Saturday, October 22, 2011

Yale Students Develop Rapid Bacteria Detector

A team of engineering graduate students at Yale is working to create a new technology that could help prevent widespread food-borne illness and lead to quicker diagnosis of bacterial infections. The device, called alpha screen, is a portable, rapid, pathogen scanner that can detect as few as one bacterium.

“A rapid pathogen screener is a device capable of detecting microorganisms in near real-time, without the use of cultured colonies or a traditional microbiology lab,” PhD student Monika Weber told Security Management. Weber leads the development team.

Development of the device started as an assignment while under instruction of Prof. Mark Reed, the Harold Hodgkinson professor of engineering and applied science. The assignment was to develop a device that had marketable potential.

“We were thinking about what kind of device would be beneficial….We started asking doctors what areas of medical science could be improved,” Weber said.

Doctors told them that bacteria diagnostics would be a promising focus area because of the current time and costs of culture growth – which can sometimes take days or require separate labs. Their comments resonated with Weber, who recently became lactose intolerant because of a bacterial infection. Testing using the alpha screen is 10 to 50 times faster than the current methods—culture growth and polymerase chain reaction. The current cost of one test is estimated at $1-20 times less expensive than current testing methods. Weber says a quicker diagnosis could have prevented her condition.

Weber’s isn't the first endeavor focused on rapid bacteria detection. Researchers in the past have spent years developing similar technologies, but Weber says variations of the alpha screen are being developed to address all areas that have the need for bacteria detection.

The low cost of testing using the alpha screen will be beneficial to developing countries, Weber said—countries where a timely diagnosis could mean the difference between life and death. The device will allow doctor’s offices to make diagnoses in real-time.

For the food industry, the alpha screen has very promising applications as well, she said. “We hope that the alpha-screen technology…will help prevent disease outbreaks, like the 2011 Listeria outbreak in the U.S. and the E. coli outbreak in Germany. We anticipate that alpha-screen will have dramatic implications for domestic and international food security,” she said.

The alpha screen is still in its development stages. The basic version is a battery-powered device about the size of a coin. It can only detect one or two types of bacteria. “However, to meet the needs of prospective customers we have also designed a larger unit, which could detect simultaneously over 100 different types of bacteria,” Weber said.

The team is working to raise funds for further development and prospective commercialization. Earlier this month, they moved $20,000 closer to that goal after winning the Create the Future design contest. The contest awarded prizes to recognize research efforts in engineering focused on benefitting humanity, the environment, and the economy. More than 900 product ideas from 50 countries were entered.

Weber said she hopes the publicity for their work will pay off for the project.

“We hope to attract people and institutions that would sponsor research and investors to move the project along,” Weber said.

Weber’s isn’t the first endeavor focused on rapid bacteria detection. Researchers in the past have spent years developing similar technologies.

Wednesday, October 19, 2011

Debunking Rapid Method Myths at PDA

During the very popular Urban Myths session, I presented an overview of the myths associated with the inability of our industry to effectively implement rapid methods. Although a number of companies have successfully validated RMMs, they only represent a fraction of the industry that can benefit from using these alternative technologies. So what are the myths associated with RMMs? Here is a short list:

- Rapid methods are not accepted or understood by regulatory authorities, nor do they support QbD or PAT
- They will never replace pharmacopoeial tests
- There is little validation guidance
- RMMs offer no return on investment
- RMM use will result in exceeding specifications and action levels, which will translate to an increase in batch rejections
- Changing acceptance levels will not be allowed

During my presentation, I debunked each of these myths and many more, and provided support for rapid methods from global regulatory authorities in terms of validation, implementation and submissions. I also addressed where rapid methods can be used in contamination control programs, Quality by Design (QbD) and Process Analytical Technology (PAT) strategies. Much of what I discussed is already available for review in the Regulatory, Validation, and Return on Investment (ROI) sections at

Briefly, the regulatory authorities not only accept and understand RMMs, but also have embraced and encouraged their use for a number of years. When formal changes to existing regulatory dossiers is required, both the FDA and EMA provide guidance and policies on how to accomplish this. And the Australian TGA and Japanese PMDA also support the implementation of RMMs as well.

Rapid methods can be used to support QbD and PAT strategies, including, but limited to, in-process bioburden testing, environmental monitoring, and process water and endotoxin analyses. Many companies have already validated RMMs as alternatives to compendial finished product release testing, such as USP and Ph. Eur. sterility testing (publications on the use of RMMs for finished product testing can be reviewed on our References page). And firms can and have realized significant cost savings and cost avoidances when implementing novel microbiological technologies. Finally, rapid methods can also support a comprehensive contamination control program, and when contamination arises, RMMs can be used as investigative tools, providing results much faster than traditional means.

In summary, most if not all RMM myths are not true, the regulatory authorities want to see RMMs implemented and encourage their use, RMMs are directly aligned with the future state of pharmaceutical manufacturing, QbD, PAT and continuous process and product improvement, there is validation guidance available, and the cost of implementation can be a good investment.

Tuesday, October 18, 2011

Rapid Methods Session 2

The first speaker during this session was Claudio D. Denoya, PhD, Adjunct Professor Department of Molecular and Cell Biology, University of Connecticut. Dr. Denoya discussed the current microbiology curricula taught within academic institutions and the role pharmaceutical science and modern microbiological methods should play in these programs. The focus was to understand whether microbiology students and the courses they take are appropriate in preparing individuals for a career in microbiology within the pharmaceutical industry.

When reviewing the microbiology curriculum, many courses focus on biochemistry and very few will cover the application of microbiology concepts in industry, especially pharmaceuticals. Additionally, one of the areas that are absent in the academic curricula is how to assess, validate and implement alternative, molecular and rapid microbiological methods. Because alternative methods will play an important role in the future of contamination monitoring and control, and QC microbiology activities, it is critical that these concepts be included in academic courses, especially since this enhanced knowledge and associated skill sets will be the expectation for pharma microbiologists who will want to move up the technical career ladder.

As a case study, Dr. Denoya stated that out of 65 graduates from a scientific masters program in applied genomics (at the University of Connecticut), 63 of them obtained careers in their first choice within the pharmaceutical industry. The remaining students returned to school for advanced degrees.

The next speaker was Rudolf Gilmanshin, PhD, Vice President, Advanced Platform Research, Pathogenetix, who presented on a single-molecule technology for broadband detection and identification of bacteria. The technology is based on genome sequence scanning, or GSS. Generally, genomic DNA is obtained from a sample under evaluation, and is tagged, linearized and detected in a microfluidic chip. The DNA travels through areas of focused laser light (within the microchip) and the responses are compared against an internal database.

Long fragments of DNA are required from the sample (60-350 kb or 20-115 um), must be double stranded and free of nicks, be free floating in solution, be able to hybridize with the fluorescent tags, and be of high purity. Following lysis of the sample organisms, preparation occurs in about 3 hours in an automated washing and purification instrument. The fragment is then stretched within the microfluidics chip, and will then pass through separate lasers, which will excite the fluorescent tags. Organism concentration for use in the system is between 10^7-10^9 cells.

Restriction enzymes and the tags generate fluorescent signatures used for fingerprinting. Because of the uniqueness of underlying genomic sequences, hybridized tags generate the unique signals. The system is extremely sensitive, with a detection level of 0.1% sensitivity against the internal database. Beta systems will be placed in hospital clinical labs in Q4, 2012.

The last presentation in this session was made by Alessio Fantuzzi, PhD, Microbiological Project Supervisor, and Michele Bosi, Quality Control Manager, Chiesi Pharmaceutical. They presented a case study in development and qualification of an alternative method for the release of non-sterile and sterile products (e.g., sterility testing).

For sterility testing, they perform the compendial sterility test; however, they have added an additional reading phase during the test incubation period using the ATP bioluminescent Pallchek system. The use of the Pallcheck system represents a qualitative assessment of the growth of organisms if they were present in the original sample. To demonstrate equivalence against the compendial methods, they followed the validation guidance as specified in USP 1223 and Ph. Eur. 5.1.6.

For non-sterile product the incubation period lasts for 24 hours, which is the time it takes for the system to detect the number of organisms that is at a threshold level higher than background noise. For sterile product, after a 48-hour incubation period, they filter the media and assess the media for microorganisms using the Pallchek system. To support this rapid sterility test, they also utilize an enhanced environmental monitoring and risk assessment program, and the total sterility testing program has been reduced from 14 days to 8 days.

The success of these validation programs have prompted their company to implement rapid methods ofr all of their future products/formulations.

Rapid Methods Session 1 at the PDA Global Microbiology Conference

This is the first of two rapid method sessions at the PDA Micro Conference. The first speaker was Michele J. Storrs-Mabilat, PhD, Global Scientific Partnerships Manager, Industrial Microbiology Division, bioMerieux, Inc. She presented information on a novel rapid and automated prototype system for the microbiological monitoring of sterile pharmaceutical environments. The Midass system was introduced, and Midass is an acronym for microbial detection in air system for space. This technology was originally developed for use by astronauts on route to and from Mars, where the air in the space capsule will recirculate for a period of up to 3 years, and there will be a need to assess the microbiological state of the capsule environment during the journey.

For the pharma industry, the Midass system is a complete system for monitoring surfaces, personnel and air. The system utilizes a peppermill-type collection device for air sampling, cellular lysis and nucleic acid purification. A separate NASBA card, which contains primers and probes/beacons, is used to amplify the purified rRNA targets. NASBA is used instead of conventional DNA/PCR amplification because RNA is a better predictor of cellular viability, is not susceptible to contamination by extraneous DNA, and the amplification reaction is carried out at a single temperature instead of multiple temperatures as is required by PCR. Amplification takes place in 60-90 minutes, and the system will detect both bacteria and fungi. The time to result is 3 hours, but there is an opportunity to reduce this time in the future. A table-top instrument is used to process the peppermill and the amplification card.

Total viable counts are obtained not in the form of colony forming units (cfu), but in gene copies or genomic equivalents (Geqs). Sensitivity is estimated at 1 cfu (or 1 Geq) per cubic meter of air or per 25 square cm for fungi, and 20 cfu (20 Geqs) per 25 square cm for bacteria (work is still underway to determine the sensitivity for bacteria in air). Initial testing shows encouraging equivalence between a cfu and a Geq. Finally, the system is considered to be non-destructive, where the purified nucleic acid material may be stored for further analysis, such as microbial identification.

The second speaker was Gene Zhang, PhD, Principal Scientist, Bayer Healthcare Pharmaceutical, who presented a Case Study on Validating a Microbial ID System to Meet the New Regulatory Requirements for Part 11.

There are a number of microbial identification rapid methods systems available and many are operating via computerized systems. The pharma industry is now expected to ensure that the data management capabilities and electronic records for these types of systems meet Part 11 compliance. In fact, FDA warning letters have included reference to computer systems that have not been validated against the expectations to Part 11 requirements. Dr. Zhang reviewed how a firm can meet these requirements and used a rapid nucleic acid amplification identification system based on 16S rRNA sequencing as an example.

USP Update on Rapid Micro Methods and Chapter 1223

Radhakrishna Tirumalai and James Akers both provided updates to the USP Microbiology Expert Committee activities. Of note, the following rapid method topics were discussions.

During last year’s PDA Micro meeting, the USP stated that they were going to update/revise the existing USP 1223 informational chapter and provide additional guidance with respect to the use of alternative micro methods. First, we must be reminded that the use of RMMs as a replacement for existing methods is nothing new, as the USP provides guidance on the validation of alternative methods. More importantly, USP micro methods are intended to be referee tests (i.e., adjudicative) for the analysis of monograph products and compendial articles, and they were not intended to be QC release assays or in-process tests. Actually, USP referee tests were not intended to be used as QC assays without modification, and this modification is the responsibility of the method user.

USP <1223>, the informational chapter that provides guidance on validating alternative or rapid methods, is under revision. Some of the changes from the current version may include enhanced guidance on method selection and qualification, and more specific content than what is currently provided. Additionally, the committee is concerned that RMM implementation is being held up because users are too concerned at arriving at a perfect definition of method equivalence. Therefore, we may see some changes in how the USP recommends how to determine whether an alternative method is as good (or better) than a compendial method that is current in use.

Next, the expert committee is now exploring the development of a new referee sterility test; however, the referee method cannot be sourced from a single, patented technology. The committee will initially y will focus on biologics (cytotherapy/regenerative medicine products) and radiopharmaceuticals. And they will get support from the USP biologics committee as well as scientists from CBER (please see my August blog posts for additional guidance form the FDA on the use of RMMs for sterility testing).

Finally, the committee plans on providing validation-useful information for the development and validation of HPLC methods for antibiotic assays for products where micro methods are still being employed.

Monday, October 17, 2011

Dr. Fung Discusses a 30 Year Review of Rapid Methods in the Food Industry

Welcome to the 6th Annual Global Conference on Pharmaceutical Microbiology! There are a record number of attendees this year, so the conference is sure to provide excellent opportunities for interaction with microbiologists from across the industry. Over the next few days, I will be blogging on presentations related to rapid and alternative microbiological methods.

The opening keynote address is being presented by a world-renowned microbiologist and subject matter expert in rapid methods for the food industry, Dr. Daniel Y.C. Fung. Dr. Fung is Industry Professor, Food and Science, at Kansas State University. His presentation focused on Global Developments of Rapid Methods and Automation in Microbiology: A Thirty Year Review and Predictions into the Future.

Rapid methods and automation in microbiology is a dynamic area of technological advancement sustaining a stream of emerging technologies. Rapid microbial methods continue to offer unique opportunities for improving product quality assurance and economy of quality control and manufacturing operations. Almost ten years ago, improvements in microbial isolation, rapid detection, characterization, and enumeration lead to his prediction “…companies that aren’t converting to rapid methods won’t be in business in 10 years…”

Dr. Fung reviewed the use of rapid methods within the food and medical sectors since the 1960’s. Methods have included modifications of traditional, growth-based procedures using conventional medium, including a double tube agar method Dr. Fung developed himself. In this procedure, growing C. perfringens was able to be viewed within a few hours. And over the years, more automated systems were being introduced. For example, impedance microbiology procedures have been around for more than 30 years, as well as methods for the detection of ATP.

Immunological dip-sticks then came on the market, which provided results on the presence of food-borne pathogens in as early as 10 minutes. Today, we can utilize a wide variety of molecular and nucleic amplification systems, including automated, real-time PCR, as well as novel biosensors, microarrays and nanosensors.

Within the food processing sector, it was projected that more than 740 million micro tests were performed in 2008 by more than 40,000 food processing plants, and it is estimated that the worldwide market for micro testing is more than $2 billion. And the market for food microbiology testing continues to grow, year over year. For example, the rate of growth of micro testing from 2008 to 2010 was more than 6%. But Dr. Fung also stated that the use of rapid methods can also provide considerable cost savings, depending on the method being utilized.

The take home message from Dr. Fung’s keynote is that the number of microbiology assays associated with the monitoring of food will continue to increase, especially in light of recent contamination events, and that rapid technologies will play a very important role in protecting the world’s food supplies.

When asked what the pharmaceutical industry can learn from the food industry (in terms of the adoption of rapid methods), Dr. Fung stated that the expectations for microbiological safety is much higher in the pharmaceutical industry than in the food industry, and that we can benefit greatly from the implementation of rapid methods. Interestingly, the food industry looks up to the pharma industry for guidance on excellence in microbiology testing. Their perception is that we pharma microbiologists strive for perfection, and that we are always looking at ways to implement new technologies. Unfortunately (from my point of view), our industry has been extremely slow to adopt rapid methods for a number of reasons, and that the food industry is actually well ahead of where we are today. This will be a topic of discussion during my rapid methods presentation tomorrow afternoon.

Saturday, October 15, 2011

Live Blogging from the PDA Microbiology Conference

As I did last year, I will be blogging live from the PDA 6th Annual Global Conference on Pharmaceutical Microbiology. Here are the following rapid micro method presentations that I will be attending on Monday and Tuesday:

Keynote Address: Global Developments of Rapid Methods and Automation in Microbiology: A Thirty Year Review and Predictions into the Future. Daniel Y.C. Fung, PhD, Industry Professor, Food and Science, Kansas State University

A Rapid and Automated Prototype System for the Microbiological Monitoring of Sterile Pharmaceutical Environments. Michele J. Storrs-Mabilat, PhD, Global Scientific Partnerships Manager, Industrial Microbiology Division, bioMerieux, Inc.

Advanced Microbiological Systems—A Case Study on Validating a Microbial ID System to Meet the New Regulatory Requirements for Part 11. Gene Zhang, PhD, Principal Scientist, Bayer Healthcare Pharmaceutical

The Benefits of Developing an In-house Disinfectants Qualification Process: Understanding, Responsiveness, and Control. Eric Myers, Senior Supervisor - QC Microbiology, Pfizer, Inc., Claudio D. Denoya, PhD, Adjunct Professor, Department of Molecular and Cell Biology University of Connecticut

Single-Molecule Technology for Broadband Detection and Identification of Bacteria. Rudolf Gilmanshin, PhD, Vice President, Advanced Platform Research, Pathogenetix

Rapid Micro Project in Chiesi Pharmaceuticals Group: Development and Qualification of Alternative Method for the Release of Non-sterile and Sterile Products. Alessio Fantuzzi, PhD, Microbiological Project Supervisor, Chiesi Pharmaceutical, Michele Bosi, Quality Control Manager, Chiesi Pharmaceutical

Debunking the Myth’s Surrounding Rapid Microbiological Methods and Their Impact on Pharmaceutical Manufacturing and the Quality of Medicinal Products. Michael J. Miller, PhD, President, Microbiology Consultants, LLC.