Microbiological Testing of Gelatin Capsules

Introduction

Two-piece capsules have been used for almost a century in the pharmaceutical field, and gelatin has been adopted as the main material of these capsules due to its excellent characteristic as a gelling agent. The gelatin dissolves under high concentration into water of a high temperature and quickly gels in room temperature. The thickness of the film made by the gelatin becomes uniform.

The gelatin capsules consist of gelatin, plasticizers and water. Modern day shells may, in addition, consist of preservatives, colors, opacifying agents, flavors, sugars, acids, enteric materials etc. A mixture derived from pork skin and bones is used in capsules.  Pork skin gelatin contributes plasticity while bone gelatin gives firmness.

One important reason for the exclusive use of gelatin for making hard and soft capsules is its solubility characteristics in stomach fluids. It absorbs cold water readily, though the rate of absorption depends upon moisture content of gelatin. Bloom Strength is an empirical gel strength measure which gives an indication of the firmness of gel. The plasticizers used are glycerin, sorbitol etc.  Preservatives, if included, are generally a mixture of methylparaben (4 parts) and propylparaben (1 part) to the extent of 0.2%. Flavors, if added, should not exceed 2% and are generally ethyl vanillin or essential oils. Sugar, if included, may be up to 5% to give the gelatin shell desirable chewable characteristics.

Microbiology Testing

Each incoming lot of capsules needs to be tested using USP <61> and USP<62>. The total aerobic bacterial count should not exceed 3,000 cfu per gram, the combined yeast and mold counts along with Bile-Tolerant Gram-Negative Bacteria should not exceed 300 cfu per gram.  Material must also meet the requirements of the tests for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.

Challenges of Current Methodology

Testing gelatin capsules for microbiology might result in several challenges.  The 1:10 dilution of the product has high viscosity and is sometimes difficult to pipette. Many capsules have vivid colors that interfere the reading of the plates. 

The current methods used in microbiology originated over 100 years ago.  There have been limited improvements in methods used for microbiological testing in the last decade. The current USP methodology is slow, requiring up to 5-7 days for product release, is manual, and in many cases is inaccurate.  Paper-based QC laboratory processes can be expensive, error-prone, time and labor-intensive.  

Rapid microbiological methods (RMM) offer a cost effective alternative to USP methodology.  With an RMM’s high degree of automation, significant reduction in time to results, faster product release, and improved process control, while providing enhanced accuracy, better repeatability, and total automation

BioLumix Alternative

The BioLumix System simplifies testing, expedites time to results, reduces the testing cost and accelerates product release while providing better control of microbial contamination.  The system can be used to automate microbial testing with a more cost effective and streamline manner. The system reduces the error rates produced by paper-based activity recording and batch data entry.  The BioLumix system also helps automate microbiological quality control processes.

The BioLumix technology is based on continuous monitoring of changes in color or fluorescence as a result of microbial metabolism in ready to use test vials. The results are presented as soon as detection occurs without any involvement of the operator. The fully automated system offers a paperless operation with increase efficiencies.

The key to the technology is the two-zone ready to use vials which eliminates any product interference.  Many types of gelatin capsules were tested in the BioLumix system for total aerobic bacterial count, combined yeast and mold counts, Bile-Tolerant Gram-Negative Bacteria, and for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.  As can be seen in the figure, there is no product interference even with capsules containing the most vibrant colors.  All assays yielded clear results that correlated well with the USP methodology.

Key: Dark Blue-clear capsule;Green- Brilliant Red Capsule; Light Blue-Brown capsule;Red- Teal capsule; Purple-Inoculated Brilliant Red Capsule.

The BioLumix system is validated against the USP methodology.  All assay results are complete in 48 hours with an automated Certificate of Analysis; resulting in faster product release.

Come and See us at Supply Expo West in Anaheim March 11th – 13th

This marks our third year exhibiting at the Supply Expo West trade show located at the Anaheim Convention center March 11th – 13th and thanks to our growing customer base we are rapidly becoming a recognizable solution to today’s cGMP microbial requirements.  I wanted to take this opportunity to invite you to our exhibit (booth #708) at the show and express what’s new at BioLumix.

Currently, the majority of our clients are manufacturers of Nutraceutical and Dietary Supplements. In the past year we have doubled our customer base, many of which are recognizable names in the industry who will also be exhibiting at this year’s Supply Expo West and Natural Products Expo.

At BioLumix we continue to innovate, growing our assay repertoire and capabilities – allowing you to perform all required assays on raw material, finished products, as well as test environmental samples and processing water. We are also expanding our offering into the Pharma – OTC, Cosmetic, and Dairy Industries.  Come to our booth (#708) or contact us directly (734-984-3100) to learn more about the exciting new developments at BioLumix; such as:  our environmental monitoring and water testing solutions, as well as our labor and material saving Preservative Efficacy Test (PET).

FDA Meeting

The BioLumix rapid, automated microbial detection system complies with cGMP regulations and comes with a full validation package against the standard USP methodology.  BioLumix representatives met with FDA officials of the Center for Drug Evaluation and Research (CDER) to present its technology, answer questions, and demonstrate our Rapid Microbiological Method (RMM).   The BioLumix technology was warmly received by the CDER representatives and we are encouraged by our ability to submit a Drug Master File (DMF). This will help to simplify and speed up the regulatory review process for companies adopting our technology.

Fast Return On Investment

An automated Certificate of Analysis is generated in just 48 hours!  Streamlining your microbial testing with our reliable, easy-to-use system will save you time and money.   In most cases a very quick return on investment can be proved.  We invite you to contact us and inquire about our Return On Investment calculation.  We have shown in most cases the BioLumix System will pay for itself within the first 6 months of ownership.

Hear What Our Customers Are Saying

Don’t just take our word for it, talk to our customers.  We have recently conducted a customer survey and are proud to share we have 100% customer satisfaction and that all of our customers would recommend the BioLumix.  Unanimously, our customers agree – the main features of the BioLumix system are:  Speed to results, simplicity of use, accuracy and clarity of data presentation. You are free to contact our customers to discuss their first hand experience.

Free Product Trial

Give us your most difficult samples and we will test them for free.  We will provide you with a detailed report which includes a side-by-side comparison to USP methodology along with a sample of the BioLumix 48 hour automated Certificate of Analysis.  The data generated is strictly confidential and is only used to help validate the system for your company should you decide to purchase.

We look forward to working with you and earning another satisfied customer!

Rapid Detection of Coliforms in Yogurt

What are coliforms?


Coliforms are a group of bacteria commonly found in the environment, including soil, surface water, vegetation and the intestinal tracts of warm-blooded animals. Most coliforms do not cause disease, but a small percentage can cause illness in people, especially young children, the elderly, and those with weakened immune systems. Coliforms are rod-shaped Gram-negative non-spore forming organisms. They can ferment lactose with the production of acid and gas when incubated at 35-37°C. Coliform bacteria are not a traditional taxonomic group, like Salmonella, Escherichia coli, or Listeria. Instead, the coliform bacteria are a collection of strains in the Enterobacteriaceae family. E. coli, Enterobacter spp., Klebsiella spp., and Citrobacter spp. are the most common coliforms.

How do coliforms get into milk?

Coliform bacteria are normally shed in the feces of healthy livestock, including dairy cattle. Thus, poor herd hygiene, contaminated water, unsanitary milking practices, and improperly washed and maintained equipment can all lead to elevated coliform counts in raw milk at the dairy farm. The milking of cows with wet and manure-soiled udders and inadequately cleaned milking equipment are the most common ways for coliform bacteria to enter milk on-farm.

Coliforms in milk: What does it mean?

The coliform bacteria count is used as an index of the level if sanitation and/or water quality employed in the handling and processing of milk products. In dairy products, the process of pasteurization easily kills coliform bacteria. Therefore, the finding of coliforms in pasteurized products indicates some level of contamination has occurred after pasteurization during product manufacturing or packaging. Hence, coliforms are used as a general indicator of sanitary conditions in dairy production and processing environments.

Traditional methods for testing Coliforms


Traditionally the agar plate count method using VRBA (Violet Red Bile Agar) and the MPN methods are being used.  The plate count method takes 24 hours to perform and can use 1.0 ml of 1:10 dilution and as a result has a sensitivity of <10 cfu/gram.

When higher sensitivity is required the MPN (Most Probable Number) method can be used.  Multiple tubes (typically 3) are inoculated for each dilution.  The method involves the inoculation of at least 3 decimal dilutions (a total of 9 tubes) of LST (Lauryl Tyrptose Broth).  Any LST tube that shows growth and gas production is transferred to BGLB (Brilliant green lactose bile), the MPN is calculated based upon the positive BGLB tubes.  This assay is very labor intensive and takes 3-5 days to complete.

Rapid High volume Automated Method for the Detection of Coliforms

Direct inoculation of Product and Speed to Results


In a recent study 25 different types of yogurts were tested by the BioLumix method and the plate count method.  The BioLumix ready to use vials were directly inoculated with 1.0 ml of various yogurts and monitored in the BioLumix system for 12 hours.  Figure 1 shows the curves obtained.  Key:  Dark blue – Grape yogurt, Green – Grape yogurt inoculated with Citrobacter, Lt Blue – Raspberry yogurt, and Red – Raspberry yogurt inoculated with E. coli.

 All products tested using the automated BioLumix assay for coliforms yielded results in a considerably shorter test time (typically 10-12 h) than the conventional Violet Red Bile Agar (VRBA) method (24 h to 72 h with confirmation). The BioLumix method can detect <1 cfu/gram of product, being 10 times more sensitive that the plate count method.  The BioLumix coliform medium had better selectivity in detecting coliforms, eliminating the need for the confirmation step.

Labor Savings

The BioLumix System offers a significant reduction in time to obtain results while reducing hands-on labor due to its automation and simplicity of use.  The procedure involves 2 simple steps:

  1. Add 1.0 ml of product directly to the ready to use vial
  2. Add the sample information to the computer

The system automatically will run the system and generate the desired reports. Therefore, there is no sample preparation required no media preparation or counting of plates.  Due to its simplicity the assays can be performed by less skilled personnel.

Automation

The BioLumix system is a fully automated system offering automated data achieving, and automated reporting.  It offers the ability to operate the laboratory as a paperless operation. Due to its connectivity through the Intranet it allows for automated transfer of product release information to the warehouse.

Free Product Trial

Interested in a free product trial?  Contact BioLumix today!  P.734-984-3100.

Rapid Microbiological Method or Conventional Method? What Should You Use?

In the get-it-done-yesterday environment companies are finding that the traditional microbiological methodologies and especially sending microbiological samples to outside labs cost them time, money and opportunities1.  Internalizing the microbiology testing and especially adopting rapid microbiological methods (RMM) can significantly speed up the time to results from 7-10 days to 24-48 hours.  The time saved creates opportunities for faster response to contamination, quicker product release, faster delivery of product to the marketplace, reduced warehouse space, and enhanced product quality.  Advantages of RMM can include: Greater accuracy, better sensitivity, increased sample throughput, automated data capturing allowing easier data handling, and reduced cost for product release.

However, like any other investment it competes for company resources.  Therefore, there is a need to calculate the financial benefits of the investment in RMM.

Dr. M. Miller, an authority on RMM, has written extensively on this topic and suggests that before adopting a new RMM or internalizing Microbiology the key steps required include2, 3, 4:

  1. Review of existing technologies
  2. Understand the technical and financial benefits of RMM
  3. Develop the business case for RMM

This will include the use of financial models that can compare the overall costs associated with the current microbiology method with the costs and savings associated with the purchase, qualification, and implementation of the RMM

Return on Investment (ROI)

ROI is the ratio of money gained or lost on an investment relative to the amount of money invested.  According to Dr. Miller3 for RMMs, the cost of performing the conventional method (CM) is compared with the cost (and savings) of using the new method. The information is reported as a percentage and usually represents an annual or annualized rate of return. The ROI is calculated using the following formula4:

CM costs can include:  cost of consumable; reagents and supplies; sample preparation time; data management documentation and record retention time; as well as cost of labor and overhead.

Potential investment in RMM can include:  Capital cost; and validation cost.

Potential cost savings of RMM can include:  Reduced time for product release; Lower headcount; lower re-processing fees; reduction in plant downtime; increased production yields; reduced raw materials, in-process and finished products inventory holding; reduction in back orders; increased production flexibility; and better protection of company image.

Payback Period (PP)

The PP is the time required for the return on an investment to “repay” the sum of the original investment. In the context of implementing an RMM, this would be the time (usually in years) required to realize sufficient cost savings to pay for the initial investment of the RMM capital equipment as well as for qualification and implementation activities3, 4.

Requirements from RMM

While Rapid Microbiological Methods (RMM) offer high degree of automation, significant reduction in time to results, faster product release, ability to employ non-microbiologists to operate the system, and improved control; there are three basic objectives that must be meet:

  1. Comply with FDA and cGMP regulations
  2. The RMM must be validated against the standard USP methodology using a protocol similar to USP <1223>.
  3. Ensure a swift return on investment.

Additionally, the ideal system should be capable of:

  1. Performing all the microbiological assays required to be performed by lab on a single platform.
  2. The technology should eliminate or at least minimize any product interference.
  3. Allow for a paperless microbiology laboratory-allowing the laboratory to operate without handwritten data entries
  4. Automate the system reporting
  5. Real-time communication to locations outside the lab to provide early warning of contamination

Required Commitment from RMM Vendors

Validation:

To make the transition to a rapid method the vendor should help in the system validation to make the transition to the new system smooth and “painless”. It should include Installation Qualification (IQ); Operational Qualification (OQ), and help with the Performance Qualification (PQ). PQ is the most extensive portion of the validation and sometimes companies considering RMM are deterred by the amount of work required for validation. Vendors can generated most of the data and submit it to the FDA in a form of a DMF.  The same information can be supplied to new users together with unique data pertaining to their products.

Software:

The software must be compliant with 21 CFR Part 11.  21 CFR Part 11 regulations are established requirements to ensure that electronic records and electronic signatures are trustworthy, reliable and generally equivalent substitutes for paper records and traditional handwritten signatures. Any instrument system containing software must be compliant with this regulation.

Technical Support:

Instruments suppliers should guaranty live technical service that is fast, knowledgeable, and readily available.

The BioLumix system is an example of a system that fulfills the requirements listed above.  Its straightforward streamlined design offers rapid accurate results leading to reduced material-holding time for faster product release.  Early warning of contaminated samples as well as sample release information could be automatically communicated through your intranet, significantly improving your company’s efficiencies.

To evaluate the value proposition of RMM systems BioLumix offers a free assessment of your specific ROI and Payback Period.  This will help you determine whether there are sufficient cost benefits of adopting a RMM system.  Past ROI calculations demonstrated that in many situations the ROI obtained showed that a RMM, like the BioLumix system, improved the company’s bottom line and satisfied the financial expectations of site management.

References

  1. 2009. Gadal, P.; Yvon, P. Rapid Microbio ROI – Calculating scientific benefits as return on investment dollars. Pharmaceutical Formulation & Quality. 11(3): 44-47.
  2. 2009. Miller, M.J. Breaking the rapid microbiological method financial barrier: A case study in RMM return on investment and economic justification. BioPharm International. 22(9): 44-53.
  3. 2009. Miller, M.J. Ensuring ROI from your RMM. Pharmaceutical Manufacturing. 8(6): 32-35.
  4. http://rapidmicromethods.com/files/roi.html
  5. USP <1223> Validation of Alternative Microbiological Methods.

Ruth Eden, PhD

President

BioLumix. Inc.

BioLumix to Submit DMF to FDA for its Rapid Microbiology Method in Pharma

BioLumix Meeting with FDA


BioLumix met with FDA’s Center for Drug Evaluation and Research (CDER) personnel to present its new technology, answer questions and demonstrate how the BioLumix rapid, automated microbiology can be used in the Pharmaceutical industry.  The technology was warmly received by the CDER and BioLumix is encouraged by its ability to submit a Drug Master File (DMF). This will help to simplify and speed up the regulatory review process for companies adopting its technology. 

The DMF is a technical document that contains support data for specificity, limit of detection, robustness, ruggedness, and equivalence to USP methodology.  The DMF can be used to streamline the validation of the BioLumix system, saving companies that wish to adopt the technology time and money.  For over the counter drugs the documents can serve as the basis for the validation package.

FDA’s Support and Encouragement of New Technologies

Dr. David Hussong, FDA’s Associate Director for New Drug Microbiology, Office of Pharmaceutical Science, CDER stated that “FDA actively encourages use of new technologies including rapid microbiology methods” (RMM).  During the 2007 PDA 2nd Annual Global Conference on Pharmaceutical Microbiology, Dr. Brenda Uratani, consumer safety officer for the CDER, described the benefits of using rapid methods in microbiology.  Drs. David Hussong and Robert Mello (New Drug Microbiology Staff at CDER) published a paper and stated the following: “New microbiology methods can offer advantages of speed and precision for solving microbiological problems associated with materials or environmental influences.  Neither Corporate economics nor regulatory attitudes should be a barrier to the use of new testing technologies or different measurement parameters.  In fact, if we are to increase our understanding of quality-based products and processes, then quality by design principles and risk analysis methods must be extended to the development of new microbiological technologies. This approach will drive process engineering to yield real, measurable gains in microbiological product quality assurance.”

Dr. Bryan Riley, New Drug Microbiology Staff at CDER, published a paper describing the opportunities for RMM’s within the pharmaceutical industry, “The use of rapid microbiology methods by the pharmaceutical industry should offer many advantages. Receiving microbiology test results sooner will provide for better control and understanding of the manufacturing process via faster feedback”.  He continued stating that “There are many exciting potential uses for rapid microbiology methods in the pharmaceutical manufacturing process, and industry should not feel that FDA will be a hindrance to the appropriate use of these methods.”

Why New Rapid Automated Methods Are Desired?


Newer microbiological methods can improve company’s quality of testing and result in significant financial savings.  Such methods can reduce cycle times resulting in a leaner and more responsive supply chain.  At every point where microbial testing is done (raw materials, work-in-progress, or finished products) batches may be held in quarantine for up to seven days before they are pronounced ready to move to the next stage.  Waiting for microbial results can tie up working capital and results in storage expenses and a delay in supplying products to the market.  The faster results can identify a contamination and enable implementation of corrective action, and cost savings.  Consequently, an increasing number of pharmaceutical companies are becoming interested in adopting RMM’s.  By utilizing rapid methods for microbiology, manufacturers can dramatically slash the amount of time products must be held for microbiology testing and drive new efficiencies with less capital tied up in finished goods inventory, and reduced warehouse space requirements.

Fast, Reliable Preservative Efficacy Test and Microbial Detection for Cosmetics

Microbial contamination of cosmetic products is important to the industry and can be a cause of both product and economic losses. The water and nutrients present in the product can make them susceptible to microbial growth; although, only a few cases of human injury due to contaminated cosmetic products have been ever reported. More often, microorganisms are the cause of alterations in product appearance and smell; such as changes in viscosity and color or offensive odors.

The Challenge of Cosmetic microbiologists stretches beyond providing clean consumer products to be sold in stores, they need to provide safe products which will be reused following subsequent consumer self contamination.  Every time a brush, a sponge, a hand or other cosmetic applicator is wiped from one’s self and reintroduced to the product, microorganisms (which exist on everyone’s skin, eye lashes, lips, etc) are being transferred into the product. Cosmetic microbiologists need to develop product preservative formulations which are capable killing these newly introduced microorganisms before they multiply to harmful levels.  The demand to develop consumer friendly as well as environmentally friendly formulations that are less conspicuous to consumers and still protected against microbial contamination is greater then ever.  

To assure that the cosmetic products are going to be safe even after re-contamination by the user a “Preservative Efficacy Test” (PET) or “Challenge Test” is performed. The PET is performed by artificially and heavily contaminating a product with representative microorganisms. After defined periods of time, a specifically defined reduction in the microbial populations must take place.  The current plate count method requires numerous dilutions, sample plating, incubating, not to mention a great deal of plate handling and reading, along with tedious manual data transcription.

Resent trends in this field not only include research for fast, reliable alternative methods to detect microbial contamination, they include efforts to automate Preservative Efficacy Testing. To date there has not been an effective automated method developed for the Preservative Efficacy Test (PET).    

The BioLumix Alternative

BioLumix now offers an automated solution for both PET and microbial contamination on a single platform saving labor and consumables while reducing time to results.

The BioLumix Optical Technology is based on continuously monitoring changes of color / fluorescence as a result of the microbial metabolism in test vials. The results are presented as soon as detections occur without any involvement from an operator.  The technology utilizes a CO2 sensor that is a universal detector of aerobic bacteria, yeast and mold metabolism (Remember the Kreb’s cycle? Part of this metabolic pathway involves the conversion of carbohydrates, fats, and proteins into water, CO2 and energy).   

The procedure utilized allows for an easy estimate of the initial inoculum with a single ready-to-use vial that can replace multiple plates.  After the product interaction with inoculated product the system automatically calculates if the appropriate reduction in numbers of organisms was achieved.

The system is fully automated with automated data archiving, data maintenance in databases, and automated report generation.  It is paperless, increasing efficiency and saving on disposables, time and space, and best of all the system is unaffected by product interference.  Please call us for a free product trial.

Kevin LaBrecque

Fast, Accurate Dairy Microbiology Testing For Yeast and Mold

Cultured dairy products are the fastest growing area of consumer dairy consumption.1 Rapid dairy microbiology testing for yeast and mold, the most common contaminants in these products, is critical to the industry and consumer safety. Yeast and mold can cause substantial economic losses and produce harmful metabolites.2

Traditional dairy microbiology testing requires 5 days to complete using the plate count methodology. Sending samples to outside labs adds to the cost and time of testing. A microbiological system developed by BioLumix has cut dairy microbiology testing time to as little as 24-48 hours. With the BioLumix microbiological system, manufacturers can streamline existing dairy microbiology testing operations or internalize their outsourced testing for reduced costs and improved efficiency.

In-house microbiology testing with the BioLumix system is simple. Samples are placed in ready-to-use, disposable vials that contain a transparent solid sensor at the bottom. These sensors detect the production of CO2 by all yeasts and molds. This means that if yeasts and molds begin to grow in the medium above the sensor, they will produce CO2 that will diffuse into the sensor and change the sensor’s color. Only gases can penetrate the sensor, which means the optical readings cannot be masked by any liquids, microorganisms, and/or particulate matter.

The BioLumix microbiological system can then detect any optical changes, presenting the results of the assays as soon as detections occur with no need for involvement of an operator or a microbiologist, providing significant savings on laboratory labor. Any out-of-spec samples are flagged in red, demanding attention. The greater the contamination level, the faster the result, ensuring a rapid warning of poor-quality raw materials, finished products or any equipment line issues. The speed of these dairy microbiology results ensure that dairy product manufacturers don’t have delays from testing so they can move products from the warehouse to the marketplace quickly and without premature spoilage.

A recent dairy microbiology study assessed the BioLumix microbiological system with various cultured dairy products that were clean, naturally contaminated, or inoculated with different yeast or mold strains. This included products with high levels of active starter culture or probiotics.3 The BioLumix microbiological system demonstrated 100% accuracy in detecting over 100 different combinations of clean contaminant products, which was comparable to plate count methodology, the current standard in dairy microbiology. However, the BioLumix microbiological system reduced detection time from 5 days to 24 hours for most yeast and 48 hours for mold.

The BioLumix microbiological system simplifies and automates your laboratory procedures by delivering shorter sample preparation, automated real-time communication, fast product release, and early warning of contamination, as well as automated data entry, data archiving, report generation and product release. With reliable dairy microbiology results in just 24-48 hours, dairy manufacturers can focus on efficiency and product quality. For a full reprint of the study or any further information on the BioLumix microbiological system, visit www.mybiolumix.com or call 1-734-984-3100.

References

1. Cogan TM, et al. Advances in starter cultures and cultured foods. J Dairy Sci. 2007;90:4005-4021.

2. Hussein HS, Brasel JM. Toxicity, metabolism and impact of mycotoxins on human and animals. Toxicology. 2001;167:101-134.

3. Eden RF, Brideau R. Rapid automated detection of yeast and molds in cultured dairy products. International Association of Food Protection Annual Meeting 2009.

Karen F. Vieira, Ph.D.

Environmental Monitoring in the Pharmaceutical & Nutraceutical Industries

FDA expects manufacturers to be in control of the environmental conditions within the manufacturing facility. Controlling the environmental condition is not only a regulatory requirement but also part of protecting and producing a quality product. Environmental monitoring of Pharmaceutical & Nutraceutical manufacturing facilities provides assurance that the environment is in control, and in compliance.  There is substantial evidence establishing a direct relationship between the level of environmental control and the final quality of the product.

A variety of methods are available to measure total particles in the air, Total Organic Carbon (TOC), and ATP (Adenosine Tri-phosphate).  These methods while very fast to perform do not correlate with total bacterial count or any specific group of organisms in many cases.  Therefore, these results do not tell us when we have viable organisms in the environment and on production lines. The standard plating methodologies can take several days for results.  Rapid microbiological methods can provide the solution

Why Not Monitor ATP?

Adenosine-Tri-Phosphate (ATP) is an energy molecule stored in all microorganisms and therefore an indicator of life.  ATP bioluminescence began to gain traction over plate count approaches in the late 1980s, especially in the food industry.  It is basically a “dirt” detector rather than a microorganism detector.   It is a common misconception that the results received from ATP testing systems in relative light units (RLUs) for surface samples should correlate with a microbial total plate count result.

The lack of correlation between ATP and plate counts means that samples can have high ATP readings and no bacteria or conversely have low ATP readings and high bacteria count.  For example, the inside of a tomato is sterile and doesn’t contain bacteria, yet it contains large amounts of ATP.  Likewise, it is possible to have bacteria and still get pass from an ATP based test, because 10,000 bacteria/swab are required for detection.  To complicate the matter further, there are different levels of ATP in different cells on the surface. Yeast, for example, contains 100 times the ATP amount of Coliform bacteria.

The standard used for post-cleaning surface contamination is typically 100 – 1,000 bacteria / 100cm2, and 10-50 yeast and molds which is lower than the detection limit for the ATP test.   Therefore, ATP results do not tell whether the surface is microbiologically acceptable.  In other words, it does not tell how many or what kind of microorganisms or chemical contaminants are present on the surface—only that there is organic matter present on the surface in which microorganisms might be able to grow.  Also, sanitizer residues on food contact surfaces or certain food components may interfere with the ATP reaction.

The BioLumix Advantage

BioLumix is an optical rapid, automated system that simplifies microbiological testing.  The system can be utilized for simplified monitoring of the manufacturing environment. Three assays were tested:  Total Aerobic Count, Total Combined Mold and Yeast Count, and Bile Tolerant Gram Negative Count (Enterobacterial Count).  A study was performed to compare and validate the BioLumix system as an alternative to the standard plate count method in detecting microbial contamination in manufacturers’ production surfaces.  Five different surfaces were used to simulate production surfaces.  10X10 cm coupons were inoculated with microorganisms, allowed to dry and then swabbed.  The swabs were inserted into a diluent (buffer or TSB) and analyzed by both the BioLumix and plate count assays.


A total of 550 coupons were tested, 290 were inoculated with various levels of contamination from <10 to <5,000 cfu/swab. The swabs containing microorganisms above the specified levels showed a very good correlation between the BioLumix and the various plate count results, with an overall agreement for samples above spec of 97.2%.  Almost all the discrepant swabs had plate counts very close to the specified level. None of remaining 260 swabs which contained counts below the specified levels did detect in the BioLumix system.  Consequently there was 100% agreement between the two methods.  The system also has the utility to detect for objectionable organisms such as E. coli, S. aureus and Salmonella.

Therefore, the BioLumix system offers:

  •  Automation of results
  •  Speed
  • Paperless
  • Detects microorganisms and not indicators
  • Can detect multiple types of organisms
  • Increases operation efficiency and consequently, improves quality and reduces costs                 
  • Enhanced reporting and ability to track trends

Rapid Microbiological Methods in the Era of Validations

Any new technique or equipment needs to be validated prior to entering its use in the commercial arena. The validation assures equivalency of the new method to the reference method. This means that the new technique or device is giving us “real results” that are reproducible and that can be trusted. USP <1223> Validation of alternative microbiological methods and ISO 16140 defines the general principle and the technical protocol for the validation of alternative methods in the field of microbiological analysis In the industrial world there is a pressing demand for rapid microbial detection technologies in order to improve the quality of products and their safety and speed up time to results. Their benefit could include significant reductions in time-to-result over conventional methods, improved sensitivity, specificity and accuracy, benefits of automation, reduced requirements for staff training, rapid product release, and lower inventory.

There are several new rising technologies that provide rapid microbial detection; such as the exceptional one developed by BioLumix. However, to be broadly used and accepted these new technologies need to comply with USP standards and fulfill the validation process. In order to get the validation assurances a series of pre-established analyses need to be done and compared the data generated by the new technology with the USP data to show equivalence. How valid the classical analyses are, it is a matter of historical use and common acceptance.

There are three required qualification components in a validation package; these are: installation, operation, and performance qualifications. The assurances for installation and operation qualifications deal with how reliable the setting and handling are; and that the proper conditions are given for the device or technique to work properly. To ascertain the reliability of performance qualification intuitively important are the internal positive and negative controls and well-known standards. Controls are the building blocks of any scientific approach to test hypotheses and to answer specific questions accurately. The controls and standards contain known amounts of the substance tested by the new technology. Under good laboratory practices isolated microbes are used in the manufacture of these controls and standards. Controls and standards are then used to validate the detection and quantification of unknown samples by the specific technique or device. In addition, controls and standards allow the validation of various important performance criteria, such as: accuracy and precision, specificity, and range, limits of detection and quantification, along with ruggedness and robustness. Accuracy is how close the results to the true value are. The precision deals with the agreement among individual tests. Specificity is given when detection of a particular microbe occurs even in a mix of different microbes without cross detections. The range is the upper and lower microbial count level limit that gives precise and accurate measurements. Limit of detection are the lowest microbial counts that can be detected. Ruggedness is related with the reproducibility of the results considering different equipment, personnel, laboratories, etc. And robustness indicates the capacity of the device or technique to absorb small variations such as: incubation conditions, media brands, reagents quality, etc.

The validated BioLumix system is an alternative to the classic USP methodology, which is highly time consuming and requires trained technical personnel. Using the BioLumix system, in just 48 hours a certificate of analysis is generated from samples that do not require special handling neither sophisticated processing. Hence, one of the best advantages of the BioLumix system is that does not require highly skilled personnel. BioLumix system ought to be used by a wide variety of manufacturing industries, which need to accurately certify the microbiologic status of their products in order to ensure their safety. Remarkably, the BioLumix system is capable of detecting a wide variety of microbes that raise health concerns such as: E. coli, Salmonella, Staphylococcus, yeast and molds, etc. BioLumix is committed to fulfilling the high demand for saving industries money and time in this fast pacing era of assurances and validations.   

A Rapid+Easy Method for Microbiological Testing of Dietary Supplements Complying w/cGMP & USP

Nutraceutical and dietary supplements consist of vitamin and mineral supplements, amino acids, herbs, essential oils, enzymes, homeopathic remedies and other natural products. For years dietary supplements have been unregulated by the U.S. Food and Drug Administration (FDA). In 1994 the Dietary Supplement Health and Education Act (DSHEA) was enacted, which stated that dietary supplement manufacturers must ensure supplements’ safety before marketing them.1

In 2007 the FDA issued the final rule for the implementation of current good manufacturing practices (cGMP) for supplements. 2 The FDA 21 Code of Federal Regulations (CFR) Part 111 required the implementation for large manufacturers in June 2008 and that by June 2010 all dietary supplement manufacturers test their products for identity, purity, contaminants and microbiology quality assurance, like pharmaceutical companies do. 3

To meet these new requirements, dietary supplement manufacturers must test their products for microbiology quality assurance and generate a certificate of analysis for each batch. To do this they either send product samples to contract laboratories for USP microbiological testing or test them internally. To reduce the time to get results, increase efficiency and reduce expenses, BioLumix offers a fast, automated, cost effective way for nutritional and dietary supplement companies to perform USP microbiological testing. The BioLumix system is an in-house method to easily perform accurate and automated microbiology quality assurance assays on finished products, in-process and raw materials and environmental work surfaces. This system can cut quarantine time for microbial testing from 5-6 days to 35-48 hours.

Samples are placed in ready-to-use, disposable vials inserted into an instrument that determine the microbial quality of the product. The system can perform all FDA-required tests such as total aerobic count, yeast and mold count, Enterobacterial count, and objectionable organisms such as E. coli, S. aureus, Salmonella and P. aeruginosa. The BioLumix testing system also includes a step-by-step process to optimize the installation, operation and performance so that it is easy for nutraceutical and dietary supplement manufacturers to do microbiology quality assurance.

The BioLumix testing system allows for faster sample preparation plus provides automated real-time communication, fast product release and early warning of contamination via USP microbiological testing. Because the BioLumix system is automated and its software generates the results, it is not necessary to have an employee dedicated solely to running the microbiology quality assurance tests. This makes the BioLumix system a cost-effective way for dietary supplement manufacturers to meet the new FDA cGMP requirements for microbiological testing. For more information on the BioLumix cGMP USP microbiological testing system, visit http://www.mybiolumix.com or call 1-734-984-3100.

References

1. Dietary supplements. FDA Web site. http://www.fda.gov/food/dietarysupplements/default.htm.

2. FDA Issues Dietary Supplements Final Rule. FDA Web site.

http://www.fda.gov/bbs/topics/NEWS/2007/NEW01657.html

3. FDA 21 CFR part 111. FDA Web site.

http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=111&showFR=1FDA

Karen F. Vieira, Ph.D.