New techniques can reduce a 14-day safety and quality checks to four hours
Faster and earlier results are the mantra of biopharmaceutical companies when it comes to the tests required before they release a batch of products. Rapid microbiology systems, which can eliminate time-consuming culture procedures and improve the breadth of testing, have become the leading technology in the race to get products out the door, cutting the time to test results from 14 days to four hours.
“It takes too long for the pharmaceutical industry to get results from microbiology to release a batch of product,” said Philippe Gadal, PhD, CEO of AES Chemunex Inc., a rapid microbiology system maker based in Cranbury, N.J. “They have to keep a ton of product before they get results and can release it.”
Rapid microbiology tests aim to relieve that bottleneck. Some tell simply whether or not a microorganism is present; others, like the DNA-based test from DuPont Qualicon in Wilmington, Del., go to the next level by characterizing the organism. Even with those advances, the new rapid systems will require validation at customer sites and broader use before customers become completely comfortable with them.
“With rapid screening microbiology, you get results in a matter of hours. The question is if it will withstand the test of time,” said Fran McAteer, vice president of quality at Microbiology Research Associates Inc., a lab in Acton, Mass., that specializes in United States Pharmacopeia (USP) testing. He said he considers rapid microbiology technology to be in the beta-test stage.
Users of the new systems may find themselves in a catch-22 situation: Although they are attracted by the rapid test response, they must get U.S. Food and Drug Administration (FDA) approval for each use of the product, a process that can take from two months to two years.
“The FDA validates each specific application, by product and on site,” said Dr. Gadal. “And you need to submit plenty of data. So if you have 10 products, you need to make 10 applications [to the FDA].”
That makes the technology easier to deploy for new products, because drugs already on the market would require revalidation with the new systems. “This isn’t an easy task for customers,” said Karen Mullen, senior market manager for biopharma at bioMerieux Inc., a rapid microbiology system company in Durham, N.C. “Biotech companies like those using stem cells and cell transplants are probably close to putting this technology into routine use. It’s easier to do this with new products.”
Mullen said customers get a return-on-investment by being able to release products earlier. There are a number of rapid microbiology systems on the market, she said, but “there is no one size fits all. What you use depends on the expertise of the person running the system and what you are trying to find.”
Tweaking Existing Tests
BioMerieux, which already has several testing products on the market, launched a new version of its BacT/Alert sterility test system at the Parenteral Drug Association (PDA) conference in early October. Ray Turnley, senior product manager for BactT/ Alert, described the new BacT/Alert 3D Dual-T dual-temperature product as an innovation in the process of sterility testing.
“USP<71> customers have to test at two temperatures and with two media,” he said. The new product tests at 32°C and at 20-25°C. The prior version of the product tested at 32°C with two different media, one aerobic and the other anaerobic. The two cultures and temperatures more closely mimic USP<71> and European Pharmacopoeia’s EP 2.6.1 testing, he said. The dual product is being validated at two sites.
The BacT/Alert 3D Dual-T automates the compendial sterility test method. It can save three to four days over traditional manual methods, said Turnley. The manual methods use a raw material production sample and incubate it at two temperatures in two different media; it is then checked from day seven to day 14. BioMerieux’s automated system checks the samples every 10 minutes, performing impedance detection to see if there is any microorganism growth. Turnley added that the system can detect more organisms at a lower temperature, and it can test for yeast and mold.
The company also recently announced a new version of its Media Fill Test for validation and control of aseptic processes. The current practice requires technicians to manually check from 5,000 to 50,000 vials filled with culture media to look for turbidity. “Turbidity reading can be subjective,” said Chad Wilkey, product manager for culture media at bioMerieux. Human reading of so many vials is another bottleneck, often requiring the hiring of temporary help.
The new test, used in the filling process, changes the color of a contaminated solution, making it easier to spot. Wilkey said a great deal of research and development went into identifying a color that would work with the large potential number of microorganisms. “We test 150 different commonly isolated strains,” he said. “A color change happens 98% of the time. In the other 2%, the affected products turn turbid.” The new test is being evaluated.
Improved Sterility Test
AES Chemunex launched its Stereal-T sterility test at the October PDA conference. The test, done in real time, is specified by USP <71> and harmonized with EP 2.6.1 and the Japanese Pharmacopoeia. Dr. Gadal said the new test has improved discrimination, and he hopes to cut false positives by half. The test is done using the ScanRDI rapid analyzer for microbial control of filterable products, which has a sensitivity down to one cell. It is based on flow cytometry and uses fluorescent labeling of single cells with laser excitation. It distinguishes microorganisms based on their electronic and optical properties and keeps them live even after labeling.
“The critical limitation of the traditional sterility test is it takes 14 days for cell growth,” said Dr. Gadal. The improved test does not require cell growth, and it uses a different indicator targeting microbial viability: enzyme activity. The results are available in less than four hours. He added that the test has enhanced sensitivity compared to growth technology, along with improved recovery of injured, stressed organisms.
To combat the electronic noise issues inherent in flow cytometry, the company uses more than 20 discriminants to tell what is noise, mold, or yeast, for example. Up to eight tests per day can be performed, with results typically available within fewer than four hours from filtration.
Going forward, Dr. Gadal said the next step is to increase the level of automation on the ScanRDI and then look at adding capability to identify the microorganism. Current test requirements only require a “yes” or “no” for the presence of organisms to release a batch of pharmaceuticals.
DNA-Based Tests ID Pathogens
Another approach can both confirm the presence of and characterize an organism. DuPont Qualicon makes a rapid microbiological test that is DNA-based. The RiboPrinter system, an automated version of a Southern blot, provides strain-level characterization and identification information within eight hours by comparing an organism to 7,000 fragment patterns in the system’s database.
If there is contamination in a plant, areas as well as workers can be sampled to find a match, locate its source, and target remediation, said George Tice, global director of research and development for DuPont Qualicon. The system can be used to develop a microbial map of the facility to locate problem areas and spot trends, such as a pathogen that keeps reappearing.
The test results give a percentage of similarity of the pathogen to other fragment patterns in the database. To get a match, the similarity has to be at least 85-90%. Even when there is no match, users can generate their own libraries by storing samples for possible future use.
Testing begins with isolating a colony and preparing samples. The automated process starts when the cells are lysed to release the DNA. A restriction enzyme cuts the DNA into sequence-specific fragments, which are then separated by size using gel electrophoresis. The sample is transferred to a membrane, and fragments are hybridized with a DNA probe and mixed with a chemiluminescent agent. Light emissions are captured as image data using a digitizing camera. The system then extracts the RiboPrint pattern from the data, and the pattern is compared to the database for characterization and identification. Eight samples can be handled at a time.
The system can discriminate at the strain level. Alcon, the contact lens solution products company, used the system when a contaminant was discovered during water sterility testing. The contaminant turned out to be Staphyloccoccus. One sample was isolated from water and another from the garment of a line worker. Tice said it would have been logical to conclude that the line worker was the contamination source, but the system showed two different Staphyloccoccus strains. According to Tice, information below the species level cannot be attained using conventional testing methods. He said the company plans to add capabilities to the system, including testing for yeast and mold.