March 1, 2011 (Vol. 31, No. 5)

Established Practices Like Lyophilization and Six Sigma Are Finding Novel Uses in Biotech

Contract manufacturers are embracing new uses of established techniques to give them an edge in an increasingly competitive environment, according to companies exhibiting at the “Interphex” conference later this month in New York City. Examples of such practices include lyophilization, single-use manufacturing, and established decision frameworks like Six Sigma.

In some cases, the advances are being made slowly, balancing the gains with the extreme caution that is inherent in this industry. Six Sigma methodology is a case in point. It was developed in 1985 by Motorola to help it compete with Japanese telecommunications companies. Yet it is still working its way into biopharmaceutical companies.

Baxter was one of the early adopters and has reaped the benefits for at least the past decade. A few years ago, when Baxter’s BioPharma Solutions business ran into difficulties with variability in a client’s project, it applied Six Sigma methodology to identify the problem and the solution.

As Reuben Vandeventer, a DMAIC program manager at Baxter’s BioPharma Solutions facility in Bloomington, Illinois, recalls, “We had run some test batches but the process still yielded high particulate in the product vials, with no obvious reason. We executed a Six Sigma project, which uses a DMAIC progression (define, measure, analyze, implement, and control) as the central framework of the methodology. We found evidence of the problem and also how to solve it by using Six Sigma statistical analysis and problem solving.” That allowed Baxter to implement a control process that ensured the problem wouldn’t recur with that client or any other.

“The remedy involved measuring the process by collecting and microscopically analyzing solution samples at the beginning, middle, and end of the filling process, measured in parts per milliliter. Those samples are compared in real-time against a historical control chart to determine the appropriate actions based on historical decisions associated with the control charts. Now, if particulate matter exceeds the parameters in that chart, action is taken,” Vandeventer says. Baxter also uses Six Sigma to improve its forecasting accuracy to stabilize variability.

The Six Sigma approach is used by many industries concerned about process variability and instability. At Baxter, getting buy-in on the Six Sigma process begins with an internal marketing campaign that brands the project, generates excitement, and touts the successes.

Vandeventer advises companies implementing a Six Sigma approach to identify “quick wins” in each project and go for those first. Then, once people see results, apply Six Sigma approaches to bigger, lengthier projects. “There are no silver bullets, and people need to be coached,” he stresses. Therefore, “you need someone with Six Sigma experience to be the champion.”

“You also need training to minimize the pitfalls.” One of the key stumbling blocks, he says, is scope creep—trying to solve too much at one time. Another, is becoming derailed by an early setback. “Organizations get disappointed with results from the analysis phase,” if their basic assumptions are proven inaccurate.

Although scientific data tends to be straightforward, business process improvement data is anything but, he emphasizes. “Look at the data. If it doesn’t make sense, tweak the study to ensure that it really is measuring the process you’re trying to improve.”

Quality by Design

Ben Venue Laboratories applies quality by design methods to its lyophilization (freeze-drying)operations, “providing scientists with a more thorough understanding of the defined space to thereby develop the most robust and efficient lyophilization cycle possible,” according to Timothy McCoy, senior formulation scientist.

“One of the advantages is that scientists can design the cycle around the defined critical process parameters to develop an optimal primary drying step, which is key to producing a robust product,” McCoy says. Ben Venue is also investigating ways to better control supercooling, using nucleation technology to lower the moisture content and create more homogeneous crystal structures.

New technologies are also improving the development and manufacturing services, McCoy says. For example, “adding a sample thief to one of our development-scale lyophilizers enables us to accurately monitor moisture levels as a function of secondary drying conditions and time. That information is used to model and confirm the secondary drying stage of the cycle in one run.” That is particularly important with large, complex molecules to ensure that their activity is retained in the dried state.

“We also have designed a new pilot plant with six development-scale lyophilizers representing bench-, mid-, and large-scale units, letting us confirm the performance of our developed lyophilization cycles at a comparable scale before entering cGMP manufacturing,” thus accelerating process development, McCoy adds.


Lyophilization is a mature technology that is finding new uses in biotech. Oregon Freeze Dry is using this low temperature, low-pressure preservation method with biopharmaceuticals, specialty chemicals, medical devices, active pharmaceutical ingredients, and finish delivery formats to provide novel delivery solutions. It is beneficial when heat, oxygen, and/or shear prevent efficient production or efficacious final products.

“We are finding limitless opportunities to preserve the form and functionality of materials for topical, implantation, or oral delivery of specific moieties,” according to Walt Pebley, vp.

Currently, Oregon Freeze Dry is working with PATH, an international nonprofit health organization based in Seattle, to evaluate lyophilization as a delivery vehicle for oral vaccine quick dissolve, thus eliminating the cold chain and therefore extending the benefits of certain vaccines to developing countries. This technology also makes it possible to give full dosage of vaccines to babies without using needles. “The efforts require the fusion of vaccine culturing with cryopreservation, freezing, lyophilization, and unique packaging,” he says.

Oregon Freeze Dry is also assessing lyophilization as a way of stabilizing scaffolds for wound care. “We are creating emulsions of hydrogels, collagens, and specialty chemicals for dispensing into unique freezing apparatus that control ice nucleation, to form a crystal structure for the resulting lyophilized scaffold,” Pebley explains. “The next step is to incorporate compatible active ingredients homogeneously into the scaffold matrix, either before or after lyophilization, depending upon the stability requirements.” That approach provides “an elegant method for preserving form and function.”

In the specialty chemical area, Oregon Freeze Dry is using lyophilization to “preserve or maximize the internal surface area of materials to increase the productivity of catalysts, activated carbon and nano materials,” Pebley adds. “It appears that the principals of lyophilization can be applied across a much broader spectrum of applications than we previously thought.”

Lyophilized collagen scaffolds are being developed at Oregon Freeze Dry to hold vaccines and other therapeutics, allowing for elimination of the cold chain.


Microbial contamination is a key concern of many biopharmaceutical manufacturers, yet “there is a reluctance on the part of nonmicrobiologists to take action about microbial issues,” according to Joseph McCall, senior group leader, sterile operations for DSM Pharmaceuticals. The main areas nonmicrobiologists should focus on, he says, are surface and equipment design, cleaning parameters, and disinfection and sanitization.

“Identifying the contaminant is easier said than done,” McCall admits, “but it may lead to identifying the source of contamination.” At that point, he advises examining the cleaning strategies. “Experiment and demonstrate effectiveness against your known contaminants. Personnel-borne contaminants like Staphylococcus require a different strategy than those introduced by a system or process.”

“Disinfection and sanitization processes must be tailored to the unique situation,” McCall stresses. “Understand what it is capable of doing within the system. For example, a biofilm that coats the interior of pipes may need chemical obliteration rather than steam sterilization.”

McCall also recommends looking closely at the facility’s physical layout. “Walk through the engineering layout to identify dead legs—areas of piping in a circulating system where liquid can collect without being recirculated.” Also, look at piping slopes. “Bacteria don’t walk. Gravity and orientation play a role. For example, if you identify a dead leg, but the area of concern is four stories up, with bends in the pipes, it may be relevant.”

The message, McCall says, is this: “Understand that wear and tear exists, and systems that were once validated may no longer meet those standards.”


Xcellerex’ FlexFactory® is a bioprocessing manufacturing platform based upon single-use solutions, designed as modules and optimized cleanroom space. The FlexFactory is designed for a specific need, validated, and operated, and then may be transplanted to a new site without issues with proprietary cell lines, media, and technology that may limit a biotech’s future options. Xcellerex operates its own FlexFactory and also sells FlexFactories to clients.

“The automation system used to operate our FlexFactory is a purpose-built, application-specific tool for bioprocess operations, covering both development and manufacturing,” Ken Clapp, senior director of global marketing and product management, explains. “The software integrates the typically separate up- and downstream unit operations and associated equipment into one comprehensive automation environment.”

The result, Clapp says, is a manufacturing facility that can be built and deployed quickly and less expensively than its traditional counterpart. “Integrating automation with disposable manufacturing, with access to contract development and manufacturing services, helps organizations become more productive by aligning manufacturing capacity more closely with regulatory approval,”helping production to start sooner.

Comprehensive Services

One of the concerns in outsourcing aspects of development and manufacturing is the risk that inefficiencies will creep into the system and that critical knowledge will not be transferred successfully. Brian Arnold, brand and marketing manager at Cook Pharmica says that working with a CMO that provides comprehensive service at a single location minimizes wasted time, redundancy, information loss, and the number of relationships that must be managed.

Cook Pharmica’s high-speed syringe line under barrier isolator

European CMO News

RecipharmCobra Bio recently received European patents for its Xer-cise technology, which genetically modifies bacteria without leaving antibiotic resistance genes on their chromosomes. As Rocky Cranenburgh, Ph.D., head of molecular biology and inventor of Xer-cise, explained, “The system uses Xer recombinases, which are native to bacteria.”
The Xer-cise cassette consists of an antibiotic resistance gene flanked by dif sites—the recognition sequence for Xer recombinases—and regions homologous to the chromosomal target locus. That cassette is inserted into the target bacterium by homologous recombination.
The Xer recombinases resolve the two directly repeated dif sites to a single site, excising the intervening antibiotic resistance gene. “That reduces the steps involved,” Dr. Cranenburgh says, because, “once inserted, you don’t have to transform the bacteria with an additional plasmid carrying a foreign recombinase gene, which subsequently needs to be removed from the cell.”
“Xer-cise has enabled molecular biologists at RecipharmCobra and elsewhere to rapidly construct bacterial strains that are not antibiotic resistant, and are therefore advantageous for the commercial production and delivery of biologics,”adds Simon Saxby, vp of biologics.

Richter-Helm BioTec

Richter-Helm BioTec reported that it has modified its bioreactors to use Pichia pastoris. “That technology is very much in demand now,” because of its ability to secrete products and to manufacture  complex proteins, such as new antibody formats, said Kai Pohlmeyer, Ph.D., head of business development. The firm also implemented advanced technology to process and manufacture plasmid DNA, which increases yield and maintains highest quality, Dr. Pohlmeyer noted.
 Last autumn, Richter-Helm added expanded bed adsorption (EBA) chromatography to its technological mix. “With the implementation of EBA chromatography, clients can bypass initial recovery steps because the molecule of interest selectively binds to the EBA material, even from crude broth. We also optimized our E. coli expression systems and feeding strategies for fermentations, increasing yields of 10–20 g/L,” he continued.
To further process the increased amounts of raw protein, large-scale  chromatography equipment was added.

Boehringer Ingelheim

Boehringer Ingelheim is honing its global strategy, fielding a local team of representatives to be closer to the customer, and making its technologies more widely available to customers through bundling, according to Barbara Esch, Ph.D., marketing communications business intelligence.
Those technologies include the development of new expression systems. The E. coli genome integration system, for example, expresses recombinant proteins by integrating the gene of interest into the genome instead of placing the corresponding DNA onto an expression vector, Dr. Esch explained. Therefore, when the E. coli cells divide, each genome retains the gene of interest and expresses it at induction.
“We also have downstream technologies,” continued Dr. Esch, and adapted the crystallization purification step long used in chemistry for applications with stable entities, including interferon and some antibody fragments.

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