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Spectrophotometric Equipment for Microbial Fermentations

BioPharm International, May 2011 Issue (PDF)

Editor's Note: Part I of this two-part series ran in the March 2011 issue of BioPharm International. That article put forth the following premises: 

Organizations that recognize quality by design (QbD) as a necessary component of an integrated enterprise-wide strategy will have a distinct competitive advantage over those who do not. Competitive advantage potential is not confined to manufacturing, but rather, it permeates all critical aspects of enterprise performance from research and development (R&D) through commercialization. The broad  cross-functional implications of effective integration and the downside potential of ineffective integration and functional coordination make leadership by executive management essential. Functional managers who understand the science and technical implications must be able to translate that knowledge into the language of financial, operational, marketing and strategic advantages that is the

 of CFOs and other top leaders. In short, managers must be able to develop a business case. This article discusses that general business case in more detail. 				

A business case for implementing a QbD approach essentially involves development of a cost-benefit analysis encompassing the following: 					

Organizational readiness to capitalize on the emerging compliance paradigm

Clearly defining how QbD reduces risk and cost for the enterprise (i.e., product pipeline versus the organization's historical cost and speed profile).

Beyond the general business case lie the challenges of engaging the larger organization in undertaking and developing the specific business case for the company's particular situation and embarking on a QbD initiative.  Those challenges include:   					

Enrolling management in a more rigorous assessment of costs and benefits 

Developing a phased, integrated path to organizational competence

Recognizing and achieving new paradigms in cross-functional coordination.

By making the general business case for QbD and mobilizing executive management to undertake its careful consideration, you can not only help demonstrate to management the way to untapped business value but also greatly increase the scientific rigor and competence of the organization's technical functions.  

ELEMENTS OF A QBD COST-BENEFIT ASSESSMENT

For the purposes of this article, we are defining QbD as the submission to FDA of a design-space based validation as opposed to a traditional three-batch validation. The assessment that follows, therefore, involves gauging the current state of the organization in terms of potential financial benefits of implementing QbD and the potential costs to realize those benefits. 

The objective is to achieve a strategic competitive advantage in the marketplace. Defining and implementing such a strategy will require leadership from the highest levels of the organization. Any assessment must, therefore, rigorously address the fundamental concerns of top management across all functional silos.  

There are many who would argue that the strategic benefits of QbD are self-evident and, given the trend in industry and FDA's endorsement to support the approach, organizations ought to make the transition as quickly as possible. Be that as it may, it is still incumbent upon management to determine the cost and benefit of such a transition if for no other reason than financial planning.  

The following section outlines some major factors to consider when estimating potential costs, benefits, and return on investment (ROI) with a QbD approach. Given the potential change-management issues that may arise during assessment, it is recommended that a  cross-functional governance structure involving upper levels of management be put in place to direct the effort.  

Too often QbD benefits are described in broad, idealized, hypothetical terms. This is exactly what we don't want to do if we are committed to rigorously addressing the fundamental concerns of top management and enrolling them in leading a transformation. Specifically, both the potential financial benefits and the potential costs to position the organization to effectively execute against a QbD strategy need to have sufficient rigor to obtain the buy-in of the finance team. Regardless of perceived benefit from the technical wing of your organization, if the CFO isn't on board, any effective integration of QbD into the organization's overall strategy and its associated organizational transformation impact is jeopardized before it begins. 

A credible current-state analysis to identify and quantify potential financial benefits needs to involve an analysis of the organization's historical performance vis-à-vis the management of risk and life-cycle costing. A candid, forensic assessment tracing current products through research and development (R&D), scale-up, technology transfer, and commercial production will identify cases in which unexpected delays and costs were incurred which could have been avoided if the organization had been employing a QbD strategy.  

Essentially, this is an estimate of the cost of poor quality (COPQ) associated with the organization's historical pattern of deviations and corrective actions but, unlike traditional COPQ assessments, the scope of the analysis is expanded beyond post-commercialization issues in production to include costs associated with regulatory filings, process development, and technology transfer early in the process of product development. 

Products currently in development or undergoing technology transfer provide good examples for examination because these types of changes and actions are fresh in the minds of those involved. 

The forensic assessment described herein need not capture every event in every product's life cycle to provide a credible estimate of opportunity. A few examples in which cost can be reasonably estimated can be more effective than many loosely quantified anecdotes.  

Failure Modes and Effects Analysis (FMEA), in combination with COPQ, can be particularly helpful as well to extrapolate and estimate potential risks and associated recovery costs which could reasonably be avoided if the organization were to integrate QbD into its overarching competitiveness strategy.  

FMEA is applied prospectively to an organization's product pipeline and late-stage development activities and considers the expected complexity of formulas/processes and projected regulatory filing requirements. Historical performance patterns established in the COPQ phase of the assessment inform the probability, detectability, and impact of failure in this FMEA exercise. 

ASSESSING POTENTIAL COSTS ASSOCIATED WITH IMPLEMENTATION

Running parallel with the benefits assessment is the assessment  of potential cost in the cost-benefits analysis. The same issues apply relative to building a credible estimate to enlist top management, particularly finance, in the opportunity QbD presents. 

The assessment of cost is more challenging to nail down than the estimate of benefits, however. When assessing benefits,  a company has the advantage of historical perspective of costs incurred due to delays and product losses in areas with well documented budgets.  

In assessing implementation costs, one  needs to estimate the cost of transitioning the organization from its current level of technical and organizational readiness to that which will be necessary to effectively execute a QbD strategy. 

The FMEA developed in the benefits assessment can be useful in assessing potential costs as well.  Identifying mitigation strategies to address potential failure modes and risks identified in the FMEA will, in turn, highlight gaps in organizational readiness and design for which corrective action costs can be estimated. 

Figure 1 illustrates the major considerations in functional coordination associated with mitigating risk and executing a transition to a QbD strategy. Each organization will be at different places on the continuum of readiness to implement these elements and each has its own associated costs. 

Figure 1: Cross-functional coordination involves support across all divisions.			

Considerations include not just the gap in technical know-how, but also issues related to change-management and the speed with which the organization can make the transition. Organizational transformation to adopt QbD will probably involve changes in organizational design and in  cross-functional interactions. Such changes can encounter strong resistance by those attached to the 

Temporary outside assistance to smooth these issues and facilitate change may help accelerate transformation and reduce cost. Particularly complex products or processes may require extensive experimentation and more time in development to adequately characterize the process and present acceptable data. Inexperience both internally and within FDA may cause delays in first-time QbD-based submissions, a potential cost that should not be overlooked.  

Finally, upgrades of software and data-management systems may be required to allow integration of historical data and to facilitate cross-functional coordination. These potential costs need to be considered as well. 

In the weighing of costs and benefits, it has been our experience in working with many life sciences companies that the organization-specific benefits of QbD almost always far outweigh the costs, yielding an ROI that continues to grow year after year. 

However, the great magnitude of the potential benefits does not mean that the organization should immediately undergo massive transformation, a giant step that many leaders and decision-makers may be unwilling to take up front. Instead, one can adopt a phased approach that allows the company to move confidently toward a fully integrated QbD strategy. 

Organizations can begin by building technical competence in basic process characterization, designed experiments, and hypothesis testing ahead of full scale QbD-submission preparation. Once those methods and techniques have been mastered, the organization can undertake QbD process development but also can continue to file with FDA in the traditional way. In the final phase of development, a company can embark on QbD-based filing. 

It is important to understand, however, that there is a balance which needs to be achieved. Yes, there are costs and risks associated with organizational transformation but the full benefits of QbD will not be realized until the final phase, in which filings are fully QbD-based. Organizations that evolve slowly will reap some benefits along the way but will remain at a competitive disadvantage to those with a more aggressive transition strategy.

  is a managing consultant at Tunnell Consulting, 

Compliance Notes

A rigorous cost-benefit assessment can help to chart a cost-effective path forward.

Quality by Design: The Case for Change (Part II)

Current purification of monoclonal antibodies (mAbs) as therapeutic agents relies on protein A affinity chromatography as one of its key process steps (1–4). The rapidly growing market for this class of high-value therapeutic agents makes its production process an important target for improvement. As cell lines have been engineered to produce increased titers of mAbs, the actual purification process increasingly becomes a bottleneck. 

Protein A resins have greatly simplified the mAb recovery process but they are expensive, often costing millions of dollars, and their current binding capacities are insufficient to meet the needs of cell lines producing higher titers of mAbs. Currently used protein A resins are made of recombinant protein A or recombinant multiple Z–binding domains comprising derivatives of protein A that are both recombinantly produced by bacterial fermentation, purified and then chemically cross-linked to premade agarose beads. The implementation of multiple and laborious steps in the production process of protein A resin is reflected in its high retail price. The current manufacture of protein A resin inherently contributes to its limited performance. The chemical cross-linking of the protein A ligand to agarose leads to a random orientation of the ligand, and hence a loss of accessible binding sites, and a reduction of the mAb binding capacity. The protein A sepharose as porous affinity resin requires diffusive flow of the mAb solution that, in turn, increases the residence time of the mAb. Increased processing time not only impairs the economics of the production process but also facilitates enzymatic breakdown of mAbs (5).  

COST-EFFECTIVE, ONE-STEP PRODUCTION OF NOVEL PROTEIN A–BASED RESINS

A new expression platform technology has been developed for the one-step production of protein A–based ligands already cross-linked to polyester beads. 

 has been successfully engineered to manufacture polyester beads that display multiple copies of the protein A–derived and antibody-binding Z domain (6). This new technology harnesses the natural polyester storage granule formation process inside bacteria for the production of ligand coated polyester beads (7) (see Figure 1). The polyester synthase (PS), which mediates polyester synthesis and bead formation, remains covalently attached to the polyester bead surface. PS was engineered to efficiently display multiple Z domains. A hybrid gene encoding the PS–Z domain fusion protein was constructed and introduced into 

 genes mediate conversion of the central metabolite acetyl–CoA to the activated polyester precursor, which is converted to polyester beads by the activity of the PS–Z domain fusion protein. This genetically engineered 

 enabled efficient production of polyester beads displaying the PS–Z domain fusion protein at high density and functionality. These polyester beads (PolyBind–Z) were extensively analysed using gas chromatography-mass spectrometry and denaturing gel electrophoresis combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). Initial performance testing using Enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and batch purification combined with protein quantification revealed a specific and high binding capacity for human immunoglobulin G (IgG) and various mAbs (6–8).  

Figure 1: Transmission electron microscopy image of engineered E. coli showing accumulation of intracellular polyester beads (PolyBindâZ).			

Current commercial technologies for the manufacture of protein A resins require the fermentative production of protein A or its derivatives, the production of a carrier (i.e., agarose beads), and ultimately chemical cross-linking of the target protein to the carrier. This usually leads to random orientation of the protein accompanied by a decrease in specific activity/function, and requires the tedious removal of the often toxic cross-linker. Homogenous functional orientation and elimination of the need for cross-linker removal suggest further advantages of this new polyester bead-based technology. The upstream and downstream processing (see Figure 2) along with the superior functional performance of PolyBind–Z in high affinity bioseparation of antibodies will be discussed.  

Figure 2: Upstream and downstream production schematic for PolyBindâZ.			

OBTAINING HIGH CELL DENSITY CULTURES FOR ECONOMIC PRODUCTION OF POLYBIND–Z

Commercialization of biopolyesters and biopolyester-derived products has been slow because of the excessive cost of production. However, production of biodegradable polyesters as commodity products has become economically feasible due to a significant amount of applied research and engineering on bacterial production strains and fermentation methods, allowing high cell density 

 fermentations up to 190 g/L dry cell weight (7, 9–11).  Today, 

 genetically engineered to overproduce biopolyesters as renewable commodity plastics have been reported to accumulate polyester levels approaching 80% of the dry cell weight.  Further, volumetric productivity for commodity plastics using commercial technologies has reached levels above 3 g/Lxh.   

Here, the authors describe a new platform technology that uses biodegradable biopolymer beads as the carrier for the presentation of a functional PS–Z fusion protein, increasing the complexity of the product. Given that this platform technology incorporates functional proteins presented on the surface, productivity assessment of the PolyBind–Z beads inherently includes functionality. Initial fermentations produced low yields of PolyBind–Z beads (25%–30% of the dry cell weight) that displayed IgG binding functionality in the range of 30 mg/g of drained beads. To increase 

 cell density, productivity, and functionality of the PolyBind–Z beads, the fermentation conditions, as well as the IgG binding fusion protein, were examined and modified.This resulted in a scalable industrial fermentation process for high-performance PolyBind–Z. Using a fed-batch strategy and incorporation of a defined media lacking animal products, the volumetric productivity of the PolyBind–Z beads has reached levels of approximately 50% when compared with commercial commodity bioplastics production processes. Further, the specific IgG binding functionality (discussed below) of the PolyBind–Z beads was also significantly improved to 100 mg/g of drained beads.   

While significant advances have been achieved in the production of highly functional PolyBind–Z beads, the current production process holds the promise to be further optimized.  To increase the productivity of our system and approach levels obtained by the commercial commodity bioplastics industry, additional fermentation optimization studies using quality-by-design are underway. 

CURRENT DOWNSTREAM PROCESSING: LYSIS AND REMOVAL OF IMPURITIES

Although this platform technology has allowed the development and production of tailor-made highly functional biobeads, a substantial practical and financial hurdle must be overcome. As with many products generated that use microorganisms in their production process, the intracellular polymer or protein must be extracted and purified from the host cell components by an economically feasible process.  Fortunately, there are a number of scaled extraction technologies currently used by both industries that can be applied to the extraction of PolyBind–Z beads. These technologies include both mechanical disruption and chemical/enzymatic extraction. However, in contrast to the commodity bioplastics industry, extraction and purification of the PolyBind–Z beads requires retention of protein function.  Inherent to the platform technology is a significant proportion of covalently linked surface protein. As a result, the chemicals/solvents applied by the commodity bioplastics industry to extract and purify biopolyester, such as chloroform, butyrolacetone, and sodium hypochlorite, are not viable with this platform; such chemicals would damage the functional proteins displayed on the surface of the biopolymer beads. However, incorporating some of the advances in industrial scale cell disruption and extraction techniques, we have developed a method that uses several different technologies to efficiently disrupt 

Once the PolyBind–Z beads are extracted from the 

 a stringent purification process is required. Given the resin purity required for the mAb production process, purification of the PolyBind–Z beads to remove host cell proteins (HCPs), nucleic acids, and endotoxins is critical.  Informed by already designed and industrially-used protein inclusion body purification processes, we have developed scalable purification steps to remove the majority of the HCPs, nucleic acids, and endotoxins (see Table I). The impurities and contaminants are continuously monitored using sensitive biopharmaceutical standard methods as we further develop the purification process to reduce the impurity levels associated with the beads to levels acceptable by the industry (12).  

Table I: Current PolyBindâZ impurity profile.			

POLYBIND–Z: AN UNUSUAL PROTEIN A RESIN AND ITS APPLICATION PERFORMANCE  

For decades, the biologics industry has relied on packed-bed chromatography as the gold standard for antibody capture. Well-known, understood, and characterised materials and processes have enabled antibody manufacturers to manage the capture step of antibodies with predictable outcomes. The primary issue with traditional protein A sepharose media is that it is costly, requiring multiple cycles to reduce the overall cost contribution of the media to the final product. The need for these additional cycles has further complicated the process by requiring the introduction of wash, regeneration, sanitization, and validation steps to bring media costs to an acceptable level. Standard, packed-bed chromatography operates with pressures in the 0.1–0.4 MPa range, which is enabled because of the size, rigidity, and porosity of their solid supports. Typical commercial offerings are composed of beads that are usually >100 μm in diameter and that allow sufficient interstitial space for adequate flow rates. Balancing the high flow rate of larger particles and maintaining sufficient binding capacity is achieved by managing the porosity of the solid supports, effectively increasing surface area as well as increasing binding times due to diffusion of the target into the pores.   

The functional beads can be delivered in a variety of formats in addition to bead slurries, based on initial experimental data One goal of our work has been to process the beads in such a way that their morphology is controlled, avoiding some of the limitations of the basic small particles, such as  unsuitability for packed-bed columns or expanded-bed chromatography. In experiments, we processed the particles into fibres to be used as coatings, or cast into membranes. This work opens up a range of potential formats for the material for use alone or in combination with other materials, and processes. In addition to the use of the material for capture of antibodies, the same material could be used to remove impurities, or to catalyze reactions through the surface display of other binding proteins or enzymes, respectively.  

We have developed simple batch purification protocols that allow the basic beads to be used in research or small-scale settings. Because of the surface display of the ligands, target binding is rapid and efficient, typically binding greater than 90% of the antibody in less than 30 seconds. This demonstrates the advantage of convective interaction over the current diffusive flow processes. Centrifugation at low speeds, <5000 x g, allows the beads to be resuspended and washed.  A disadvantage in packed-bed chromatography, the beads' compressibility is an advantage when used in Eppendorf tubes because after binding, washing, and antibody release from the beads, they can be spun down (10,000 x g) to a point where they do not readily resuspend, allowing removal of the supernatant without aspiration of any beads.  

For commercial-scale application, we have seen early promise with the beads in a cross-flow filtration system. Initial experiments indicate this approach would be suitable for production- scale application of the particles in the capture of antibodies to produce a truly disposable harvest format.  

Figure 3A: Static Immunoglobulin G (IgG) binding capacity (Eluted IgG in mg/g of drained beads). PB represents PolyBatics PolyBindâZ, 1 and 2 represent commercial protein A agarose beads. Binding capacity is in mg of human IgG/g of drained beads.			

The engineered beads exhibit both high binding capacity and excellent specificity, with beads routinely exhibiting binding capacities in excess of 100 mg/g of drained beads (see Figure 3A). This level of binding represents a doubling of the published binding capacities of common commercial offerings, and is an artifact of the combined high surface-to-volume ratio and engineering of the fusion partners to exhibit more of the binding domains at the surface of the particles. We demonstrated the relative binding capacity of the PolyBind–Z versus two commercial offerings (see Figure 3A). In one example, we used 50 mg of drained PolyBind–Z to bind 5 mg of human IgG equating to a total binding of 100 mg/g drained PolyBind–Z beads (see Figure 3B).  

Figure 3B: Commassie stained polyacryamide gel of unbound and elution fractions from a representative static hIgG binding experiment. 5 Î¼g of purified huIgG (1 mg/ml) was incubated with 50 Î¼g of either PolyBatics control beads (Lanes 3 and 4), Protein A sepharose beads (Lanes 6 and 7) or PolyBindâZ beads (Lanes 9 and 10) then eluted with equal volumes of a low pH buffer. Equal volumes of sample were loaded into each lane. Lane 1: Biolabs Ladder; Lane 2, 5 and 8: Pure IgG (5 Î¼g); Lanes 3, 6 and 9 represent unbound fractions; Lanes 4, 7 and 10 represent elution fractions.			

In the case of the basic bead configuration, we displayed several of the Z–binding domains from protein A per engineered PS. It is also possible to combine specific binding domains, as one prototype combined the Z–binding domain of protein A with the GB1  binding domain of protein G to increase the range of IgG subclasses bound by the beads.  

The specificity with which we have been able to remove IgG from the product feed stream is as important as the total binding capacity. In the case of human plasma, the PolyBind–Z was able to capture the IgG while leaving behind high abundance proteins, such as albumin.  

POLYBIND–Z PRODUCTION COSTS AND DISPOSABLE USE

One of the value propositions we seek to develop is the overall cost reduction of using PolyBind–Z beads in antibody capture. It is estimated that 60%–80% of the buffer cost comes from cleaning of the purification resins. These costs are eliminated if disposable media are incorporated into the process. For comparison we modelled the PolyBind–Z as a single use resin in a cross-flow membrane system versus traditional packed bed methods (see Table II). What is not reflected in the relative costs is the cost of buffer preparation and storage. In the interest of consistency, we have only compared consumables and not the hardware required.  

Table II: Projected relative cost savings: PolyBindâZ used as a single use resin in a cross-flow membrane system versus a leading Protein A sepharose used in a traditional packed bed column system.			

We assume the cost of buffers and time saved in regeneration, sterilization, and validation represent the primary cost savings in the process. Fraud et al presented data comparing ion exchange resin chromatography to membrane adsorber chromatography (13). In their case study, they demonstrated that up to 95% of the buffer and water used for cleaning and sterilization are saved in disposable systems. Combined with other factors, such as smaller footprint for buffer storage, and shorter process times due to convective versus diffusive binding, the argument for shifting to a disposable system is compelling. 

is project manager, R&D, all at PolyBatics, Palmerston North, New Zealand. 

6. J.A. Brockelbank, V. Peters, and B.H.A. Rehm,  

13. N.Fraud, U. Gottschalk, J. Lim, and A. Sinclair, GREENBiopharma conference (2010). 

Features

The authors discuss an alternative to traditional Protein A resins.

Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification

If you, like me, were a science student as an undergraduate who had friends in the humanities, you had the inevitable discussion about the objectivity of fact versus the subjectivity of faith. The scientist would cite the rigor and reliability of the scientific method and the "truth" of reproducible results. The humanities student would argue that the so-called axiomatic truth of the sun rising tomorrow, for example, was just another form of faith. The scientist, argued the humanities student, was operating under the assumption that past behavior was a predictor of future behavior and so believed the sun would rise tomorrow merely because it had every day in the past for eons. The arguments are not very original, nor are they decisive, and yet they remain with me. Every so often during introspective moments, I subject myself to a sort of epistemological grilling: How do I really know what I think to be true? What data set am I using to reach my conclusions, and how was it generated? And finally, do I believe this to be true because I want it to be, because it's an act of faith or will, or is there really substantial—objective—support for this?  

 These are questions I revisit every year when we conduct our annual Bioprocessing Survey. (Thank you, by the way, to those who took the survey; and for those who did not, there's always next year.) The biopharmaceutical manufacturing sector has over the years been exploring the benefits of single-use, disposable components in its operations versus those of traditional stainless-steel components. Opinions about the relative merits of one kind of equipment over the other abound. And they diverge, depending on whether one actually uses disposables. 

 In many cases, users of all-stainless equipment underestimate the advantages of single-use and overestimate the challenges. For example, only 35% of all-stainless users (and 31% of those using hybrid systems–systems that combine stainless and single-use components) perceive a cost advantage to all-disposable systems as opposed to 54% of those who actually use all-disposable systems. In another example: only 18% of respondents who use all-stainless equipment think that process reproducibility is an advantage to all-disposable equipment. In contrast, 31% who use all-disposable cite reproducibility as an advantage. Users of all-stainless may also overestimate challenges to all-disposable manufacturing lines. Twenty percent of respondents who use all-stainless equipment think that all-disposable equipment increases the risks of contamination. No one—0%—of those who actually use all-disposable equipment does.  

The survey cannot determine how firmly held are these beliefs or what it would take to dispel them. But it's an interesting object lesson to me and perhaps to others who see science and scientists as purely objective.  

I had another reason to consider attitudes toward disposables in particular. During the INTERPHEX 2011 conference, I hosted a panel discussion around continuous manufacturing. The discussion focused on traditional small-molecule, solid dosage forms. I learned that one of the hurdles is finding an automated method for moving powders from one unit operation to another—the answer to which for some products may be to keep all the chemistry in solution. It then occurred to me that a completely soluble process would start to look a lot like the process for biopharmaceuticals and could therefore benefit from the use of disposable components. If that's the case, then the "objective truths" of a whole new group of process developers and engineers will have to be overcome. I wonder how our survey results will look then.

From the Editor

Those who doubt there's faith in science, should check out our annual Bioprocessing Survey.

Perception and Reality

By stripping waste and other non-value-added steps from the production process, lean manufacturing has saved companies billions of dollars. But why stop there? If a company is using traditional testing methods to release its products to market, the adoption of rapid microbial methods (RMMs) is one of the most important additions a company can make to its lean initiatives.  

Lean quality is achieved by extending the principles of lean manufacturing to microbial testing. RMMs are being adopted by increasing numbers of pharmaceutical companies around the world to reduce lead times while ensuring product safety. Implementing rapid methods can unlock huge savings by shortening microbial hold times to just 24 hours for microbial limits, and cutting sterility testing time by more than 50%. This translates into an average 5–year net present value savings of $500,000 or more per facility.  

Improved efficiency also reduces recovery time in the event of contamination. During production, when so much is at risk, the benefits of rapid methods are doubled. The faster that corrective action can be initiated, the faster recovery can occur, which reduces the potential impact on customer relationships and the bottom line.  

Products that are susceptible to microbial contamination but expected to be shipped contamination free are screened before being released into distribution. That screening adds multiple days to the production cycle and extends lead times. Microscreening typically takes 3–7 days for microlimits testing and 14 days or more for sterility. That's a long time to wait when there is a good production process in place that is turning out products free from bioburden almost all the time. The wait time is frustrating, and expensive. 

Well-validated, rapid methods are just as effective as traditional microbiological methods, only faster. Results can be delivered in as few as 24 hours using a rapid method featuring adenylate kinase (AK). AK-amplified bioluminescence combines the use of adenosine triphosphate (ATP), the gold standard of rapid detection methods, with a patented enzyme technology to deliver accurate results quickly. 

All living organisms contain the compound ATP as a vital part of their energy metabolism. In the standard bioluminescence test, when ATP is detected, a reaction occurs that generates a photon of yellow-green light, similar to that of a firefly. It is a very sensitive technique, but limited by the fact that an organism contains only a small amount of the metabolite ATP.  

Adenylate kinase (AK) is another vital part of energy metabolism. When supplied with an excess of ADP (adenosine diphosphate), AK acts as a catalyst, converting ADP into ATP. Because AK is an enzyme rather than a metabolite, it can be harnessed to generate almost unlimited amounts of its product. After 25 minutes, for example, the amount of ATP can be 1000 times greater than that which the organism originally contained. As a result of this amplification and the reduced dependence upon microbial growth for detection, AK-amplified bioluminescence provides faster results when screening for microbial contamination. 

A new alternative to bringing a rapid screening system in-house is to partner with a contract analytical lab that offers rapid detection as a service. With overnight shipping widely available, there's still much to be gained by being able to release products within two days. 

Look for an accredited lab that is certified in current good manufacturing practices.  Such labs will understand the demands of the regulated environment. Ask about their experience with validation services and, if there's a future possibility of bringing testing in-house, with method transfer. In addition to having a robust rapid screening system, the lab should be equipped to perform follow-up testing on any samples that test positive. 

Some laboratoies are capable of mapping organisms at the genetic level. This strain typing process uses repetitive extragenic palindromic sequence polymerase chain reaction (rep-PCR) to identify and track organisms. Because it can be used to catalog any mutations or seasonal changes over time, strain typing can be a significant benefit to facilities plagued by a recurring bug.   

As a growth-based method, AK-amplified bioluminescence has the advantage of being able to test products with a wide range of physical characteristics. This includes clear, opaque, highly pigmented, acidic, and viscous products, whether filterable or not. Both AK-amplified and traditional methods rely on batch or lot sampling, enrichment, and incubation. While the traditional method depends on laboratory technicians visually inspecting hundreds of samples one at a time over multiple days, the rapid method assays up to 120 samples in about one hour using a light-measuring instrument called a luminometer.  Results are objectively recorded and presented in clear, color-coded tables and graphics, so no "judgment calls" are needed to interpret the results. 

 Transitioning pharmaceuticals and other regulated products to a rapid release method is straightforward and is, in fact, encouraged by many global regulatory bodies, including FDA. A contract laboratory with experience in this method should be able to assist its clients in validating their product or group of products for routine release using a rapid method. This typically entails side-by-side testing to ensure the rapid method is as sensitive as the traditional method. Once this comparability protocol is completed and the validation data is submitted, the RMM is effectively implemented.  

A good RMM provider may have regulatory compliance expertise on staff, as well as drug master files (DMFs) accepted by FDA. DMFs include data for specificity, limit of detection, robustness, ruggedness, and equivalence, and can be used to supplement or streamline the validation of a company's rapid system. This may save significant time in both preparation of regulatory filings, and in FDA's review and approval process. 

is senior director of biologcial sicences at Celsis Analytical Services, St. Louis, MO,  

Rapid microbial screening provided by contract laboratories can save companies time and money.

Outsourcing Lean Quality Manufacturing

The pain and suffering afflicting innocent children from a rare genetic disease, and its conquest through biotechnology, would hardly seem to be a winning formula for a blockbuster at your local Cineplex. So it is surprising to see that this formula has been adopted twice in films with big-name casts and large budgets. The films' implications are important for the public's vision of modern medical science, especially at a time where science literacy among the public is said to be at an all-time low.  

 (Universal Pictures; Directed by George Miller) and 

 (CBS Films; Directed by Tom Vaughn) both depict families who attempt to discover and implement cures for their children's genetic diseases when they find no remedy in current medical science. Both are "based on actual events" and  both are beautifully filmed and edited. Both present a great amount of scientific detail and make a powerful case for the lavish support that medical research enjoys. Both have feel-good endings that, amazingly enough, turn out to be largely accurate.  

On the down side, both have a lot of contrived conflict situations, invented characters, juxtaposed events, and hyperbole. But perhaps the most interesting aspect of the films for those in the field of biotechnology is the acknowledgement of the tremendous progress biomedical science has made during the past two decades. 

, released in 1992, portrays the life of Augusto and Michaela Odone as they struggle to find a treatment for adrenoleukodystrophy (ALD), an X-linked recessive disease in which the myelin sheath covering the nerve axons deteriorates. Because of a mutation in the peroxisomal membrane transport protein, there is a failure of beta oxidation of long-chain fatty acids (VLCFAs) that accumulate in the circulatory tract, causing the loss of myelin and leading to fatal disease. The Odones discovered that a mixture of erucic and oleic acids (combined with dietary restrictions) could lower the serum levels of the VLCFAs. While the clinical benefits of the treatment were in doubt initially, long-term studies have confirmed that the oil can indeed prevent, but not reverse, some damage caused by the mutation. 

In the coda of the film, a group of young boys are shown with a voice-over claiming that they benefited from the oil. At that time, there was no solid evidence to support this claim. Miller, the director, was criticized for playing "fast and loose" with the facts. But later, long-term studies seemed to back the claims of the therapy's benefit. 

There is another fascinating epilogue to the story. Hugo Moser, the father of ALD research, wrote a critical review of the movie in which he took Miller to task for introducing a non-existent controversy between researchers and the ALD parental support group. However, in later writings, he emphasized the positive effects of the oil treatment, based on long-term studies. 

 hit the theaters. It received mixed reviews and did not do well at the box office. This film portrays the Crowleys, a real-life couple who, once again, buck the medical establishment in an effort to save their children from the ravages of a rare genetic disorder, in this case, Pompe's disease. Harrison Ford plays an eccentric and irascible molecular biologist, Robert Stonehill of the University of Nebraska, who was patterned after the real-life investigator, William Canfield of the University of Oklahoma Health Sciences Center.   

Much of the film was shot in Portland and Seattle, even though Canfield's company, Novazyme, was based at an incubator at Presbyterian Health Foundation Research Park, in Oklahoma City. Crowley joined Novazyme in March of 2000 as CEO and pursued the research that eventually played a role in the successful therapy of the disease. In 2003, the affected Crowley children received the enzyme replacement therapy for Pompe disease developed by Genzyme. The children are still on respirators today. Crowley and Canfield sold the company to Genzyme in Boston in 2001.  

Crowley is now the CEO of a highly successful biotechnology company that specializes in drugs that cause errant enzymes to fold properly. 

 was made at a time when the human genome, routine gene cloning, knockout animal models of genetic disorders, and microarray screening did not exist. The Odones' discovery was not based on current technology and their experimentation on human subjects would be inexcusable today. Their work, however, was credible as a literal "mom and pop" therapy. 

Today's high-tech universe in which one can manipulate molecules based on three-dimensional computer models, gene identification and cloning is rapid and routine. In addition, human-subjects committees and ethical guidelines for clinical trials are all pervasive have greatly improved?.  

Future breakthroughs in the treatment of genetic disorders will come from big laboratories and legions of scientists in white lab coats. Although an angry loner battling the system provides a much more romantic plotline than an executive who works within the framework of the establishment to achieve his goals, there are legions of genetic disease waiting to be cured and their obliteration will be not documented on the big screen. But this industry's work and achievements will continue to save lives.

, president of Newport Biotech Consultants in Newport, KY, 

Final Word

Two films portray, in surprising detail, the hard work and determination of the biopharmaceutical industry.

Big Science Hits the Big Screen

Drug-development pipelines have shrunk; fewer new products are being approved for market; and the prospect of significant declines in revenues due to the looming "patent cliff" is prompting pharmaceutical companies to scale back research and development (R&D). Pfizer rocked the industry in February by announcing a major cutback in R&D spending, including plans to shutter its long-time research facility in Sandwich, United Kingdom, and to reduce its Groton, Connecticut, research facility. As part of a move to cut its $9-billion R&D budget to some $6-7 billion by 2012, Pfizer is moving antibacterial research from Groton to Shanghai, China, and will use its Cambridge, Massachusetts, research operation to form more links with small biotechnology firms in the area.   

The crisis in the pharmaceutical industry is generating a serious search for new business models. Manufacturers are looking to partner more with small biotechnology firms and academic research institutes, to shift research and production operations overseas, and to streamline operations and reduce waste wherever possible. The National Institutes of Health (NIH) proposes to ramp up support for translational medicine that will shepherd basic research through the R&D "valley of death" to yield new therapies. Patient advocacy groups are consulting with and providing funding for public and private therapy development programs. America's position as the world leader in biomedical R&D is "under siege today," and facing its biggest threat in 65 years, commented former Congressman John Porter, at a forum in March 2011, sponsored by ResearchAmerica. "Is America going to put progress on hold?" he asked in calling for decision-makers to consider the importance of science and innovation in making spending decisions. These trends are shaping many aspects of biopharmaceutical development, production, and marketing.  

Some of the blame for longer, more costly drug development falls on the shoulders of FDA. Stepped-up demand for more safety and efficacy data, prior to approval as well as after a drug comes to market, can add to development costs, and ultimately weaken less robust pharmaceutical R&D programs. Moreover, success rates remain notably low for new drugs in clinical development, despite years of efforts to better inform the clinical-research process to avoid wasting millions of dollars on unsuccessful studies. The Biotechnology Industry Organization (BIO) reported in February that only one in ten new drugs make it from Phase I studies to FDA approval, based on an analysis of thousands of drug-development efforts from 2003 through 2010.  

Most disappointing is an actual decline in new drug approvals by FDA last year, a troubling shift after two years of slight increases. The agency cleared only 21 new molecular entities (NMEs) in 2010, down from 25 in 2009. Even more discouraging are reports that fewer applications for innovative new therapies were filed with FDA last year, squelching optimism about an upturn in product approvals in the near future. Yet, FDA is caught in a hard place, as patient advocates demand earlier access to promising therapies, while policymakers and consumer groups insist on more scrutiny of test products to better detect potential safety problems.  

The approval downturn kept several highly touted experimental products from the market. Cardiovascular safety issues prompted FDA to reject new diabetes therapies and kill several new weight-loss drugs, while also pulling Abbott's Meridia product from the market. More recently, a promising fast-acting inhaled insulin product (MannKind's Afrezza) was put on hold following FDA requests for more data on product usage and safety. The agency drew heat for turning down an application from Cell Therapeutics for pixantrone, a treatment for non-Hodgkin's lymphoma that showed some efficacy, but not enough for FDA approval. Cancer advocates continue to oppose a move by FDA to narrow its approved indication for Avastin, insisting that the benefits outweigh new evidence of serious side effects. 

There are some bright spots amidst these disappointments. Last month, FDA approved a new treatment for melanoma, Bristol-Myers Squibb's (BMS) Yervoy (ipilimumab). The drug is the first to prolong lives of patients with this deadly skin cancer and a vast improvement over existing therapies. Even better for the manufacturer is an expectation that this new monoclonal antibody, which enlists the body's immune system to attack cancer cells, could lead to similar treatments for other cancers.  

FDA also made headlines earlier this year by approving the first new treatment for lupus in more than 50 years—Benlysta (belimumab), developed by Human Genome Sciences with support from GlaxoSmithKline. The drug's discovery relied on information resulting from human genome mapping and represents the first real success in this field after multiple failures. 

BMS cites its new cancer drug as evidence that an increased focus on pharma R&D can yield big dividends. "R&D pays," stated CEO Lamberto Andreotti in an interview with the 

 noting that the firm is investing research money very carefully in a range of disease classes and expects to have four more new therapies approved by FDA in another year. Merck similarly told Wall Street analysts in February that it's not cutting back on its $8 billion R&D spending plans, even though the decision may result in missing long-term profit forecasts.  

Last year's slim drug-approval list included several notable products. Amgen won approval for osteoporosis treatment Prolia (denosumab), and Roche's Genentech brought out Actemra (tocilzumab), an intravenous drug for rheumatoid arthritis. Boehringer Ingelheim won the race to bring to market a new blood-thinner Pradaxa (dabigatron), although others may catch up soon. Probably the most exciting new product was Dendreon's therapeutic prostate cancer vaccine Provenge (sipuleucel-T). Other new vaccines for meningococcal disease and pneumococcal disease also were approved by the Center for Biologics Evaluation and Research (CBER).   

FDA officials say that drug approvals are returning to former on-time schedules after sagging in recent years as the agency struggled to implement a host of new requirements established by the FDA Amendments Act (FDAAA) of 2007. Last year, the Center for Drug Evaluation and Research (CDER) began to benefit from staffing increases and regulatory clarification, which helped employees achieve review timeframes more steadily. Now the agency is moving into a "period of consolidation," says CDER Director Janet Woodcock, as it completes multiple FDAAA initiatives, negotiates a new Prescription Drug User Fee (PDUFA) program, and establishes a quality-management system for  more efficient 21

Woodcock reported in December 2010, at an FDA/CMS  summit, that the agency once again was meeting user fee timeframes for processing applications, particularly submissions for NMEs, and that the "big wave in missing goals is coming down." There also are more first-cycle approvals, a key benchmark for both sponsors and regulators, and the rate of first launches in the US is holding steady.  

At the same time, FDA is looking to improve the regulatory process in ways that encourage new product development. Combination therapies, for example, stand to benefit from draft guidance issued last December on codevelopment of investigational drugs used in combination, which is particularly germane to formulating new cancer therapies.  

The looming reauthorization of PDUFA in 2012 is prompting a re-evaluation of the agency's Risk Evaluation and Mitigation Strategies (REMS) program to meet industry concerns about too many diverse REMS formats; lead proposals are to have less burdensome controls for REMS that only require distribution of medication guides, and to devise common formats for such documents. FDA recognizes, says Woodcock, that REMS requirements should not delay product approvals.  

FDA-industry user-fee negotiations also seek to make presubmission meetings more productive and to support new strategies for streamlining product testing and application review. Discussions regarding generic-drug user fees, moreover, are moving forward. A main objective for FDA is to gain additional support for more timely plant inspections in the face of a notable rise in foreign sourcing of active ingredients as well as generic-drug production.  

Another area of focus is FDA's accelerated-approval process, which is designed to avoid delays in moving important new therapies to market. The system has been criticized because manufacturers often fail to complete agreed-on confirmatory trials in a timely manner, and some follow-up studies have shown limited efficacy and serious side effects, as with Roche's Avastin. FDA officials have proposed that sponsors launch confirmatory trials before the agency grants fast-track approval to ensure that additional studies are performed according to plan.  

Great difficulties in developing new drugs for broad patient populations, such as diabetics and the obese, are prompting collaborative efforts to better understand approval requirements. Woodcock and her staff recently met with a group of obesity experts to discuss standards for bringing weight-loss drugs to market. The scientists proposed that regulators consider the broader health benefits of weight loss, such as reduction in sleep apnea, in assessing potential side effects from drug therapy.  

FDA Commissioner Margaret Hamburg continues to stress the importance of advancing regulatory science in order for FDA to be able to support the translation of science into real-world therapies. New biomarkers for toxicology can identify drugs likely to fail much earlier in the process and also better target therapies to individuals most likely to respond, Hamburg noted. And innovative clinical-trial designs can yield answers using fewer patients and less money. The conventional thinking is that new discoveries from biomedical research will lead to new products. But, she explained at the ResearchAmerica forum, there is a regulatory science gap that can prevent new opportunities from coming to fruition.  

The changing biomedical research landscape and cutbacks in industry R&D programs are encouraging more public support for pharmaceutical research around the world. The Innovative Medicines Initiative in Europe is building a schizophrenia database of industry-sponsored clinical trials to better identify signals of patient response to test drugs. The United Kingdom's Medical Research Council has established the Developmental Pathway Funding Scheme to support basic research on drugs and medical devices, and the Wellcome Trust's Seeding Drug Discovery initiative is funding efforts to take drug candidates through early clinical trials. Both US and EU scientists are wary of being left behind by soaring Chinese investment in R&D.  

At home, NIH Director Francis Collins launched a high-profile campaign to promote translational medicine as a way to spur development of new medical treatments that can benefit patients. In December, an NIH advisory committee recommended establishing an NIH National Center for Advancing Translational Sciences (NCATS), a move engineered by Collins to bring together several NIH programs that provide resources for translating basic discoveries into new medicines and diagnostics. These include a program that supports development of therapies for rare and neglected diseases, along with NIH's national network of research sites at academic medical centers supported by Clinical and Translational Science Awards.  

As the former director of NIH's Human Genome Project, Collins is optimistic that new genetic discoveries can chart pathways for discovering new medical treatments, and that the emergence of more well-validated genes will be useful in "identifying drug targets in unprecedented numbers," he said in an interview. The scientific enterprise is yielding up a lot of new ideas about therapeutics, he observed, yet "traditional private sector efforts to capitalize on that are taking a hammering." NCATS aims to bolster the funding of research projects at a time when biotech and pharma companies face serious financial challenges. Along these lines, the initiative also will encourage more collaboration between academic researchers and biopharmaceutical companies and to strengthen ties with FDA to ensure that NIH-sponsored studies provide the data needed to support registration of new products.  

The project envisioned by Collins will help re-engineer the drug-development pipeline by investing in new assays that can screen thousands of molecules to find ones that will hit defined disease targets. Another objective is to improve assessment of toxicity, which may involve shifting from the use of animal testing to identify potential problems.  

As part of the process, Collins' translational science campaign aims to convince Congress and the American public that the federal investment in biomedical research can pay off in terms of new, life-saving therapies. The Obama administration has proposed a very slight increase in the NIH budget for fiscal year 2012, which would just barely maintain current funding levels. Even during the Republican budget cutting campaign of the mid-1990s, NIH retained several strong GOP advocates on Capitol Hill and largely escaped the chopping block; that kind of support seems to be lacking among current Republican leaders. 

Collins believes that today there is  greater private-sector interest in NIH-funded preclinical and clinical testing, as well as in compound rescuing or "repurposing." Pharmaceutical companies have long lists of compounds that have been abandoned along the way, maybe because a business plan changed or the money ran out or clinical trials failed to show efficacy, Collins notes. "We have been talking with leaders in the pharmaceutical industry about an opportunity to open the freezers and make such compounds available, with appropriate intellectual property protection for them," Collins explains. "This isn't a giveaway, but could be a win-win if such a compound were found to be active for a different application than originally considered." NIH will not move into drug development 

, Collins emphasizes, but will hand promising compounds off to private sector sponsors.

Washington editor, Chevy Chase, MD, 301.656.4634, 

Regulatory Beat

FDA, NIH, and industry seek new strategies to spur drug development and promote access to therapies.

Filling the Pharma Pipeline

The tragic earthquake and tsunami in Japan in March rocketed supply-chain issues to a prominent position. Even the most sophisticated supply-chain managers were caught off guard by the event, which caused the shutdown of many manufacturing sites due to earthquake damage or power-supply interruptions. What is striking about the impact of the Japan earthquake on the supply chain is the widespread production disruptions caused by supply interruptions of simple components. Automobile manufacturers were forced to shut down manufacturing operations around the world due to component shortages. Production of Apple's iPad2 was threatened by the shutdown of a supplier in northeastern Japan making a connector costing just a few cents.  

The automobile and electronics industries are known for their supply-chain sophistication, including their ability to source components globally and have them delivered just-in-time to the manufacturing floor. The fact that their production would be disrupted by such minor components speaks to just how complex effective supply-chain management really is. 

Reports so far suggest that the bio/pharmaceutical supply chain has only been marginally affected by the events in Japan. We have heard of only two interruptions. One was from a supplier of glycine, an ingredient in some common solid-dosage products, which indicated that production may be shut down for up to 24 months. We also heard of disruptions in some supplies of gelatin used in softgel capsules, for which alternative suppliers are readily available. 

Nevertheless, the bio/pharmaceutical industry has not been without its own supply-chain difficulties in recent months. A major fire destroyed a warehouse attached to Catalent's commercial packaging facility in Corby, UK, and problems stemming from glass-vial delamination resulted in a recall of many injectable products.  

The pharmaceutical industry has done surprisingly little to protect itself from supply-chain interruptions. PharmSource recently conducted a survey about a widely used component for solid-dosage drugs, which typically requires preformulation compatibility testing, and found that few companies have exhibited the foresight to qualify second or third suppliers. 

For component-assembly industries, such as automobiles or electronics that are relatively unregulated, the interruption in the supply of a minor component will be disruptive and relatively short-lived. In most cases, it could be a matter of weeks to redesign the component or have substitute suppliers step in for secondary supply. In the bio/pharmaceutical industry, however, the long-term interruption of a minor component could be catastrophic. Even a relatively minor excipient can cause changes in a product's characteristics, thereby requiring full preformulation analysis, stability testing, and testing of  production batches. Add to that the supplier qualification and regulatory-filing requirements, and the production time lost can be considerable.  

The bio/pharmaceutical industry's lack of supply-chain sophistication spans the entire length of the chain, from input sourcing, through production and inventory management, to distribution. At the front end of the chain, we need only be reminded of the heparin disaster of a few years ago to appreciate the weaknesses in quality assurance and supplier management for key inputs. At the distribution end of the supply, the bio/pharmaceutical industry has been under attack regarding counterfeiting and security of supply. A recent article in 

 magazine recounted last year's theft of $75 million of finished drug product from an Eli Lilly warehouse in Enfield, Connecticut (1). The article highlighted the lax security at the warehouse, including failure to monitor security cameras that could have caught the theft while it was taking place. 

Supply-chain management weakness also can be seen in the way the industry manages its inventories. On average, global bio/pharmaceutical companies turn their inventories a little more than twice a year. They typically have inventory on hand equivalent to about 180 days' worth of sale. By contrast, a best-practice consumer products firm, such as Procter and Gamble, turns its inventory about six times a year and has about 60 days' worth of inventory on hand. As global bio/pharmaceutical companies try to maximize their cash-on-hand to help weather the patent cliff and take advantage of licensing and acquisition opportunities, keeping so much cash tied up in inventory is a competitive disadvantage. 

The supply-chain management challenges facing the industry represent a major opportunity for providers of contract services, especially those in manufacturing and packaging. Because of their critical position relative to both the upstream and downstream segments of the supply chain, CMOs and packagers are well positioned to help bio/pharmaceutical companies manage their risk and squeeze cost and inventory out of the system. 

We have seen isolated instances of contract-service providers responding to supply-chain management opportunity (e.g., Patheon's recent efforts to promote its services as a backup source of supply and Almac's short-run packaging services). We've also seen major providers of third-party logistics services (3PL), such as UPS, make greater efforts to address the bio/pharmaceutical industry.  

However, the big opportunity will come when service providers figure out how to increase the flexibility and responsiveness of their own operations. For instance, every day that a CMO can knock off the manufacturing schedule lock-in requirement from its traditional three months can save its bio/pharmaceutical company clients millions of dollars in cash tied up in inventory. The internal process changes necessary to achieve those scheduling improvements are likely to deliver substantial cost savings and increased throughput. CMOs have traditionally focused on additions to processing technologies, equipment and gain-to-gain incremental volume. Given the growing supply-chain challenges, a focus on operations excellence in the context of supply-chain management is likely to yield greater performance for both the client and CMO and yield market share gains going forward.

 is president of PharmSource Information Services, Inc., Springfield, VA, 703.383.4903, 

http://features.blogs.fortune.cnn.com/2011/03/31/drug-theft-goes-big/

Outsourcing Insights

Supply Chain Pain

Biotechnology processes depend on key equipment to aid in process decisions. The lifespan and support period for equipment can sometimes come to an end in the midst of commercial production. When this happens, alternative equipment must be identified and qualified to replace the original equipment without impacting the process outputs. This article provides the approach used during a recent exercise to identify a replacement spectrophotometer used to make process decisions for microbial fermentations. Three spectrophotometer models were tested in these experiments. First, instrument and process linear ranges were determined for each spectrophotometer. Next, a calibration model (margin of error) was used to determine the accuracy and precision of each spectrophotometer. These results were used to select the replacement spectrophotometer. Finally, the selected spectrophotometer was evaluated in bench-scale fermentations in order to confirm the capability of the instrument to serve as a viable replacement.

Spectrophotometers measure optical density (OD) at a particular wavelength, and are used in microbial fermentation processes to monitor culture growth, make process decisions (e.g., initiate feeds and induce production at specified OD), and to measure performance parameters (final OD). With the current company-wide platform spectrophotometer (Model A) being phased out by the vendor, the authors set out to identify a replacement spectrophotometer for microbial fermentation processes. Ideal characteristics for the replacement spectrophotometer included the following: 					

An expanded measurement linear range relative to Model A.

Minimal to nonexistent bias relative to the OD output of Model A.

Similar or better measurement precision relative to Model A.

The second criterion was especially important because the replacement would then be "like-for-like," meaning that minimal updates to existing manufacturing procedures and process-related documents would be required. 

Spectrophotometer technical specifications

Three test spectrophotometers, manufactured by different vendors, were evaluated. Table I compares the technical specifications of the test spectrophotometers (Models B, C, and D) to those of the current spectrophotometer (Model A).  

Table I: Technical specifications of test spectrophotometers.			

Spectrophotometer models A and C have a tungsten/deuterium light source that provides continual reading of sample absorbance. Model C is the next generation model of spectrophotometer to Model A. Spectrophotometer models B and D use a xenon light source that does not continually read the sample absorbance, and operate by a press-to-read method. All spectrophotometers have a small footprint, which is desired because of space constraints in large-scale manufacturing areas. The normalized model A linear range is 0.2–1.6 dilution OD. 

National Institute of Standards and Technology (NIST) OD standards were purchased from GFS Chemicals in the 0.2–12.0 normalized OD test range. NIST OD standard solutions were used to determine the instrument linearity of each spectrophotometer. 

Culture broth samples were taken from bench-scale, fed-batch fermentations using an 

expression system. The process linearity experiment samples were frozen prior to testing to allow all samples to be analyzed at one time. All dilutions were performed using a saline diluent. 

Figure 1 shows the multiphase experimental strategy used to screen, select, and qualify the new spectrophotometer. The first set of experiments determined the linear range of each spectrophotometer. Linear range testing was performed in three parts, including: instrument linear range determination, spectrophotometer accuracy and precision testing, and process linear range determination.  

Figure 1: Spectrophotometer screening, selection, and qualification experiments. (ALL FIGURES COURTESY OF THE AUTHORS)			

The instrument linear range was determined by measuring NIST standard solutions over the normalized range of 0.2–12.0 OD, regressing the measured OD to the NIST OD standard values, and reducing the range until the statistical lack-of-fit test against a quadratic model had a 

–value >0.05. A calibration model (see below) was then performed using inputs from the instrument linear range testing to assess the accuracy and precision of each spectrophotometer in the measurement of NIST OD standards over the instrument linear range. 

Process linear range was determined by measuring a variety of dilutions of fermentation culture broth samples taken from multiple time points across the fermentation. The process linear range for each sample point was estimated by visual inspection of calculated actual OD (dilution factor times dilution OD) linearity when plotted against the dilution OD range. The output of these experiments led to setting an overall linear range for each spectrophotometer acceptable for use in microbial fermentation processes. 

In the second set of experiments, the accuracy and precision of spectrophotometer models B, C, and D were assessed in comparison to those of Model A using a calibration model. The inputs of the calibration model included culture broth dilution OD sample measurements from each spectrophotometer from multiple time points across fermentations. The output of the accuracy and precision testing was the basis for selecting the replacement spectrophotometer.  

Following spectrophotometer selection, bench-scale fermentations were conducted to confirm that process decisions based on the replacement spectrophotometer resulted in similar process performance as did the Model A spectrophotometer. 

The accuracy and precision of the spectrophotometers relative to either the NIST OD standard or to the Model A OD were assessed using a calibration model (1). This analysis was performed by regressing the measured value of a sample from a given spectrophotometer (Y) against the true standard value (either the NIST OD standard value or Model A value, X) (see Figure 2).  From this model, both the accuracy (bias) and the precision can be estimated for each spectrophotometer using a 95% confidence interval.  

Figure 2: Graphical representation of the calibration model.			

Figure 3 shows the regression of measured OD versus NIST Standard OD for each spectrophotometer. Using the reduced regression models, the instrument linear range was set for each spectrophotometer (see Figure 3). The instrument linear range of Models A, B, and C were comparable, whereas the linear range of Model D was reduced.  

Figure 3: Regression of measured optical density (OD) versus National Instiutes of Standards and Technology (NST) standard OD used to determine instrument linear range.			

Accuracy and precision of NIST  OD standard measurement

Fermentation samples are more variable than standard OD solutions. Therefore, it was necessary to determine the fermentation process linear range using each spectrophotometer. A calibration model assessed the accuracy and precision of the spectrophotometers to measure NIST OD standards over the common instrument linear range of 0.2–4.0 OD. Figure 4 shows the precision and accuracy of each spectrophotometer relative to the NIST OD standard values. Each spectrophotometer precisely measured the NIST OD standards over the linear range, as represented by small vertical bars. However, the measurement accuracy to the NIST OD standard values varied across the linear range. Model A had the highest accuracy and best matched the value of the NIST OD standard values. Model B and C had slightly less accuracy, and the predicted values were slightly higher than the NIST OD standard values at the high end of the linear range (2.8–4.0 OD). Model D diverges from the value of the NIST OD standard values at 1.2 through the linear range up to 4.0 OD.  

Figure 4: Calibration model of each spectrophotometer to National Institute of Standards and Technology (NIST) OD standards performed over the normalized linear range of 0.2â4.0 dilution OD.			

Process linear experiments were performed on samples at the start of feed, start of induction, and final OD. Figure 5 shows a representative example of this analysis. For visual reference, Figure 5 shows the average Model A data within the current historical linear range (0.2–1.6 OD ± 1 standard deviation). These data show an expanded actual OD linear range (0.2–6.0 dilution OD) for Models A, B, and C. Model D showed a reduced actual OD linear range (0.2–4.0 dilution OD), and an offset of measurement accuracy compared with the other spectrophotometers tested.  

The authors outline qualification procedures for a process-critical piece of equipment.

BioPharm International-05-01-2011