Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

The characteristics of particles–whether formulated into a suspension, capsule, or tablet–can have a tremendous impact on the performance of a drug product. Particle analysis is, therefore, a critical tool for drug manufacturers throughout the drug development and commercialization process. Ian Treviranus, product line manager for particle characterizationwith HORIBA Scientific, spoke with Cynthia Challener, editor of the 

Pharmaceutical Sciences, Manufacturing & Marketplace Report

, about different particle-characterization techniques and recent developments in the field.

Pharmaceutical Sciences, Manufacturing and Marketplace Report:

 Why is particle-size analysis important in the pharmaceutical industry? 

 Particle characteristics can have a tremendous impact on the behavior of an API and/or a formulated drug product. Smaller particles tend to dissolve more rapidly and thus have higher bioavailability. A uniform particle size (narrow particle-size distribution) will similarly improve performance compared to drugs with a random distribution of particle sizes. The appropriate surface charge is important for preventing aggregation and agglomeration of particles in suspensions and emulsions, and therefore impacts stability. The surface area of a solid dosage drug plays a role in its dissolution profile. The properties of coatings applied to particles (i.e., thickness, uniformity) also affect drug performance. Finally, processability can be impacted, such as in tableting systems, where particles with certain shapes exhibit much better flow behavior than others.

 For what applications in the pharmaceutical industry is particle-size analysis useful/valuable and what technologies are typically used?

 Particle characterization is critical throughout the entire drug development and commercialization process. Because particle properties play such a major role in the performance of both APIs and formulated products, these characteristics must be determined in the earliest stages of drug development. Once particle properties are defined in an FDA submission, the manufacturer must ensure that those specifications are met as the drug progresses through clinical trials and on to commercial production. Often, not only the specified methods, but the same instruments (makes and models) are used at each stage to ensure consistent analysis results.

The two most common technologies used in the pharmaceutical industry for particle characterization are laser diffraction (LD) and dynamic light scattering (DLS). LD is useful for particles ranging from tens of nanometers to a few millimeters while DLS is appropriate for particles that are a few nanometers to about one micrometer in diameter. Other techniques include optical image analysis, surface area analysis, and sedimentation analysis.

LD and DLS are used to determine particle size, particle-size distribution, and particle dispersion in a formulated tablet or capsule as well as to measure the properties of tablet and particle coatings. More recently, laser diffraction has proven to also be applicable to the evaluation of protein agglomeration. DLS, meanwhile, can be used to determine the zeta potential of particles in suspensions or emulsions. The zeta potential is a measurement of the difference in the charge on the surface of a particle and the background; a higher number indicates greater stability and reduced likelihood of agglomeration. Optical imaging techniques are used for particle-shape analysis while surface-area analysis provides information on the porosity of a tablet, which affects its dissolution rate.

 What recent developments in particle analysis technology are being applied in the pharmaceutical industry, and what advantages/benefits do they provide? 

 Much of the advances in particle-analysis technology occur on an incremental basis through cooperation between instrument manufacturers and their pharmaceutical industry customers. With laser diffraction, iterative small improvements in polishes, light sources, detectors, computer algorithms, and noise subtraction add up over time to new capabilities for new applications. For example, laser diffraction previously was discounted as a technique for the determination of protein agglomeration. Today, however, LD performance has been shown to indeed be suitable for the reliable quantification of protein aggregation, even at very low concentrations.

For dynamic light scattering, there have been significant improvements with respect to its use for the determination of zeta potentials. The interest in zeta potential measurements has grown rapidly in recent years, and instrument manufacturers have responded by making incremental improvements in DLS to improve performance.

The need to properly characterize nanoparticles is also driving interest in newer technologies, especially in Europe. Since 2011 when the European Commission Joint Research Center (JRC) published its definition of nanoparticles and recommended the use of number-based analyses, which involve looking at individual nanoparticles, something that LD and DLS cannot do, there has been growing interest in methods such as nano tracking analysis (or nanoparticle tracking analysis, NTA) and nanocoulter counters. There is an expectation that the JRC definition will eventually become regulation in Europe, and the US will ultimately adopt something similar. Nanoparticle characterization is an issue for any product that contains nanoparticles that will be used by consumers, including medicines that are ingested or applied to the skin in some way.

 What issues/limitations remain with particle size analysis in the pharmaceutical industry today, and what efforts are being made to address them? 

 The holy grail of particle analysis is to develop instruments that can cover a wider range of particle sizes or types, provide as much new information as possible, and do so in one instrument that is easy to use. For example, HORIBA is working to combine its experience in spectroscopic chemical identification with its particle-characterization systems. Marrying particle-size analysis with optical analysis and/or surface area analysis is another opportunity that the industry is exploring.

With respect to limitations, protein-aggregation analysis remains a real challenge. There is no one instrument at this time that can provide quantitative results over the entire size range from the smallest protein to the larger aggregate. Many biopharmaceutical customers are also looking for improvements in zeta potential analysis systems. One major flaw of this analytical method is the fact that, when the electric fields are applied to the samples, currents are generated. Pharmaceutical customers were reporting both variability in results for repeated tests and a trending of results in one direction, indicating that samples were being destroyed or degraded. Damage to sample cells is also a problem. Both can be extremely costly, particularly if it means larger sample sizes are required and the material is not recoverable for further analysis. 

This issue is one that HORIBA has chosen to tackle. By replacing the precious metal electrodes typically used for zeta potential measurements with specially designed pressed graphite electrodes, it is possible to conduct the analysis with a much lower electric field. As a result, only a weak current is generated that does not damage the sample cell or the sample, which provides consistent results that our customers can have confidence in. Furthermore, the graphite electrodes are extremely durable and don’t need to be replaced for the lifetime of the instrument. 

Articles

Iterative and multiple incremental advances in particle-characterization technology are making a difference in pharmaceutical analysis applications.

Particle Characterization is Critical for Drug Performance

Cell-culture media is critical to the production of biologic drugs, including cell and gene therapies. Initially, media makers focused on increasing yield and productivity for protein-based applications. Today, productivity is high enough that downstream processing has become the bottleneck. Media manufacturers have thus turned their attention to improving the consistency and quality of protein expression. For therapeutic cells and viruses, yield and productivity continues to be a critical area of focus in addition to downstream processing. Several leading producers spoke with Cynthia Challener, editor of the 

, about their efforts with respect to media development. Participants included Melanie Brandl, head of global marketing for cell culture media and supplements at EMD Millipore; Gautam Choudhary, associate vice-president, Gibco Cell Culture Bioproduction with Life Technologies; Willem Folkerts, clinical technologies portfolio and global marketing head, and Alexis Bossie, head of media R&D , both with Lonza Bioscience; and Bruce Lehr, director of cell sciences and development with SAFC.

Optimization of performance
Pharmaceutical Sciences, Manufacturing and Marketplace Report:

 What are the major trends in the development of cell-culture media today?

 All suppliers are moving toward chemically defined formulations that are also made from animal-origin free (AOF) components. Additionally, there is a growing trend toward the development of optimized platforms for media trains and a continued drive toward greater process yields through higher producing titer systems.

 The focus has switched to product quality and the determination of how different media characteristics affect product quality, such as glycosylation patterns. This issue is of particular importance for biosimilar manufacture, where a major requirement is the ability to demonstrate that the biosimilar matches the branded drug as much as possible. Other trends include a growing interest in flexible manufacturing processes and the use of disposable equipment and the emerging use of perfusion systems.

Media and feed platforms are developed to ease fast optimization of each fed-batch process respecting different cell-clone requirements. The success of the platform is linked to the formulation, but is also strongly dependent on the quality and safety profiles of cell-culture raw materials. Improvements in the solubility of single components are targeted to increase concentration levels and media performance.

 For protein media, which is well-established, the focus is on optimization for platform cell lines to give high yields and desired protein characteristics, such as aggregation, glycosylation, and folding. For stem-cell media, and particularly pluripotent or mesenchymal stem cells, the media needs to be designed so that the desired highly specialized cells proliferate but do not differentiate until they are supposed to. The industry is driving towards xeno-free formulations here as well. Primary cells also require highly specialized media that provides proliferation without loss of function.

Pharmaceutical Sciences, Manufacturing and Marketplace Report: 

What approaches are being adopted to address upstream manufacturing process/material issues?

 Raw materials have to undergo defined qualification processes to secure quality, origin, and consistency in their manufacturing, including evaluation of serial amplification assays to check impurity impacts, trace elements, and the effect that individual components have on product quality. Beyond standard vendor qualification, EMD Millipore applies its pharmaceutical ingredient qualification expertise to bring the raw-material topic to the next level.

Verification of raw materials is necessary to ensure that they are not only of the required grade, but also “fit-for-use,” meaning ensuring that the raw materials function as needed. Today, Lonza is doing a lot more testing, particularly related to building capabilities for cell characterization and determination of how media impacts cell performance. Some of the tests include evaluation of cell-surface markers and the production of cytokines, growth factors, and other cell products that are indicative of cell behavior.

 Pharmaceutical industry customers want the therapeutic cells manufactured in clinical trials to be the same as those produced on a commercial scale. Therefore, an understanding of the impact of culture media is very important for obtaining better consistency from batch to batch. 

 At Life Technologies, we have a robust vendor- management program. Individual components are scored for risk and processes have been put in place to mitigate any concerns. Additionally, analytical tools are employed to verify the composition of components.

 Raw materials have become critical to media manufacturing and development. Pharmaceutical companies want more transparency, so they can follow the complete supply chain. In response to this growing need, SAFC has developed novel biological assays and evaluation technologies to ensure that all raw materials are appropriate for cell-culture applications. We are also collaborating closely with our customers and suppliers and working to develop methods for identifying all of the chemicals in formulated media, which can consist of up to 70 components.

 What advances in cell- culture media can be expected in the near future? What about long term?

 Pharmaceutical companies are looking for improved efficiency with respect to the formulation of chemically defined media for fed-batch operations. They are looking for good base media that can be used throughout production with their platform cell lines and use tweaks to feeds to optimize each different cell line. With the increased use of perfusion, there is also a need for media that is formulated specifically for those processes

 In cell therapy, there is a move away from adherent technologies to the use of suspension systems that are similar to the systems used for protein production. The use of suspension systems will enable easier scale-up and also reduce the cost of manufacturing. These systems will require different media, particularly given the fragile and sensitive nature of the cells.

Base cell-culture media continue to be improved upon, but there will likely be a point reached when the incremental benefits are minimal. However, functional additives focused on product quality, titer, and product safety look to become the focus for the future. 

 Efforts are being made to achieve complete elimination of undefined components and at the same time gain better knowledge about the individual components and how they impact product quality. We also expect to see novel ingredients that dissolve easier and have better uptake profiles as well as improvements in the ability to control cell metabolism (e.g., lactate control). Other approaches are to make media safer with respect to any type of adventitious agents such as viruses, which is economical on the upstream side of the process.

 What activities has your company pursued recently with respect to cell-culture media?

 Most recently, Lonza formed a collaboration with Sartorius Stedim Biotech (SSB) in which SSB will assume global sales and marketing of media and buffers used in the manufacture of protein-based therapeutics and vaccines while Lonza will continue to carry out development and manufacturing of these products. The media will be packaged in ready-to-use, prefilled SSB sterile single-use bags. The ability to offer fully integrated single-use solutions for the entire cell-culture process provides a real benefit to customers. 

 Lonza has also been awarded two separate contracts by the National Institutes of Health Center for Regenerative Medicine to produce induced pluripotent stem cells (iPSCs) for research purposes and clinical grade under current good manufacturing practices. 

 EMD Millipore recently launched a new CHO media product as part of our platform development effort that offers a complete solution for cell-culture batch, fed-batch, and perfusion processes. Produced as a top quality dry powder in our cGMP-compliant manufacturing facility, it has shown equivalent or superior cell growth and production compared to several other commercially available media and demonstrated impressive purity and consistency, which is an important consideration in optimizing bioprocess control, especially in the rapidly emerging biosimilars production market.

 Life Technologies has developed and launched a pH-neutral, highly-concentrated additive designed to keep specific productivity high late in culture. This additive is also designed to have minimal impact on product quality. We also continue to expand our media format by adding highly-concentrated additives that not only enhance titer but are also easy to use. 

 SAFC’s Zinc Finger technology allows for genetic manipulations so that certain genes that control glycosylation (related to molecule antigenicity) can be knocked out and cell lines improved. We are also developing targeted integration as an improvement over random integration. It can speed up the process and enables control of gene placement for more controlled expression, which leads to both performance and regulatory benefits. 

To meet our customers’ expectations for more efficient media development, we have adopted high-throughput systems for the rapid screening of hundreds of combinations of media products and feeds. The use of multivariate analysis and a design of experiment approach are also making it possible to identify key components and their impacts on media performance and protein quality.

With high productivity achieved, makers of cell-culture media are working to optimize product quality through better understanding and control of raw materials and production processes.

Media Makers Focus on Quality

Although taste-masking is more often associated with pediatric and geriatric liquid formulations, the APIs used in solid dosage drugs often have a very bitter flavor as well. Tablets and capsules are swallowed whole, thus masking the taste of the ingredients involves prevention of dissolution before the pill reaches the stomach. Nigel Langley, head of technical sales for pharma ingredients and services at BASF Pharma Ingredients and Services, discussed the two approaches to taste-masking of solid and semisolid dosage drugs with Cynthia Challener, editor of the Pharmaceutical Sciences, Manufacturing and Marketplace Report.

Choice of protection
Pharmaceutical Sciences, Manufacturing and Marketplace Report:

 What are different methods that can be used to achieve a taste-masking effect for solid and semisolid dosage forms?

 There are two approaches to taste-masking of solid and semisolid drugs, and both involve coating the product in some way with a polymer. The goal of the coating is to prevent disintegration and release of the drug into the mouth or throat so the taste buds are never exposed to the API.  In one method, a sustained-release polymer system is used, and in the other, an instant-release polymer that exhibits pH-dependent solubility ensures that the drug remains whole until swallowed. These polymer coatings can be applied to the entire tablet or capsule, or to crystals, granules, or other particulates used to manufacture the final dosage form.

 What factors must be considered when selecting a taste-masking excipient for a particular API/formulated drug?

 APIs themselves can be acidic. Most polymers that have pH-dependent solubility characteristics are basic. Therefore, the choice of polymeric coating type can be influenced by the nature of the API. It is, however, possible to apply a barrier coating to the tablet or particle before the pH-dependent coating is applied if this type of taste-masking system is preferred. 

What is the chemistry of the different types of taste-masking polymers? How does each type generally work?

 Our [BASF] pH-dependent polymer is based on acrylate chemistry and is an aqueous dispersion of a copolymer comprised of methyl methacrylate and diethylaminoethyl methacrylate. This coating is stable at neutral pH, and thus does not dissolve in saliva, which generally has a pH of 6.8-7.2. Below pH 5.5, however, the polymer will form salts. Thus, in the stomach, where the pH is typically 1.2, but can be a little higher in people who suffer from acid reflux, the coating reacts with biosalts, becomes soluble, and the drug is released. In fact, the coating operates in the opposite manner to an enteric coating.

Sustained-release coatings work because they can be formulated with both water- insoluble and water-soluble polymers, the latter of which is referred to as a pore former. The sustained release behavior of the coating can be tuned by varying the amount of pore former and therefore customized to meet the release profile required for a specific solid dosage drug.

  Different polymer types have been used as sustained release coatings, including cellulose-based resins. BASF’s polyvinylacetate water-insoluble film former is offered as a 30% dispersion in water. Our water-soluble pore former is a polyvinyl alcohol-polyethylene glycol graft copolymer with polyethylene glycol (PEG) units incorporated into the backbone that impart flexibility.

 What specific new technology has BASF developed and how is it an improvement over current products? What might be expected in the future?

 The instant-release technology from BASF is unique in that, even though the acrylate copolymer is basic, it has a very bland odor. At least one other pH-dependent taste-masking product on the market smells strongly of ammonia, which is quite offensive in the manufacturing setting. In addition, BASF’s polymer hides very bitter tastes at a reduced coating thickness compared to other products on the market. It also prevents moisture uptake, which is an added benefit for sensitive solid dosage formulations. Furthermore, because it is a dispersion in water, no organic solvent is required for spraying of the coating.

These advantages, in fact, led Colorcon to recognize our acrylate technology as the best-in-class reverse enteric polymer for taste-masking, according to general manager for film coating Kamlesh Oza. As a result, BASF and Colorcon have been in a collaboration since October, 2011 for the development of film-coating systems using BASF’s polymer. Colorcon has developed a preformulated additive system for use with the BASF coating that lowers the number of materials to be dispensed by 50% and reduces the preparation time by almost 40%, thus enabling efficient preparation and application for taste-masking of solid and semisolid dosage forms. 

The pH-dependent technology was introduced two years ago and has attracted a lot of interest. It takes quite some time for pharmaceutical customers to adopt a new excipient and get a product approved by the FDA, but we are working closely with our customers to reach that goal.

The trick to taste-masking in solid dosage forms is to never let the taste buds have a chance.

Ignorance is Bliss in Solid Dosage Taste-Masking

In parenteral manufacturing, filtration is used for particulate control, depyrogenation, and sterilization. Russell Madsen, president of the Williamsburg Group, group leader of the Parenteral Drug Association (PDA) Filtration Interest Group, and member of the editorial advisory board of 

, discusses why filtration is important in parenteral drug manufacturing and related initiatives of the PDA Filtration Interest Group.

Pharmaceutical Sciences, Manufacturing & Marketplace Report:

 Why is filtration such a critical issue for parenterals?

 Patient safety requires parenteral products to be sterile and free from objectionable levels of particulate matter and endotoxins.  Filtration is used to remove particulates, for depyrogenation, and for sterilization of aseptically produced parenteral products.  Each of these functions has different control parameters and processing requirements. From a risk-based perspective, sterility assurance is the most critical of these filtration processes. Filtration also can be used to remove viruses from biopharmaceutical process streams and products.

 What are the major techniques that are used for filtration of parenterals, and what are the benefits and limitations of each?

 Broadly, there are three major techniques: depyrogenation, prefiltration, and sterilizing filtration.

Depyrogenation is accomplished by means of ultrafiltration, reverse osmosis, microfiltration using charged filters, and charge-modified depth filters. Prefiltration is used to control the particulate and microbial levels reaching the final filter. 

 Prefiltration can occur in several stages; for example, a depth filter can be followed by a membrane filter. Controlling particulate and microbial levels prior to sterilizing filtration minimizes the potential for blocking or clogging the final filter in a properly designed system with adequate capacity, [thereby] ensuring filtration efficiency and minimizing changes between pre- and postfiltration diffusive flow integrity test values. The use of two sterilizing-grade filters in series is a special case where the first sterilizing-grade filter acts as a prefilter to stringently control the microbial level presented to the second, final filter.

Sterilizing filtration is used to remove all viable microorganisms from the product stream when other sterilization methods, such as moist-heat terminal sterilization, cause unacceptable levels of product degradation. Stringent controls, including knowledge of the prefiltration bioburden, sterilization of the filter, its housing and associated tubing, and filter-integrity testing, are required to ensure sterility of the filtrate. Validation studies demonstrating the ability of any particular combination of filter, product, and processing conditions to produce a sterile filtrate are required. Typically, these validation studies employ 

 (if viable in the product) at a challenge level of 1 x 10

 of effective filter area in the product to be filter-sterilized.  If the standard challenge organism is not viable, other options are available. (See 

, PDA Technical Report No. 26 (Revised 2008) for details and recommendations.)

Presterilized, single-use filter assemblies

 What recent advances have been made in filtration technology and how are they addressing some of the aforementioned issues? What might be in development?

 Regulatory agencies, such as FDA and EMA, have stressed the need to have detailed knowledge of the prefiltration bioburden to demonstrate microbiological control of the manufacturing process. The presterilization bioburden should not exceed the limit established during product development and validation of the sterilizing filtration process.

The use of presterilized, single-use filter assemblies simplifies the set-up and sterilization of the filtration system, especially where multiple filters are used in series. This approach is consistent with the increasing use of single-use processing equipment, such as chromatography capsules, processing and holding bags, and sterile connectors, to minimize the risk of cross-contamination while providing operator protection from potent and toxic biopharmaceutical process streams.

Presterilized capsule filters minimize the risks of improper set-up and assembly of filter housings, leaking O-rings, and eliminate the need for sometimes complex and difficult-to-validate steam-sterilization of the assembled units. In addition, single-use sterilizing filter capsules are increasingly available in sizes, configurations, and membrane materials that directly replace conventional stainless-steel housings and cartridge filters while providing increased sterility assurance and minimizing risk.

The future will see increasing use of single-use equipment, including sterilizing filters, tangential flow units, and purification columns. It is conceivable that someday sterile, out-of-the-box systems will be available for parenteral production encompassing compounding to filling.

Prefiltration poststerilization integrity testing

Pharmaceutical Sciences, Manufacturing & Marketplace Report: 

Why is prefiltration poststerilization integrity testing so important? What are the issues that must be addressed?

 While the European GMPs require prefiltration poststerilization testing of sterilizing filters, the US aseptic processing guidance is largely silent on such testing.  However, both documents require post-use integrity testing.

Pre-use poststerilization integrity testing (PUPSIT) is intended to show that a filter in its housing, having been subjected to a sterilization process, passes a validated integrity test and has not been damaged during sterilization.  In most cases, the filter has already been subjected to one or more presterilization integrity tests: by the filter manufacturer prior to sale and by the user after having been placed in its housing and flushed prior to sterilization. The purpose of such testing is to demonstrate that the filter is integral and has the appropriate pore-size rating.

However, PUPSIT often requires exposing the downstream side of the filter to atmospheric pressure, potentially compromising the sterility of the unit. From a risk-based perspective, this approach therefore makes little sense as long as the sterilization process for the filter has been validated and shown not to damage or cause loss of integrity of the sterilized filter. If, in fact, the sterilization process damaged the filter, the damage would be detected during post-use integrity testing, minimizing the importance of PUPSIT.

PDA published an article “Pre-use/Post-sterilization Integrity Testing of Sterilizing Grade Filters” in the September–October issue of the 

PDA Journal of Pharmaceutical Science and Technology

.  The article recommends that the PUPSIT decision should be “. . . made by the filter user upon thorough, documented risk-based analysis in accordance with ICH guidelines.” PDA is planning a research project designed to evaluate the risks associated with PUPSIT and the likelihood that damage to the filter caused by sterilization would not be detected in post-use integrity testing.

 What issues is the US PDA Filtration Interest Group tackling in 2013? What do you anticipate might be issues that the group will address over the longer term?

For 2013, the next US PDA Filtration Interest Group (IG) meeting will be held on April 16 at the Peabody Orlando Hotel in Orlando, Floridia.  There will be four presentations followed by a discussion of topics of general interest.  Monica Cardona, Pall Corporation, will present “Safety Considerations for Gas Filtration in High Temperature and Oxygen Enrichment Applications.” Mandar Dixit, Sartorius Stedim North America, will discuss “Optimizing Total Cost of Ownership for Membrane Filtration.”  The title of Lia Jeffrey’s presentation, EMD Millipore Corporation, is “Retention of Mycoplasma and Small Microorganisms,” and Leesa McBurnie, Meissner Filtration Products, will provide an update on the activities of PDA’s 

Longer term, the IG [Interest Group] is likely to continue to address PUPSIT issues and support discussions on that topic with European and American regulatory agencies.  Single-use filtration technologies and devices will be prominent topics for discussion.

Russell Madsen, group leader of the Parenteral Drug Association (PDA) Filtration Interest Group, discusses technical and regulatory considerations in filtration for parenteral drug manufacturing

Filtration Critical Issue for Parenterals

Conventional microbial analyses require extensive manual operations. In addition, the necessity for visual determination of the growth of microbial colonies leads to longer assay times. The result is a lengthy process that can take anywhere from three days to more than two weeks and that can dramatically impact production and commercialization times. Automated systems providing rapid results are helping to reduce that time, but the key to acceptance is staying consistent with the compendial method. Cynthia Challener, PhD, editor of the 

, spoke with Julie Sperry, chief commercial officer of Rapid Micro Biosystems, to gain further insight into the application of rapid microbiological testing.

 For what applications in the pharmaceutical industry is rapid microbiological testing appropriate and why? What benefits/advantages does it provide? 

There are many areas where microbial analysis is critical in pharmaceutical manufacturing. Raw materials, work-in-process material, and final products all must undergo microbial testing. In addition, microbial monitoring of the manufacturing environment (i.e., personnel, surfaces, and air) is an ongoing process and critical to compliance. As a result, pharmaceutical quality-control laboratories often process hundreds, if not thousands, of tests per day, much of which involves manual manipulation of the assay plates for reading as well as data logging and other activities.

Rapid analysis with automation can, therefore, reduce the burden of manual plate counting and data entry on these laboratories and allow the workforce to be redeployed to address other important tasks. In addition, the level of human error can be reduced, and because the automation can provide the data immediately, decisions can be made quickly and definitive actions on process development issues and product releases can occur sooner.

Elements of a rapid microbiological system

 What are the critical factors that contribute to a successful rapid microbiological analysis system? 

 The most critical factor is the use of the established, or compendial, method, so that the analytical method doesn’t involve a major process change and can be validated without any need for extensive validation work.

Second, it is important that the method is nondestructive. If contamination is detected, then the microbes must be identified. Therefore, the test must be designed so that it doesn’t destroy or cause any change in the microbes. There are several ‘alternative’ technologies available, but many involve destruction of the microbes. In addition, many of these other methods don’t report the results in colony-forming units (CFUs), which is the standard unit for microbial testing. Therefore, the data have to be related to CFUs, which require validation of the data correlation and can introduce uncertainty.

: How is it possible to develop an automated system that can meet these requirements?

The key is to use a growth-based method but [also to] develop a visual analysis system that can detect colony formation much sooner than is possible with the human eye. Our system is based on the natural auto-fluorescence of the microbes. Samples are prepared using media that is typically used for conventional analyses. A certain wavelength of light is shone on the sample, and if there is microbial contamination, the microbes auto-fluoresce. A very sensitive camera detects and captures this fluorescence.

To establish the growth rules for the software, extensive tests were conducted on many different types of microbes, including bacteria, molds and yeast, in order to develop reliable software that can detect colony growth and disregard non-growing debris, such as lysed cells or particulate components. The system was validated by comparing the results of automated determinations with determinations made using the conventional method.

The system takes readings every four hours and looks for differences. Often contamination can be seen within 8-12 hours, so in many cases, a problem can be identified after just two to three readings. The system then sends an alert message indicating that the colonies detected have exceeded a preset alert limit. With this type of automated detection system, it is possible to reduce the time needed for microbial analysis to about half of what is required for the conventional method.

 How is automated microbiological testing being improved? What recent advances have been made?

 One of the issues for some of our pharmaceutical customers is the need for separate systems for sterile-product testing. Others, on the other hand, want a system that can handle the whole range of different analyses, including sterility, bioburden, and environmental monitoring.

We have taken these customer requests very seriously and have developed a line of second-generation products, which will be released mid-year, that address these issues. One of the new instruments is specifically for high-throughput environmental monitoring and water/ bioburden analyses and is designed for high throughput.

The second new system is a specialized instrument for use in sterility-testing applications. This system includes the ability to test for both aerobic and anaerobic microbes. The system has three cassettes, one of which creates an anaerobic environment with a system sensor to detect and confirm that the cassette maintains an anaerobic atmosphere throughout the testing. The emphasis here is not only on time to results, but also proper sample preparation and handling.

The third new system is a multifunctional instrument designed to handle all of the different types of microbial analyses required in pharmaceutical manufacturing and whch can simultaneously run multiple analyses: sterility, environmental monitoring, water, or bioburden. This system is useful for smaller companies that can’t afford several individual instruments, but is also suitable for larger manufacturers that want to have the flexibility to validate a range of applications and then spin the validation out to their various sites.

Most importantly, the same growth-based method and detection technology is used across the product line. One added feature, though, is that these systems can be connected to a laboratory information-management system (LIMS) for improved sample tracking.

 What limitations remain with respect to rapid microbiological testing? What might be done to address these challenges? 

 There are several issues related to sample preparation and the subsequent identification testing that play a role in the turnaround time for the overall microbial testing process. Logistics, for example, can account for a significant amount of time and resource consumption. Often the testing is done in one location, but samples are collected elsewhere. Pharmaceutical companies can make efficiency gains with automated systems that can be operated in or closer to the manufacturing facility, particularly for environmental monitoring analyses. The capabilities of our new system facilitate this testing shift. Longer term, we are interested in finding ways to help our pharmaceutical customers speed up sample preparation and microbe identification so that they can maximize the increases in productivity and decision-making that rapid, automated microbial analysis systems can provide.

It can take up to two weeks or more to obtain results from traditional microbiological testing methods. Rapid, automated testing can reduce that time in half.

Advances in Rapid Microbiological Testing

A high proportion (> 50%) of the active pharmaceutical ingredients (APIs) approved by FDA during the past 10 years were manufactured using chiral technology, according to industry estimates. Asymmetric transformations are thus a critical aspect of drug manufacturing today, and catalysis has become the primary method for the efficient and selective synthesis of single enantiomer products. Development chemists now have a choice between enzyme-based biocatalysts and transition metal-based chemocatalysts (some organocatalysts are attracting interest as well but are not discussed in this article).

 Chris Senanayake, vice-president of chemical development in the US for Boehringer Ingelheim Pharmaceuticals, Ian C. Lennon, senior vice-president for global business development with Chiral Quest, Antonio Zanotti-Gerosa, team leader with Johnson Matthey Catalysis and Chiral Technologies, Ephraim Honig, chief operating officer with Strem Chemicals, and Wataru Kuriyama, a chemist with Takasago International, discuss important aspects of chemocatalysis for asymmetric synthesis.

Considering the state-of-the-art catalytic toolbox of today, when are homogeneous chemocatalysts advantageous over other types of catalysts for chiral transformations?

In preparing the initial batches of API for preclinical studies and early clinical trials, the simplest, not necessarily the best and most economical, method is used due to time constraints. That may be a well-known stoichiometric method using a chiral auxiliary or a resolution-based approach. In parallel, we investigate chemocatalytic routes to determine if there are any cost-effective, scalable alternatives, considering all aspects, such as the metal (availability, cost), ligand (intellectual-property issues, cost), yields, selectivities, and catalyst loadings. Enzymatic routes generally take much longer to develop and often require the use of outside expertise for the engineering of the desired reactivity, so they tend to be used more often in second-generation processes in late-stage drug development.

 Chemocatalysts can be classified into two major types, homogeneous catalysts and heterogeneous catalysts. Heterogeneous catalysts have advantages in recovery and reusability; however, generally, homogeneous catalysts show better reactivity and selectivity. In addition, the flexibility of ligand design can improve selectivity, and in asymmetric reactions, high selectivity is required. Therefore, homogeneous catalysts are predominantly used. 

Zanotti-Gerosa (Johnson Matthey Catalysis and Chiral Technologies):

 The use of biocatalysis is growing, but the choice of catalyst technology must be made on a case-by-case basis. Chemical catalysts offer some real advantages. Many are available off-the-shelf and do not need to be engineered for a specific reaction. They have high activity, often require only a simple workup, and most importantly, have high-volume efficiency. What we see is that the speed of development that is possible with chemocatalysis is also very attractive because it enables more rapid development of production solutions. All of these factors have to be considered in order to find the most cost-effective catalytic solution.

Choice of method—such as between chemocatalysis and biocatalysis—will also depend on the technology that is possessed by the CMO. Chiral Quest, for example, specializes in asymmetric hydrogenation.  There are also areas where biocatalysis is not yet effective, such as the asymmetric synthesis of α-amino acids. Asymmetric hydrogenation, on the other hand, is exceptionally good for the commercial-scale manufacture of both aliphatic and aromatic α-amino acids. 

Ligand, substrate, recovery and cost challenges 

 What are the major shortcomings/limitations of current homogeneous chemocatalysts for chiral transformations? What major issues would you like to see addressed in next-generation catalyst technologies?

 The synthesis of chiral ligands is often very difficult and can require the use of cryogenic conditions and resolution steps.  On the positive side, in many cases, both enantiomers can be used, and the catalyst loadings are very low, so large quantities of the ligands are not needed. Obviously, having much cheaper and easier to make ligands while still maintaining excellent enantiomeric excess and high activity is a good goal.

On the substrate side, there are also still a number of compound classes that are challenging. For asymmetric hydrogenation, imines, oximes, 

-substituted double bonds, and aromatic systems fall into this category. Some advances are being made in the asymmetric hydrogenation of heterocycles, and this area holds much promise as it will lead to the manufacture of complex chiral molecules in very few steps and is a reaction that cannot be achieved using enzymes.

 Removal of the metal and reuse are issues that must be addressed, but there are processes where homogeneous catalysts are recycled. Rh-BINAP in our 

-menthol process, for example, is recycled from the distillation residue. However, there still is much room to improve metal and catalyst removal. Next-generation catalysts should have the advantage of both homogeneous and heterogeneous catalysis as well as high selectivity and recyclability.

 The catalyst cost is the biggest issue. Most chiral catalysts for asymmetric synthesis contain precious metals and very complex ligands, both of which are very expensive. Therefore, a cost-effective process requires very low catalyst loadings (ideally 100,000:1). Going to such low levels also eliminates the need to recover the catalyst.

Scalability is the biggest issue. Often with catalytic reactions, the catalyst does not seem to behave the same way on a very large scale as it does on the bench scale, and this issue can be more pronounced with asymmetric catalysis. This change in behavior can be due to catalyst stability issues or problems with the purity of the starting materials. Boehringer Ingelheim has established an in-house Catalysis Group to design new ligands and to develop new catalytic reactions that can address both the cost and scalability issues encountered in API development.

 What is being done to overcome these problems?

Zanotti-Gerosa (Johnson Matthey Catalysis and Chiral Technologies): 

Existing catalysts are being applied to other transformations, or being modified so that they can be effective for related reactions. For example, some hydrogenation catalysts have been shown to be effective for the reverse dehyrogenation reaction.  Other catalysts have been introduced that can do both hydrogenation and transfer hydrogenation reactions.  This flexibility has a cost impact. Development work is also proceeding with many important chiral catalysts and ligands whose patents have recently or will soon expire.

Immobilization is one solution for avoiding recovery issues. In general, immobilization can be achieved through binding of the ligands or the metal to a solid support. From the perspective of industry, immobilization of the metal is advantageous because ligands developed for homogeneous catalysts can be used as is, which reduces the cost and time of catalyst development. Rhodium (Rh) with the well-known Duphos ligand on PTA (phosphotungstic acid) for asymmetric hydrogenation is one example of this type of catalyst. Microencapsulation is another important method. Toxic OsO

 immobilized by this method is used for Sharpless asymmetric dihydroxylation and recovered without any leaching.

 In our experience, it is better to optimize the homogeneous reaction to get the lowest possible catalyst loading and the best selectivity, so that each production run takes the same time, gives the same enantiomeric excess (ee), uses the same catalyst loading, and provides the same purity of product. Consistency is especially important for GMP manufacture. Generally, making the ligand heterogeneous leads to poorer catalyst loadings (often 10 x higher or more), lower and less reproducible ee’s, and sometimes incomplete reactions.

 The key to developing a catalytic reaction into commercial scale is to gain an understanding of the mechanism of the reaction and identify the active catalytic species. It is imperative that the interactions between the catalyst and the substrate be identified. The use of high-throughput screening, combinatorial ligand discovery, computational tools, and modern analytical techniques in an integrated manner will make it possible to obtain the necessary level of understanding of catalytic behavior needed for the development of robust systems that will be effective at any scale.

 Of recently introduced chiral chemocatalysts and/or ligands, what do you believe are the most novel and why? Which ones will have the most impact on fine chemical/pharmaceutical synthesis and why? 

 We believe that our asymmetric hydrogenation catalysts RUCY and DENEB are the most novel catalysts and will have the most impact on fine chemicals. RUCY and DENEB show unprecedentedly high catalyst activity; the productivity per hour or per weight of catalyst is high enough that the cost for production can be reduced and the removal of the metal is relatively easy. In addition, RUCY and DENEB have a wide substrate scope and reduce substrates that can’t be hydrogenated by conventional catalysts. Therefore, we believe that they will have the most impact on the pharmaceutical industry, where efficient chemical transformation of compounds with a variety of functional groups is required and product quality must be tightly controlled.

 There is a lot of interest in iridium complexes developed by the Kerr group at the University of Strathclyde for the direct, flexible, and selective introduction of hydrogen isotopes to organic molecular frameworks under extremely mild reaction conditions, and these catalysts have successfully been used for the isotopic labeling of several known drug molecules. We also believe that the RUCY-XylBINAP catalyst developed by Takasago, which is effective at catalyst loadings as low as 0.001 mol%, is very promising as an industrially practical catalyst for asymmetric hydrogenation.

The Noyori ketone hydrogen technology, for which the Nobel Prize was awarded in 2001, is a very important asymmetric transformation today. In fact, second-generation catalysts that are more robust (more resistant to deactivation) and more active that are being developed by Walter Baratta at the University of Udine and others will be valuable new catalysts for pharmaceutical manufacturing. New catalysts for asymmetric transfer hydrogenation of ketones and imines being developed by Martin Wills at Warwick University are also noteworthy. 

 Asymmetric hydrogenations are widely used in industrial pharmaceutical synthesis and are least affected by scale up issues. Jacobsen epoxidation chemistry, the Buchwald–Hartwig aminations, Suzuki and other cross-coupling reactions as well as transfer-hydrogenation processes are also important transformations that are being developed on larger scales.

The ligands that will have the greatest impact on fine chemical/pharmaceutical synthesis are those that are being used today. For example, Chiral Quest manufactures many tons of products using our asymmetric hydrogenation catalysts, such as Rh-DuanPhos and Ru-C3-TunePhos. There are so many useful ligands and catalysts available that any new system will find it hard to compete.  A new ligand would need to be very easy to make, very active, and highly enantioselective or carry out a reaction that none of the existing technology can achieve.

Although the use of biocatalysis for asymmetric transformations is growing, chemocatalysis remains a highly competitive technology.