactor was characterized and compared to the laboratory system.  

The production of a high-density cGMP cell bank was initiated prior to the planned cGMP fermentation. The protocol used for creation of the cGMP cell bank was similar to that used in the laboratory. However, additional testing of the cell bank for viability, purity (bacterial and fungal contaminants), host strain identity, and absence of bacteriophage was undertaken to ensure compliance with existing guidelines.  

Figure 7: Characterization strategy for fermentation equipment. 

Equipment-related parameters for characterization were selected based on literature review, past experience with 

 fermentation, and prior knowledge of the equipment (see Figure 7) (10–16). These included certain theoretical characteristics, as well as experimentally measured characteristics of the fermentation vessels.  

Table II: Fermentation vessel specifications.  

Characterization of the fermentation equipment was initiated with a review of system specifications (see Table II). Theoretical characteristics of the fermentation systems selected to serve as comparative metrics were geometric similarity, maximum impeller tip speed, maximum power input per unit volume, and superficial velocity at 1 vvm (see Table III) (12, 15, 17, 18). The difference in the geometric similarity parameter for the 100-L vessel and 15-L vessel reflected the difference in aspect ratio of the two tanks.  Calculations for impeller tip speed indicated that higher tip speeds were achieved at the 100-L scale due to larger impeller size. To estimate power input per unit volume, a range from 5.2 to 6.5 was assumed for the power number (N

) based on the literature, and both values were used for calculations (17). Again, it was observed that for a given agitation rate, the power input per unit volume was higher for the 100-L fermenter, due to a larger impeller size. The superficial velocity was much lower for the 100-L vessel compared to the 15-L vessel for the same gas flow rate expressed in vessel volume per minute (vvm).  

Table III: Summary of characterization results. 

Features

The authors describe challenges faced in transfer and scale-up of a fermentation process.

Fermentation Process Technology Transfer for Production of a Recombinant Vaccine Component

In the course of every relationship, there arise moments for the "difficult conversation." Usually it's of the "where are we headed with this?" variety. But that's really just part of a larger question: Do we have a shared philosophy, and what is it? 

Whether we think of it that way, nations ask themselves this question routinely. Should our nation be guided by a religious philosophy, or not? Should we elect our leader as a group, or should a person ascend to leadership through inheritance? If the times require us to alter our viewpoint, do we decide as a group, or does someone decide for us, and if so, who? 

Sometimes the answer is explosive: Whoever we are, it's not working, and we need to change now.  

That's the kind of answer that can lead to revolutions. In the US, we've mostly dealt with the question of our national ethic more quietly, starting with the first national conversation, which lay out a basic philosophy in the form of a constitution. Nevertheless, there have been many difficult conversations we've failed to have as a nation. To whom do we accord full citizenship, for example? Not even the Founding Fathers could agree on this one, thereby establishing the alternative to a national conversation: the local conversation. Instead of making a national decision about which people could own land, vote, or intermarry, those questions were left to local governments.  

In these cases, challenging and sometimes overturning local philosophies (and sometimes overturning them again) has been, not through legislation, but through the courts, through litigation. That's how we've handled teaching evolution in schools, gay marriage, and abortion—locally and through litigation. 

Some conversations, it seems, are just too difficult to have nationwide. I expected the conversation about stem-cell therapies to be one of those too-difficult conversations as well, one that would also be handled case by case and concession by concession—through litigation rather than legislation.  

So it came as a surprise to learn that in mid-May, US Representatives Brian Bilbray (R-CA) and Diana DeGett (D-CO) introduced the Regenerative Medicine Act of 2011 (H.R. 1862). The Act notes that, "Regenerative medicine products have already successfully treated numerous health conditions, and have the potential to provide cures, treatments, and diagnostics for a range of diseases and disabilities including diabetes, spinal cord injury, heart disease, stroke, and various forms of cancer."  

The proposed legislation goes on to say, "A United States national strategy on regenerative medicine is critical to ensure that this technology fulfills its potential to cure and treat diseases and disabilities, reduce overall health spending, and promote economic growth."			

Never once does the Act mention stem cells, embryonic or otherwise, which are among the therapeutic agents used in regenerative medicine. It is the potential use of embryonic stem cells that makes a national conversation difficult, because, like the still-unresolved abortion debate, it pits technology against notions of personhood. Indeed, the stem-cell debate has already been the object of some well-publicized litigation. And it seemed likely that the entire debate would continue that way. 

Whatever the fate of the Regenerative Medicine Act of 2011, I hope it sets a precedent for the way we discuss novel, but potentially ethically thorny issues of technology in the future. After living together as a nation for 235 years, shouldn't we at least know how to broach difficult conversations? 

From the Editor

Which route will we take to arrive at a national stem-cell policy?

Legislation v. Litigation

The concept of using plasmid DNA encoding proteins as vaccines evolved largely from the results of experiments completed in the late 1980s and early 1990s in which DNA uptake and gene expression by cells in regenerating muscle tissues of rodents was demonstrated. The unique attribute of these observations was that the DNA was delivered without materials commonly used to mediate cellular transfection; a formulation referred to as "naked DNA." The 

 translated proteins proved to be immunogenic, and the generation of protective immune responses was soon demonstrated using several animal model systems.  

The potential advantages of this vaccine format are numerous. They include, first, the intracellular translation of proteins that can effectively deliver them to the processing pathways that generate the fragments needed to induce cellular immune responses, specifically T-cell responses; second, the ease of DNA plasmid manufacture using

and column chromatography purification methods; third, DNA plasmid stability; and finally, lack of pre-existing immunity in vaccine recipients which might limit DNA uptake. Thus, a significant amount of excitement was generated for this vaccine format in the field. 

Concerns with the possibility that vaccine DNA could become integrated into the recipient's genome, and potentially cause cancer, were allayed through a series of experiments completed in the mid-to-late 1990s, thus clearing the path for clinical testing. The levels of vaccine potency observed in research animals using naked DNA has not translated to humans and this has resulted in the use of large (1–5 mg) doses. However, the use of naked DNA as the "priming" vaccine in heterologous immunization regimens that include subsequent administration of viral vectored vaccines or proteins as the "booster" vaccines remains promising. 

Although the use of the naked DNA approach is highly attractive because of formulation simplicity, the development of products to augment DNA vaccine potency has also been an active area within the field. Vaccine design approaches include the co-delivery of genes encoding cytokines, such as Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-2 (IL-2), IL-12, or IL-15, which act directly on cells involved in generating or effecting immune responses and thus function as vaccine adjuvants. A search of the 

 website, which is managed in part by the US National Institutes of Health, indicates active clinical testing of numerous experimental products designed to induce immune responses to infectious pathogens and tumors. Thus, we can expect to better understand the safety and adjuvant activity of DNA plasmid-encoded cytokines within the next few years. 

The use of cell-surface active polymers and cationic lipid-based materials to augment transfection efficiency into cells has also received significant attention and several DNA vaccine products have been tested clinically. Two of the more advanced transfection-enhancing materials are being developed by Vical in San Diego, and include a cationic lipid, designated Vaxfectin, and a synthetic poloxamer, designated CRL1005. Vaxfectin is used in DNA vaccine formulations designed to protect against avian influenza virus and anthrax while the poloxamer-supplemented formulation is used in a vaccine designed to protect or treat cytomegalovirus infections in organ transplant patients. Phase I or II clinical testing of these products have been completed and yielded promising results in terms of safety and immunogenicity. Another promising approach for DNA vaccine delivery involves the use of devices to increase plasmid uptake or to preferentially target either the skin or muscle cells. Three distinct approaches are under development currently: the gene gun, a technology owned by Pfizer that delivers DNA vaccine adsorbed to microscopic gold particles to the skin; needle-free injection devices by Bioject Inc. that can deliver liquid-formulated vaccines to either the skin or muscle using gas pressure; and 

 electroporation devices being developed primarily by Inovio Pharmaceuticals  and Ichor Medical Devices that can deliver liquid-formulated vaccines to either the skin or muscle. 

Devices of these types are being evaluated in clinical trials currently with DNA vaccines based on virus proteins and tumor antigens. Of interest is that the devices can be used with simple DNA vaccine formulations or in combination with cytokine-supplemented vaccines and transfection-enhancing materials.  

The clinical-trial data being generated now will continue to direct the evolution of the field.  The future for DNA vaccine technologies, therefore, appears to be quite bright. 

 is vice-president of Research and Development at GeoVax, Inc, 

Final Word

Plasmid DNA-encoding proteins offer many advantages, which are now being used in clinical trials.

The Status of the DNA Vaccine Field

Value is returning to biotech. For the first time since late February 2009, the industry's collective market cap has risen above $400 billion. At that time, Genentech was contributing close to $100 billion of that total. When it was acquired, the industry's market cap dropped by about 25%. Despite the ensuing tough financial climate, it has taken just two years to recoup this loss, which is remarkable given that in the interim Big Pharma has been nibbling at the valuation by acquiring large biotech companies such as Sanofi's acquisition of Genzyme. This deal created a surge of interest and a great deal of speculation about other blue-chip biotech companies that might be in Big Pharma's crosshairs.  

We didn't have to wait long for this predicted mergers and acquisitions (M&A) activity. Canadian-based Valeant Pharmaceuticals made a hostile takeover offer for Cephalon in late March 2011. The cash offer of about $5.7 billion was eventually rejected by Cephalon who believed the offer undervalued the company's key assets and prospects. Just as investors were digesting this information came the news a few weeks later that Teva Pharmaceuticals and Cephalon had reached a definitive agreement whereby Teva would acquire Cephalon for $6.8 billion in cash.  

It wasn't only deal making that kept investors engaged. Pharmasset's shares have been on a tear, vaulting almost 60% in March (168% year-to-date), and is now one of biotech's hottest companies, with its shares currently trading at $116. Investors were encouraged by the company's data from the European Association for the Study of the Liver's annual meeting held in Berlin late in March 2011. The company reported that its candidate hepatitis C drug PSI-7977, a nucleotide analog polymerase inhibitor, was effective when added to standard therapy in 98% of patients in a 121-person clinical trial. Pharmasset also announced that screening had begun in a Phase 2b study of PSI-7977. 

Biogen Idec's stock value jumped 33% following their news announcement about positive top-line results from the first of two pivotal Phase 3 clinical trials designed to evaluate their investigational oral compound BG-12 (dimethyl fumarate) as a monotherapy in people with relapsing-remitting multiple sclerosis. Results showed that BG-12, administered either twice or three times a day, met the primary study endpoint. Biogen also reported that it had signed a collaboration with Amunix for next-generation long-lasting blood factor products. 

In the regulatory area, FDA gave the green light to the blockbuster biotech drug Benlysta, the first new lupus medicine to be approved by the agency in 56 years. Human Genome Sciences (HGS) and GlaxoSmithKline have collaborated on the drug's development since 2006. San Diego-based Optimer Pharmaceuticals' Dificid (fidaxomicin) tablets for the treatment of 

-associated diarrhea also received approval. 

Following on from the record amount of partnership dollars generated by US biotechs in 2010, the first quarter of 2011 saw a further $7.5 billion being raised through partnership deals.  

Table I: Performance of biotech initial public offerings (IPOs) completed in 2011.			

One of the top deals in annouced value was Aveo Pharmaceuticals, which signed a major cancer drug licensing deal with Astellas Pharma that netted the biotech $125 million upfront and could be worth more than $1.3 billion if all milestones are met. Their experimental drug, tivozanib, is currently in a late-stage trial as a treatment for advanced renal carcinoma, and in other studies as a treatment for other solid tumors. It is designed to optimally block the vascular endothelial growth factor (VEGF) pathway by inhibiting all three VEGF receptors, for the treatment of a broad range of cancers.   

Another of the top deals is the collaboration between Takeda Pharmaceuticals and Intra-Cellular Therapies. The two companies are teaming up in a worldwide collaboration worth up to $750 million. The companies will develop and commercialize Intra-Cellular Therapies' preclinical selective phosphodiesterase type 1 (PDE1) inhibitors for the treatment of cognitive impairment associated with schizophrenia. ITI's PDE1 inhibitors are orally available and have potential to be treatments for a variety of psychiatric and neurological diseases.  

QUIET PERIOD FOR  INITIAL PUBLIC OFFERINGS 

Only three US biotech companies priced initial public offerings (IPOs) during the past three months:   					

Specialty pharmaceutical company, Sagent Pharmaceuticals sold 6.6 million shares including over-allotments at $16.00 per share, which was at the high end of their projected $14 to $16 share price range. Since its formation in 2006, Sagent has developed an extensive portfolio of injectable pharmaceutical products.

For its IPO, Tranzyme Pharma priced 13.5 million shares at $4.00 per share. The North Carolina biotech had hoped to debut mid-March in the $11 to $13 per share range, but its underwriters couldn't get any traction for the offer until they slashed the price more than two thirds and tripled the number of shares offered. Tranzyme is a clinical-stage biopharmaceutical company focused on discovering, developing and commercializing novel, first-in-class small-molecule therapeutics for the treatment of acute (i.e., hospital-based) and chronic gastrointestinal motility disorders. 

South San Francisco-based Solazyme, which transforms plant-based sugars into petroleum-based chemicals and fuels, raised $198 million by offering 10.975 million shares at $18, above the range of $15 to $17. The company had originally planned to offer 9.975 million shares, with the upside coming from additional primary shares; in total, the company raised 24% more than originally planned.

There now have been 10 US biotech IPOs since the beginning of the year. Their improving performance is reflective of biotech's better fortunes in parallel with the strengthening of the capital markets. The IPO runway is full and it is likely we will see several more biotech companies complete their IPOs in the coming months, although to get the deals done they may have to modify their pricing strategies to offer a lower price and sell more shares, as was the case for Tranzyme. 

 is chief executive officer at Burrill & Company, San Francisco, CA, 415.591.5400, 

Burrill on Biotech

Strong pipelines, approvals, and deals drive up market cap.

Biotech Posts Strong Gains

For therapeutics administered by certain routes such as pulmonary delivery, plastic ampuls can offer many advantages over glass ampuls, vials, or syringes. Plastic ampuls are manufactured using blow–fill–seal (BFS) technology. The BFS process involves plastic extrusion, molding, aseptic filling, and hermetic sealing in one sequential operation. Unlike small molecules, biological drug products, such as proteins or monoclonal antibodies, are more prone to degradation during processing, which may result in loss of activity or safety concerns. The operating conditions for a BFS process and the nature of plastic ampuls pose many challenges to the stability and integrity of biological drug products. In this article, the authors discuss considerations in the development and manufacturing of biological products using the BFS process, including potential product exposure to elevated temperature, requirements for leak detection, and packaging operations. They also highlight challenges and strategies for BFS process characterization and validation in the context of biopharmaceutical manufacturing.

Biological drug products, such as proteins or monoclonal antibodies, are predominately packaged into vials or prefilled syringes for intravenous or subcutaneous administration. However, some biological drug products must be administered by alternative routes, such as pulmonary delivery in the form of a mist using a nebulizer. In such a case, using plastic ampuls as the primary drug container offers many advantages over vials or syringes. Plastic ampuls are convenient, simple to use, are unbreakable, and child-friendly. One example of biological product supplied in plastic ampuls is Pulmozyme (dornase alfa, Genentech), prescribed for the treatment of cystic fibrosis. Pulmozyme is a sterile, clear, colorless, highly purified solution of recombinant human deoxyribonuclease I (rhDNase), an enzyme which selectively cleaves DNA, and is available as single-use 2.5-mL ampuls. The patient self-administers the drug by simply emptying the entire content of an ampul (one dose) into the nebulizer bowl for delivery. The development of the Pulmozyme formulation and manufacturing process has been previously reported (1). 

Plastic ampuls are manufactured using blow–fill–seal (BFS) technology (2). BFS has gained wide acceptance for pharmaceutical solutions (e.g., eye, nose, and ear drops, contact-lens solutions, inhalations, oral, or topical solutions) and household chemicals (e.g., insecticides, detergents, and disinfectants) because it offers many manufacturing advantages, such as high output and reduced human intervention. The use of BFS in biopharmaceutical manufacturing, however, has been scarce. Unlike small molecules, biological drug products are more prone to degradation, which may result in loss of activity. The unique operating conditions and requirements of BFS technology also pose many challenges to the development and manufacturing of biological drug products.  

Conventional fill–finish unit operations such as mixing, filtration, and filling, and their potential impact on the product have been previously reviewed (3). This article focuses on specific challenges and considerations associated with the development and manufacturing of plastic ampuls using the BFS process, including elevated temperature, leak detection, packaging, as well as process characterization and validation.  

The BFS process involves plastic extrusion, molding, aseptic filling, and hermetic sealing in one sequential operation, as illustrated in Figure 1. The resin material for primary containers is typically received as plastic granules (e.g., low-density polyethylene [LDPE] or polypropylene [PP]). In the extrusion step, plastic granules are fed through an extruder, where they are melted at temperatures above 160 °C. The melted plastic is pressed and extruded through an orifice, resulting in a continuous hollow tube of molten plastic, referred to as a parison (see Figure 1, Step 1). The metal mold then moves to enclose the parison and forms the plastic container of desired shape, aided by either application of vacuum to the mold cavity through small orifices or by blowing sterile air into the container (see Figure 1, Step 2). Subsequently, a parison dye cut will cut the parison at the top, and the mold carrying the unclosed plastic container (at this stage referred to as an ampul) will move to the filling cabinet. In the filling step (see Figure 1, Step 3), the filling needle is inserted into the top opening and solution is discharged into the ampul. The filling needle retracts after the fill completion. The top portion of the mold then comes together to press the plastic and to form hermetically sealed ampuls (see Figure 1, Step 4). The mold then opens and the ampuls are released from the BFS machine and conveyed to the inspection station (see Figure 1, Step 5).  

 Figure 1: Overview of the blowâfillâseal process.			

THE DEVELOPMENT AND MANUFACTURE OF BIOLOGICAL PRODUCTS USING BFS

Fill–finish operations of biological drug products, particularly proteins or monoclonal antibodies, usually begin with drug substance (or concentrated bulk) supplied either refrigerated at 2–8 °C for temporary storage, or frozen at <–20 °C for extended storage. The drug substance is thawed, if necessary, at a defined temperature and process. If the drug substance has a different concentration from the final drug product, a formulation buffer can be added and mixed together with the drug substance in a mixing tank, to reach the target concentration as final drug product. The solution is then transferred into a holding tank through a filter to reduce bioburden level. The holding tank containing formulated product can be used to store the product for an extended period of time to accommodate manufacturing scheduling. These operation steps, including thawing, dilution, mixing, and filtration are typically conducted in a Grade C area. Figure 2 illustrates the process flow. 

Figure 2: The blowâfillâseal process in the context of biological manufacturing.			

Most parts of the BFS machine are located in a Grade C room; only the filling cabinet is controlled under a Grade A condition. Time-pressure filling, where a pinch valve is used to control the fill volume, is often used in the filling operation. The flow rate is determined by the pressure applied to the liquid reservoir (or surge tank), and the pinch valve opening time determines the final fill volume. During the filling process, solution sterilization is achieved by sterilizing-grade filters.  

The connection of the BFS machine with filling needles, surge tank, and sterilizing filter usually takes place in a Grade C environment. The BFS machine is first assembled and cleaned through clean-in-place (CIP) procedures. It then gets connected to the sterilizing filter, and the entire connection is sterilized under a validated steam-in-place (SIP) cycle, from the filling needle, all the way to the point of connection with the product tank.  

Aseptic processing requires contamination control and sterilization of three main aspects: drug-product solution, primary container components, and operation environment. The sterilization method for drug product solution in a BFS process is similar to a conventional fill–finish process, where typical sterilizing grade filters are used. Unlike the component processing steps in the conventional fill–finish process (i.e., washing, depyrogenation, and siliconization of vials, syringes, and stoppers), the primary container is formed in the BFS process, and the molding of plastic resin at elevated temperature provides a container "free" of viable microorganisms and with acceptable endotoxin levels. The ability for a BFS extrusion process to yield product with acceptable quality has been demonstrated by controlled challenges studies, where LDPE granulates were contaminated with characterized levels of bacteria spores and endotoxins, then pressessed through a BFS machine using Tropton Soy Broth and Water for Injection as the filling media (4). Higher fractions of contaminated units were observed with increased challenge level (or amount of spores and endotoxin in the plastic granulates). The above study demonstrated it is critical to establish appropriate acceptance limits for bioburden and endotoxin levels on plastic granulates to assure final product quality. 

The overall extrusion and molding process of the BFS operation is conducted in a Grade C area. However, the air shower for the filling shroud of the BFS machine should provide a Grade A condition. European Union  guidelines state that, "The condition should comply with the viable and nonviable limits at rest and viable limit only when in operation" (5).  There is potential risk for airborne contamination before the mold moves to the filling shroud area, where the air shower maintains a Grade A environment. Recent studies have shown that most of the airborne contamination occurs between the time when the top is cut from molded containers and when they pass under the air shower (6). The microbial and particulate controls are highly equipment and site specific. Therefore, a media fill has to be conducted for each specific machine, process, or container type. A recent survey of the BFS industry provides an excellent overview of the current practices in aseptic BFS technology, showing that the BFS process has a much lower frequency (about one-tenth) of contaminated media fills compared with conventional processes (7). 

One of the major challenges for the BFS operation compared with a conventional filling process is the high temperature involved in the plastic extrusion step. As illustrated in Figure 1, the plastic granules are melted and extruded at temperature in excess of 160 °C. The hot parison will come into contact with a metal mold, be transported to the filling shroud, and be filled with drug product. Cool water circulating inside the mold helps to lower the ampul temperature. The cooling process, however, is limited by the contact time between the ampul and the mold (a total of about 10 s). In addition, the ampul cooling is restricted by the minimum temperature needed to form a hermetic seal at the end of the filling step. The ampul temperature near the neck area has to remain high enough to ensure an appropriate seal at the end of the filling process. 

In the development phase of a biological drug product, when using a plastic ampul as the primary container, it is important to understand the temperature to which the product could be exposed. The folded conformations of proteins are only marginally stable, and a rise in temperature can denature the molecule leading to loss of activity. Elucidating the temperature profile during the BFS process will enable formulation scientists to develop more robust formulations.  

In a BFS manufacturing process, product temperature during the filling process is not an easily controlled parameter, and it cannot be directly measured. Instead, the controllable parameters are the starting-product solution temperature in the bulk tank, plastic extrusion temperature, mold-cooling water temperature, and the cycle time for each BFS step (e.g., molding, filling, and sealing).  

The authors have used a computational fluid dynamic (CFD) model (ANSYS Fluent 12) to simulate the BFS process using the Pulmozyme ampul as a model. Initial simulation results (not shown) showed that ampul-wall temperature reaches mold temperature quickly (within 1 s) during Step 2 of Figure 1. If the actual mold temperature is at cooling-water temperature (usually near room temperature of ~ 25 °C), the finished ampuls at the end of BFS process should not be significantly hotter than the mold cavity. It is the authors' experience, however, that product ampuls usually feel warm right after the BFS process. This observation suggests that the actual mold temperature is much higher than the cooling-water temperature (the actual mold temperature during BFS production is not usually measured). 

For the purpose of the CFD simulation, the authors adjusted the mold temperature to different settings. Figure 3 illustrates the product-temperature profile when the mold temperature was set at 60 °C. The simulation assumes that it takes approximately 11 s for the entire BFS process including ~3 s for the molding step, ~3 s for filling, and ~5 s for forming a hermetic seal. The upper portion of Figure 3 shows the liquid volume in the ampul, as it is being filled (from time = 3 s). The temperature profiles of the entire ampul (including wall, solution, and air) throughout the filling and sealing step are shown in the bottom sequence of Figure 3. It should be noted that in the actual manufacturing process, the filling nozzle is retracted from the ampoule after filling is complete to allow for sealing. Since this is a nonproduct contact part the current CFD simulation simply turns off the solution flow from the inlet nozzle, instead of modeling the actual nozzle retraction.  

Figure 3: Simulation of product temperature in a blowâfillâseal process.			

The simulation revealed that the ampul is cooled from the initial extrusion temperature of ~160 °C to 60 °C (assumed actual mold temperature) in less than 2 s. Forced convection dominates heat transfer during filling (from 3 s to 6 s), thereafter natural convection becomes more prominent. By the time the filled ampul was ejected from the mold (~ 11 s), only the fluid layer near the ampul walls had been heated to within 10 °C of the mold temperature. The majority of the liquid remains considerably cooler.  

In the current simulation the authors assumed a worst-case scenario of liquid being filled at ~25 °C. In the actual production process, the drug substance is typically maintained at 2–8 °C and would be introduced in the ampoule close to these temperatures. Therefore, in a production BFS process, it is expected that the drug substance would experience temperatures that are lower than these shown in the CFD simulation results. 

Based on the observation of higher actual mold temperature compared with that of the cooling water from the simulation results, it is highly recommended to monitor the actual mold temperature during the development or characterization phase of the BFS process. Monitoring could potentially be achieved by attaching thermocouples to the mold or by infrared temperature measurement of the mold surface.  

The formation of the primary container is an integral part of the BFS operation and container-closure defects can be a major problem. US regulatory authorities require that "as a final measure, the inspection of each unit of a batch should include a reliable, sensitive, final product examination that is capable of identifying defective units (e.g. "leakers")"(8). It is also stated in the EU guidelines that "containers closed by fusions, e.g., glass or plastic ampoules should be subject to 100% integrity testing"(5). 

There are multiple leak-detection technologies available, such as the vacuum-decay method, high-voltage leak detector, or near infrared leak detection. Among these methods, the vacuum-decay method is often selected for biological manufacturing becauese it has no product quality impact (even though there is no documented product quality impact from other methods to the authors' knowledge). During vacuum decay leak detection, the test article is placed inside a chamber, which is then evacuated to a known pressure. If the product container exhibits a leak, the chamber pressure will rise at a rate greater than a predetermined baseline value.  

The qualification of a leak-detection machine can be achieved by challenging the system with calibrated needle valves, or by means of "leaky" samples, created by either laser microdrilling or capillary tube insertion into ampuls. In the authors' experience, creation of "leaky" samples of ampuls is not reproducible, and needle valve is a preferred method to qualify the leak detection machine. Leaky samples created by laser microdrilling or capillary tube insertion in plastic materials have many challenges. Most plastic resins, such as LDPE, do not have the required material characteristics (too soft and poor adhesion) to support reliable mounting of a capillary or to maintain a consistent hole size. The drilling process with LDPE yields a conical-shaped orifice that results in flow rates that are difficult to predict. Furthermore, the verification process is destructive in that the flow rate is measured under vacuum, which can result in obstruction of the orifice by the product. Therefore, the actual test sample used cannot be verified prior to testing. The variables of shipping, handling, and pressure as a result of air transportation could also result in inconsistent results.  

Alternatively, the needle valves can be calibrated with a flow meter to a specific flow rate corresponding to a specific orifice size. Calibrated needle valves represent a suitable qualification approach to the system that will demonstrate the system's ability to detect an actual leaking sample with a known hole size and flow rate.  

Another main challenge with in-line leak detection operation is the high false rejection rate. Due to the high temperature required for plastic extrusion, the ampuls, immediately after BFS process, are notably warmer than room temperature. When the products are immediately placed in a vacuum-detection chamber, the heat from the ampul can cause a slight increase in pressure which the instrument may interpret as a leaky sample, which will trigger a false rejection signal. Therefore, it is highly advisable to have the product equilibrated to room temperature prior to subjecting the product to vacuum leak detection.  

Packaging requirements, extractables and leachables

The primary containers of ampuls manufactured using BFS process are plastic materials, such as LDPE or PP, which are gas-permeable to some degree. During long-term storage, water vapor may diffuse out of the ampul resulting in alterations in drug concentration. Conversely, oxygen permeation could cause protein oxidation. Gas permeation can be minimized by sealing the plastic ampuls inside laminated foil pouches (as a secondary packaging layer). For example, in the development of the Pulmozyme drug product, studies showed that protein concentration increased by more than 30% due to water loss when the naked product was stored at 37 °C for ~700 days (1). Such water loss was prevented by adding an aluminum foil pouch as a secondary packaging layer. In addition, the aluminum foil pouch protects the drug product from light-induced degradation. Furthermore, oxygen-sensitive products can be sealed into pouches under a nitrogen blanket (overlay) to provide additional protection against oxidation. 

Secondary packaging materials are typically comprised of multiple layers of polymers (i.e., polyester, PP, or polyethylene), inks, adhesives, as well as possibly unreacted monomers and oligomers derived from adhesive materials. When in direct contact with primary containers, these materials have the potential to penetrate through plastic ampuls. In addition, the direct contact between the liquid product and the primary container (even though the primary container material is typically made of much cleaner polymers) could lead to extraction of chemicals. These chemical extracts and leachates from plastic packaging materials could act as adjuvants in stimulating an immune response in the patient (9). Leachates would especially be of concern if the product is to be injected subcutaneously, as the localized concentration of product and adjuvant is more likely to stimulate the production of neutralizing antibodies than intravenous injection or inhalation (9). Therefore, it is imperative that the extractable and leachable program for plastic ampul products be conducted with both pouched and unpouched samples to understand the respective extracts and leachates from both the primary and the secondary packaging materials (10–12). 

BFS PROCESS CHARACTERIZATION  AND  VALIDATION

A biological manufacturing validation program encompasses equipment qualification, cleaning validation, and process validation. The BFS equipment suppliers and manufacturers usually have well-established practices for performing equipment qualification and cleaning validation. Process validation, however, could be a challenge for biological manufacturing using BFS technology. 

The goal of process validation is to provide a high degree of assurance that the specific manufacturing process will consistently produce product meeting predetermined acceptance criteria. Modern-day process validation programs consist of process characterization studies and full-scale qualification lots produced under GMP conditions. Process characterization can be performed on each unit operation to evaluate and characterize an acceptable range of operating conditions that yields acceptable product quality and process performance. A quality-by-design (QbD) approach, which systematically uses risk assessments and multivariate characterization studies, is the current state of the art. Process characterization may be carried out at different scales, and it may also include engineering runs to mimic the overall process flow. Based on the outcome of process characterization, full scale GMP batches are manufactured under normal settings. Both physical attributes (such as ampul appearance, integrity, fill volume, and wall thickness) and chemical attributes of the product are checked to ensure that the process performs as expected.  

It is important to recognize the uniqueness of the BFS process when applying the above process characterization and process-validation approach. The BFS machine is a custom-built machine. The equipment setup and operation often involves both automation control and manual adjustments. The operator experience or "know how" generally is very important to the proper operation of the BFS equipment. Such operator-dependent performance, to a certain extent, is in contrast to the QbD paradigm of pharmaceutical manufacturing, where critical process parameters (CPPs) are identified, understood, controlled, and monitored to achieve a robust manufacturing process. The large number of controllable and uncontrollable parameters in the BFS process makes process characterization and process validation much more challenging than conventional biological filling processes. For instance, the filling speed for a peristaltic or rotary piston pump is the typical process parameter that can be evaluated during stand-alone process-characterization studies. The filling speed, however, for a BFS process is mostly fixed, due to constraints from synchronized movement of the extruder, mold, filling, and seal operations. Only minor adjustments can be made to the duration of each of the BFS steps (extruding, molding, and filling), and such adjustment may be necessary to achieve optimum product appearance. 

Another example of manually adjusted parameters is the plastic-resin weight. The resin weight determines how much plastic is extruded to form the container and directly correlates with the container wall thickness. Too much resin may result in containers with "bulky" appearance or problems with deflash while too little resin may cause issues for container integrity or ampoule leakage. The appropriate amount (or range) of resin is typically determined from equipment qualification. It can only be manually adjusted through a few mechanical parts at the beginning of each product run. Therefore, the process and product development for BFS process needs to balance an empirical approach and a systematic approach. The biological manufacturer needs to collaborate closely with the BFS equipment supplier or contract manufacturer to design a robust program for process characterization and validation.  

Figure 4 provides an example of the potential roadmap for process characterization and process validation. Risk assessments are highly recommended for each unit operation as well as for each of the steps from the BFS operation in order to identify potential failure modes from equipment and mechanics, as well as product-impact aspects. The output from the risk assessments would be a list of process parameters that could affect the overall process or product quality. Further development and characterization studies should be designed for these parameters either as stand-alone studies or overall engineering runs. CPPs are selected based on the outcome of process characterization. Finally, full-scale qualification lots should be produced (typically at target settings of the process parameters) to demonstrate the validated state of the end-to-end process. 

Figure 4: Process flow for blowâfillâseal (BFS) process characterization and validation (CPP is critical process parameter; PC is process characterization).			

BFS technology has great potential in the field of biopharmaceutics because of reduced human intervention during the production process, convenience, and ease of use offered by its final product in plastic ampul form. The operating conditions of the BFS process and the nature of plastic ampuls pose many challenges to the stability of biopharmaceutical drug products. Biopharmaceuticals may experience elevated temperature during the BFS process. CFD could be a useful tool for better understanding the temperature dynamics during the BFS operation. The unique aspects of BFS operation call for a balanced empirical and systematic approach during process development and process validation. 

Finally, the BFS process may not be suitable for many proteins, especially large, complex proteins with multiple sites for activity and proteins that are highly temperature-sensitive.  In addition, chemical extracts or leachates from the direct contact between the product and the primary container, as well as volatile chemicals from secondary packaging layers could act as adjuvants in stimulating an immune response in the patient, which would be of particular concern if the product were to be injected subcutaneously. These factors should be evaluated and addressed in the early-development phase if plastic ampuls are selected as the product containers.   

 1. S.J. Shire, "Stability Characterization and Formulation Development of Recombinant Human Deoxyribonuclease I [Pulmozyme (Dornase Alpha)]," in 

Formulation, Characterization and Stability of Protein Drugs: Case Histories

, R. Perlman, and Y.J. Wang, Eds. (Plenum Press, New York and London, 1996) pp. 393–426. 

The authors give special consideration factors affecting blow–fill–seal technology.

Biopharmaceutical Manufacturing Using Blow–Fill–Seal Technology

For several years, Big Pharma has been cutting programs and reducing headcount as a result of mergers and acquisitions and overall cost-cutting efforts. According to industry estimates, approximately 50,000 pharmaceutical industry employees lost their jobs in 2010, and 300,000 have been let go in the past 10 years (1). Many reasons are behind these cuts: a decline in traditional research and development productivity, tougher regulatory hurdles, a plethora of government and formulary pricing controls, and significant patent expiry of major blockbuster drugs.  

Now, more than two years post the economic meltdown, the industry understands that prosperity is not around the corner. What many call the "New Normal," characterized by slower growth, tighter credit markets, a more conservative approach, less money available for investment, and tighter financial regulations, has become the new operating reality. 

It doesn't have to be all doom and gloom, however. Even in the best-run organizations, there is an untapped source of cash from what I like to call "creating your own stimulus by buying items and services better." In many companies, the cost of purchased items and services (i.e., the money paid to suppliers) is equal to between 30–70% of total company revenue. In the pharmaceutical industry, this cost is typically between 30 and 50%. What I've come to understand is that regardless of how good or how bad a company is at buying products and services, there is still plenty of cash to extract from its costs. Three methods—spend segmentation, better specifications, and online reverse marketplaces—can improve results. 

. By not applying the appropriate sourcing strategy, a company may pay between 10% and as high as 70% more for products and services, based on my experience. This differential exists because of the strategy or lack of strategy that is in place for a certain purchase. There are four types of supplier relationships that may be classified by such attributes as ease of buying, supply-market competitiveness, uniqueness, and strategic importance. These strategies may be defined as "critical," "strategic," "acquisition-oriented," and "leverage-based." A buyer's strategy needs to be in the right category to ensure that a company is paying the right price, producing the right total cost, and getting the best value associated with a particular product or service.  

For example, if a company is making a purchase where the supplier maintains the power in the relationship (i.e., "critical"), the buyer is paying an absolute premium. Typically, less than 10% of a company's spend will be designated as "critical", according to my experience. Companies with poor purchasing practices, however, often allow suppliers to act as if they were a natural monopoly. A purchaser should review its spending to move supplier relationships from the "critical" position to "strategic" or better yet to "leverage" positions. My experience suggests that on average, only 20% of spend is truly "strategic," and most of what is purchased can be highly leveraged among multiple suppliers.  

Many factors determine how much to pay for a good or service. In simple terms, supply is what is available, and demand is how much is being requested. Consumption is how much is being used at what rate, and specification is what constitutes a product or service. The interactions of these variables are the main drivers of price paid.   

Once spending is properly segmented, four focal points determine the price paid. A company's strategy affects its ability to pay less, to use less, to use something else, or not to use at all. Take a close look at specifications and carefully review the requirements. In many cases, a company will find individual requirements that are "over spec'd" or not needed at all, which lead to significant and unnecessary costs. Additionally, consumption can often be reduced by making the user community aware of the total costs involved and by policies that control overall usage, particularly for indirect items and services.   

Think about how a company buys. It is probably one of the following scenarios. The simplest is calling the supplier and accepting the price being quoted. Then there is the "haggle" method, same as the first method, but with some pressure applied to get a lower price. Then more sophistication with the "competitive bid policy," in which the buyer typically gets three bids before making a selection. Another method is to do additional negotiation after getting the three bids, an interesting approach, but it still may fall short in securing the best possible terms.   

A reverse auction, a transparent and dynamic bidding model, is different. All sellers are bidding on the exact same item or service. When using this method, the buyer must have good specifications or requirement documents available to all sellers as opposed to the haphazard way requirements are given using some of the other traditional methods. Because the buying event has great transparency, sellers can see if their pricing leads or lags. The buyer does not have to select the lowest bidder and may weigh other factors in the purchasing decision. Reverse auctions drive pricing to what the market will bear at any given time against various seller independent variables. 

Until recently, the technology required to conduct reverse auctions was costly, required considerable employee training, and presented certain internal and some external barriers for implementation. These barriers, however, are eroding. The premier reverse auction marketplace, FedBid, used by the federal government for 10 years, is now available for commercial use and offers a procurement model for commodity products and basic services. The marketplace has over 50,000 qualified suppliers. More than 50,000 "buys" were conducted in 2010 with savings ranging between 5 and greater than 50%. There is no cost to the buyer for using the technology,  which is web-browser-based. The winning seller pays a modest 3% transaction fee and is billed by the marketplace operator 60 days after the buying award is made. If the product is not currently in the marketplace, the marketplace operator will, at no charge, find the right sellers and even train a company's existing suppliers so they can compete on a regular basis in the marketplace. Reverse auctions can be good for buyers and sellers. Buyers save money, and sellers gain more opportunities to win business. Reserve auctions, along with the other sourcing practices discussed, are potential drivers for value creation. 

is CEO of Wildfire Commerce, and senior advisor for A.T. Kearney Procurement and Analytic Solutions, tel. 215.327.5739, 

 1. E. Teichert, "Top 10 Pharma Layoffs for 2010," 

Perspectives on Outsourcing

Better strategies and practices in sourcing and procurement can contribute to the bottom line.

Offsetting Budget Cuts

FDA is working to develop guidelines for documenting the similarity and interchangeability of copycat versions of biotech therapies, a process that is drawing considerable scrutiny from a broad spectrum of manufacturers. There are a host of thorny issues to resolve related to reference products, interchangeability, labeling, and exclusivity. One area of agreement is that producing biosimilars is vastly more complex and costly than manufacturing conventional generic drugs, and that FDA will require broader testing to license a product as biosimilar or interchangeable. Developing a follow-on biotech therapy will require considerable investment in time, resources, and expertise, running up a tab of some $75-$250 million and typically taking seven to eight years, according to Ameet Mallik, global head of Novartis's Sandoz biopharmaceutical division, during an investor call in May 2011. 

Even so, the prospect of biopharmaceuticals capturing a greater share of the global drug market is drawing interest from many pharmaceutical and biotech companies. The worldwide market for biosimilars is projected to grow to $3.7 billion by 2015, up from about $250 million last year, according to Datamonitor. As more biotechnology product patents expire and regulatory authorities clarify requirements, biosimilar development is projected to soar. Leading pharmaceutical companies, such as Novartis and Merck, aim to be major players in the field, and even innovator biotech companies like Amgen are eyeing the follow-on market.  

FDA officials are under pressure from Congress, government agencies, and payers to develop an abbreviated regulatory pathway for approving biosimilars, as authorized by the Biologics Price Competition and Innovation Act (BPCI), which was approved as part of the Affordable Care Act (ACA) of 2010. The legislation aims to reduce spending on prescription drugs by permitting "highly similar" versions of biotech therapies to come to market based on less-extensive nonclinical and clinical data than was required to approve the original reference drug. Manufacturers talk of marketing biosimilars at discounts of 25–30% off branded products, but reimbursement experts say that prices that are just 10% lower will be able to gain market share, particularly for therapies that have high price tags to start.  

BPCI gives FDA the task of establishing a process that will encourage development of biosimilars that meet all standards for product safety, purity, and potency. No "clinically meaningful differences" should exist between the new biologic and the reference product. Both should  use the same mechanism of action and have the same route of administration, dosage form, and strength. A biosimilar also has to be manufactured, processed, packaged, and stored in facilities that meet GMP standards.  

FDA began the formal guidance-development process for evaluating biosimilars at a November 2010 public meeting. Janet Woodcock, director of the Center for Drug Evaluation and Research (CDER), recently indicated that general guidance should appear before the end of 2011, and that requirements for human testing will vary according to how well a biosimilar can document similarity to the innovator product.  

FDA also is moving forward with a user-fee schedule for biosimilars. The agency proposes to set fees similar to those for innovator drugs and biologics, but will take a new approach to help finance FDA's extensive involvement in steering sponsors through the development process. The plan is for manufacturers to pay a product development fee of $150,000 with submission of an investigational new drug application and annually thereafter; those upfront payments would be subtracted from the eventual application fee. Performance goals for approving applications would apply to applications submitted within two years of the expiration of exclusivity for the reference product, but not to applications for products that have years to wait before they can enter the market.  

Follow-on versions of complex biologics require extensive expertise.

BioPharm International-07-01-2011