Year-end wrap up: Rapid microbiology in 2014

By Gerry Broski and Ruth Eden

To those people who still honor the tradition of sending out hard copy holiday cards, it has become quite popular for families to include a “year in review update,” which updates children’s ages, significant family events and, for those that are retired, travel destinations which have been checked off the bucket list. Electronically, this is our company update and we’ll send out the electronic holiday cards later this month.

As the year comes to a close and we review input from our customers, our market data indicates that in test method evaluation our customers are looking for higher specificity, faster time to results, and a reasonable cost-per test. They also want flexibility so that the testing platform can adapt to a number of matrices. Other factors include ease of use, robustness, and a test that is approved or can be approved. These factors above must be compelling enough for a company to switch from their existing method and adopt new technology, and it’s not just the QC or QA staff who are in the evaluation loop.

I recently went on a sales call to a company which was evaluating one of our rapid micro systems for the detection of spoilage organisms, and the conversation between the QA manager and the plant manager was enlightening. While the test would involve more work and cost for the QA department, the direct benefit would be realized for the plant manager since the product could be shipped with a much shorter quarantine period, resulting in increased throughput, and significant cost saving by compressing the sales cycle.  Summary:  Cost for QA, benefit for Operations.

That being said, automated rapid detection of microorganisms is a proven process which meets the customer criteria on several levels. It is sensitive and rapid, it can be used on almost all matrices, it involves less touch-time, takes up less space than plating or film methods, and offers a good value on a cost-per test basis. When one considers that it is essentially a “load and leave” test when interfaced to a LIMS system, the benefits are clear.

Suren Zatikyan from Wepackitall commented “When we discovered the BioLumix system, we found it gave us the opportunity to generate results in a very short period of time.  In addition, validation studies that were performed by BioLumix and later confirmed by us, proved that the system is compatible with the standard methods used by USP and other standard methods developed for microbiology labs. We use the BioLumix system to perform some of our environmental testing and monitoring programs, which is very helpful to us.”

Kristopher Flores from Wepackitall added “If the BioLumix system wasn’t in place, it would be tougher to support our needs for testing and throughput. In some instances, based on our testing and our customers’ testing, we’ve been able to validate our processes in-house and eliminate third party validation. That saves time, money, and makes an impact on the big picture for our customers.”

Neogen now offers two platforms for rapid micro detection of spoilage organisms, the BioLumix System and the Soleris System.  The BioLumix system is geared toward applications which follow USP protocols including dietary supplements, cosmetics and toiletry, while the Soleris System facilitates testing in food matrices requiring AOAC approved methods.

With hundreds of BioLumix and Soleris systems in use, Neogen is the clear leader in rapid microbiology testing in dietary supplements and nutraceutical products and very strong in many market segments in the food industry with Soleris AOAC approved tests. From initial contact to the completion of the installation and beyond, our commitment is to provide unparalleled support and total customer satisfaction.

Other advantages include:

 you can count on our sales teams to respond quickly, with the focus on your needs and goals. In addition, we can quickly and easily show you the value and the ROI.

• Expertise 
– Knowing how the BioLumix or Soleris systems will work with your products gives you peace of mind. That’s why we test samples – your samples – for you.

• Compliance validation for BioLumix systems
 – If you are concerned about validation, here is more good news.  We provide complete SOPs along with a customized validation book which includes Installation Qualification (IQ), Operations Qualification (OQ) and Performance Qualification (PQ).

• Simplicity and Ease-of-Use 
– These easy to master systems offers a new level of productivity. Sample preparation takes less than 2 minutes. The BioLumix System offers certificate of analysis generation in as little as 24-48 hours.• Support – We are committed to satisfying our customers and providing unparalleled support.

The future looks bright for Neogen and the field of rapid microbiology as we continue our ongoing commitment to the evolution to our BioLumix and Soleris platforms based on customer feedback and technology gains. That being said, I would like to take this opportunity to express my appreciation to all of our valued customers with whom we have grown significantly with through the years. With your support will continue to evolve and offer you even more valuable products.

 

The Complete BioLumix Experience! Simplified, Rapid, Automated BioLumix System: Microbiology Testing Simplified

Come to Booth #16091 @ Supply Side West 2014 and Learn More
SupplySide West ExpoThe implementation of cGMP for all manufacturers (large and small) of dietary supplements and Nutraceutical products means that all manufacturers must test their products for microbiology quality assurance and generate a Certificate of Analysis for each batch. Products must be tested in accordance with the methods described in USP <2021> and <2022>.  This includes testing for Total Aerobic Microbial Count, Total Combined Yeast and Mold, Bile-Tolerant-Gram-Negative bacteria, and objectionable organisms (absent in 10 grams of organisms such as Salmonella, E. coli, and sometimes S. aureus). To do these tests companies either send product samples to contract laboratories for microbiological testing or test them internally.

Internal testing of products for microbiology gives the manufacturer much more control over the operation, but some manufacturers are hesitant to bring the microbiology testing in-house for fear of the complexity of testing or the need for trained microbiologist(s).  Also daunting is the need for a full validation package which is required for any method that deviates from the USP methodology. Here’s the good news – BioLumix offers a solution that gives faster time to results, quicker product release, cost effective operation, and strengthens key profit drivers.   This is all achieved on a single platform that is easy to use, validated to USP requirements, and doesn’t require additional skilled personnel.

At BioLumix, we don’t just offer you a simplified, rapid, automated microbiology system… we provide the Complete BioLumix Experience!

What is the Complete BioLumix Experience?  It’s having, from initial contact to the completion of the installation and beyond, our commitment to providing unparalleled support and total customer satisfaction.
SupplySide West Expo show floor

 

In 2006, Ruth & Gideon Eden established BioLumix, Inc.  Today, because of their extensive backgrounds in science and technology combined with customer-oriented sales and support staff, we have raised the simplification, automation and speed of microbiology testing to a new level.

  • Needs Assessment - Whether you contact us direct or through our website you can count on our sales team to respond quickly.  The focus is on your needs and goals.  Our BioLumix Technology Presentation provides an in depth look into how and why our system will work for you.  In addition, we can quickly and easily show you the value and the ROI the system will bring to your company.

 

  • Expertise – Knowing how the BioLumix system will work with your samples gives you peace of mind.  That’s why we test samples – YOUR samples – for you at our laboratory in Ann Arbor, MI.  We do this for you at no cost and at no obligation!  Once completed, expect a comprehensive report from one of our expert microbiologists. That same microbiologist spends two and a half days on-site for training and installation of the system.

 

  • Validation – If you are concerned about validation, here is more good news.  We provide complete SOPs along with a customized Validation book which includes Installation Qualification (IQ), Operations Qualification (OQ) and Performance Qualification (PQ).

 

  • Simplicity and Ease-of-Use-  The easy to master BioLumix system offers a new level of productivity.  Sample preparation takes less than 2 minutes; just add the sample to the diluent and mix.  Pipette the appropriate amount into the vial and place it into the instrument and record the sample and assay information into the computer.  The rest is totally automated.  The Certificates of Analysis will automatically be generated in as little as 24-48 hours!

 

  • Support – Our jobs center on satisfying our customers and providing unparalleled support.  At BioLumix you receive 24/7 support plus a microbiologist dedicated to service you and answer your questions.  What happens if you encounter a problem? Troubleshooting can be done via the internet and in most cases problems are resolved.   If needed we provide a loaner instrument while your instrument is being fixed to minimize downtime.  Our website provides instant access to information and resources about our technology, instruments, assays, and more.  It’s also mobile-enabled to format to your handheld devices.  Links to social media, white papers, blogs, videos, and other informative contact information puts everything you need at your fingertips.

 

  • Continuous Product Improvement – At BioLumix we have an ongoing commitment to evolving and adding to our platform.   One way this is achieved is through our partnership with our customers.  Customer feedback, along with a desire to increase the breadth and depth of our offerings, helps us to make improvements to our media and our assays.  We have recently introduced two new assays – Listeria and Bacillus cereus.

 

With a simplified method such as the BioLumix System we can provide a simple cost effective method to test products for microbiology. Come visit our booth (# 16091) and take the first step into your own personal BioLumix Experience.  This year the show is being held at the beautiful Mandalay Bay Convention Resort, in Las Vegas, October 8th and 9th.

Growth Promotion and Negative Controls – Does Shipping Affect the Results?

Why Growth Promotion?

USP Chapter <61>, Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests ,there is a requirement for the testing of Growth Promotion (GP) for each batch of medium as well as testing for Negative Controls (NC).  Growth Promotion must be tested for each new batch of nutritive broth.  The purpose of the GP Test is to determine the suitability of the culture media for the growth of target organisms.  The medium is challenged with a small number (not more than 100 cfu) of microorganisms to assure its nutritive properties.

USP Chapter 61USP Chapter <61> lists the following acceptance criteria for liquid medium: “Liquid media are suitable if clearly visible growth of the microorganisms comparable to that previously obtained with a previously tested and approved batch of medium occurs.” It is recommended to use parallel testing; in parallel testing the new medium and the previously approved medium are inoculated with the same microorganism suspension, by the same technician, using the same method and same environmental conditions.  The only variable is the medium. GPT is performed by inoculating the product with ? 100 CFU of microorganisms, defined by the pharmacopoeia.

BioLumix Quality Assurance and Certificate of Analysis

For every batch of vials prepared by BioLumix a thorough testing, including growth promotion and negative controls, is done.  Growth promotion is tested performed, for each organism, by inoculating 4 vials of the new lot and comparing the detection time (DT), and amplitude of the curve to a lot that previously passed the PG. At least 2 different strains of organisms are used in each media.  Additionally at least one negative control organisms is tested and compared to the reference.  In the growth control assay the DT of the new lot has to be within a range of 50-200% from the reference vial.  A certificate of analysis is issued with each lot of vials.

Is there a need to redo GP and NC after shipping the vials to the end users?

In other words, does the shipping of the vials to the end users influence the GP and NC of the vials? The study described below was undertaken to answer this question.  BioLumix vials are always shipped UPS second day air.  The furthest destination in the USA is California.  The study was conducted by double shipping second day air to California and back.

Summary of Validation Data

Methodology-Quadruplicate vials that were shipped to California and were returned from there where compared to vials that stayed in the lab. This test was performed on three different lots of media, using three different media types (Total Aerobic Count, Enterobacteriaceae, and Yeast and Mold Vials).

Results-The data shows that the shipped vials and the vials that stayed in the laboratory results of the test show identical growth curveshad identical growth curves, falling close to each other.   There is no difference in growth promotion or negative control between vials that were shipped (actually double shipped) and vials that stayed on the shelf for any of these lots, as can be seen from the attached example.  The example compares the curves of total aerobic count vials (TAC) inoculated with Bacillus spizizenii var subtilis ATCC 6633, shipped to vials that remained in the laboratory (Key: Shipped vials: Purple, Yellow, Beige and Dark Green; vials that remained in the lab: Dark Blue, light Green, Light Blue and Red lines). As can be seen from this example the Curves are indistinguishable

Similar results were obtained for all three lots of TAC inoculated with Bacillus spizizenii var subtilis ATCC 6633; Escherichia coli ATCC 8739; and Staphylococcus aureus ATCC 6538;

A buffer was used as a negative control for the TAC vials, and no difference was seen for all three lots between shipped and vials that remained in the lab.

Similar results were obtained with combined yeast and mold vials (YM) inoculated with Aspergillus brasiliensis ATCC 16404; Candida albicans ATCC 10231; Saccharomyces cerevisiae ATCC 9763; and Staphylococcus aureus ATCC 6538 (SAT1) as negative control.  There is no difference in growth promotion or negative control between vials that were shipped and vials that stayed.

The shipping experiment was also conducted with Bile-Tolerant Gram-Negative Bacteria vials (ENT) inoculated with Escherichia coli ATCC 8739; Salmonella enteritidis subsp. enterica serovar enteritidis ATCC 13076; Citrobacter freundii ATCC 8090; and Staphylococcus aureus ATCC 6538 as a negative control. There was no difference between the data generated by the system for shipped vials, as compared to vials that remained in the laboratory.

 Conclusion

The data shows that the shipped vials and the vials that stayed in the laboratory had identical growth curves, falling on top of each other.   There is no impact of shipping on growth promotion or negative control.  The results validate that there is no reason to perform again the growth promotion and negative controls after shipping to the end user. The C of A supplied with the BioLumix vials is indicative of Growth Promotion and Negative Control followed the shipping.

Are You Ready for an FDA Inspection? Surviving the Audit

When it comes to facility audits, tensions run high, even for the most prepared.  When your facility is being audited, the documents used for evaluation come from the Code of Federal Regulations, 21 CFR 111 (Current Good Manufacturing Practice in Manufacturing, Packaging, Labeling, or Holding Operations for Dietary Supplements).  These documents review specifications, responsibilities to meet those specifications, written procedures, and laboratory operations.  Here, we focus on the microbial aspect of these requirements.

Microbiology Lab Ready for FDAWhat specifications must you establish?

For dietary supplements, specification limits must be set on contaminants that may adulterate (change) or lead to adulteration of the in process or finished batch.  Documentation as to why these specifications will help to ensure contaminants will not lead to adulteration should be in place.  It is your responsibility to ensure the established specifications are met and properly documented.

What steps should be taken to make sure specifications are met?

Appropriate testing should be in place to be sure specifications are within compliance.  You may also rely on a certificate of analysis (C of A) from the supplier of the component that you receive provided you first qualify that supplier’s certificate of analysis through testing.  If you rely on the suppliers C of A, it should include a description of the testing performed, specifications, and actual results of the test.  Periodic auditing of C of A results is required, and should be part of the microbial testing.  To ensure the finished product specifications are met, the in-process procedure should be in place to monitored and make sure that  unanticipated contamination does not occur at any stage.

Requirements for Laboratory Operations

Written procedures (Standard operating Procedures-SOP), for laboratory operations for tests and examination of product must be in place.  Lab operations should ensure that all lab material specifications and dietary supplement specifications are met.  Test methods should be well documented and validated, and sample-collecting plans should also be in place and followed.

Out of Specification Operations

If specifications are not met, a procedure (SOP) must be present and followed to correct for any error made during processing or testing.  A modified method of testing may be necessary.

Microbiology Lab RequirementsThe Validated BioLumix System

BioLumix generates a customized validation book for each of its customers that include:

Installation qualification (IQ):  Identification and validation of the system components; validation of the environmental conditions; electrical requirements; computer qualification; verifying that all installation steps were followed;  and documentation of instrument calibration.

Operational qualification (OQ):  Verifying that the equipment is properly installed, calibrated and is operational.  It includes a unique SOP for all products and assay combinations to be performed on the instrument; software characteristics and the verification that the software is 21 CFR part 11 compliant; verification that all the instrument functions operate as expected; Verification of the instrument temperature accuracy; and training records.

Performance qualification (PQ): is the most extensive part of the BioLumix validation book. It shows equivalency with USP methodology when following USP <1223> “Validation of alternative microbiological methods”.

BioLumix has some quick tips to be sure that your BioLumix system passes inspection without any fuss.

How to Use the BioLumix Validation Book?

Your customized validation book is a major component in your system, just like the instruments and vials themselves.  Make sure the person in charge of validation knows exactly where the validation book is.  Also, be sure that all pages of the book requiring signatures, are signed by the appropriate personnel, and the operator has a good grasp on the meaning of each attachment.  The training log of attachment 9 should show that appropriate training was provided to all operators.  All SOPs used in product testing must also be included in the book.

Side-by-Side Testing

Attachment 16 in the validation book shows your company’s own product testing and the data generated not only with the BioLumix system, but with the USP plate count method as well.  It generally starts with the report that was generated when samples were first sent to BioLumix for testing.  Since then, your company should have added to this data to show continuity between the BioLumix and plate count methods.  Attachment 18 is much more in depth, showing a wide variety of products, vial types, and specification limits.  This data verifies the comparability between the two methods on a broad spectrum.

Vials and Reagents

Make sure that the vials and reagents in use are within their expire dates.  Also be sure the Certificate of Analysis matches the vials.  It may seem like common sense, but when days get busy, some things may be overlooked.  Be sure all reagents are stored at the proper temperature conditions as specified by BioLumix.

Optical Calibration

It is important that your instruments be calibrated optically at least every 6 months, and full temperature and optical calibration be performed annually.  These documents should be held in Attachment 8 of your validation book.  This will show that care was taken to keep the instruments performing at optimal levels.

By following these guidelines, you should be ready for your audit.  As always, BioLumix is just a phone call away with any questions or concerns you or the auditor may have.

It is important for all to know that BioLumix customers pass FDA microbiological audits without any problems.

BioLumix Validation Book for Dietary Supplements

According to cGMP all dietary supplement products should to be tested in accordance with the methods described in USP <2021> and <2022>. The required assays might include Total Aerobic Microbial Count, Total Combined Mold and Yeast, Bile-Tolerant-Gram-Negative bacteria, and objectionable organisms (absent in 10 grams of organism such as Salmonella, E. coli, and sometimes S. aureus). To do these tests companies either send product samples to contract laboratories for microbiological testing or test their samples internally.  Many outside laboratories utilize BAM and AOAC methods that were never validated against the USP methodology for Dietary Supplement products.

BioLumix Validation BookFDA’s GMP inspection program for dietary supplements has evolved since its debut a few years ago, with the agency increasingly strict in its interpretations of the regulation and requirements.  Validation of any new microbiological method is required 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.

BioLumix generates a customized validation book for each of its customers. The book includes:

Installation qualification (IQ):  Identification and validation of the system components; validation of the environmental conditions; electrical requirements; computer qualification; verifying that all installation steps were followed;  and documentation of instrument calibration.

Operational qualification (OQ):  Verifying that the equipment is properly installed, calibrated and is operational.  It includes a unique SOP for all products and assay combinations to be performed on the instrument; software characteristics and the verification that the software is 21 CFR part 11 compliant; verification that all the instrument functions operate as expected; Verification of the instrument temperature accuracy; and training records.

Performance Qualification (PQ):

Performance qualification is the most extensive part of the BioLumix validation book. It shows equivalency with USP methodology when following USP <1223> “Validation of alternative microbiological methods”.

  • Side-by-side comparison: Side-by-side results are comparable between the BioLumix method and the reference method when testing by the BioLumix Dilute-to-Specification Protocol.   A generic study was performed testing all types of Nutraceutical, botanicals and dietary supplements for Total aerobic count. A total of 252 samples with counts in the range of 100 cfu/g to 10,000,000 cfu/g   were analyzed, including   botanical powders, botanical extracts, infusions, alcohol extracts, raw materials, and finished products. There was a 99.6% agreement between the BioLumix method and the SP plate count method. Testing similar products for yeast and mold counts in the range of 10 cfu/g to 100,000 cfu/g there was a 98.1% agreement; and for Gram negative Bile Tolerant bacteria 98.7% agreement.  Similar results are shown for all objectionable groups of organisms such as E. coli, S. aureus, P. aeruginosa, and Salmonella.  Unique customer samples are also tested and compared to USP results.
  • Specificity (Inclusivity and Exclusivity): is the ability of the media in each vial to detect a wide range of organisms belonging to the target group, and not detect using non-target organisms, which demonstrates that the method is fit for its intended purpose.  The inclusivity and exclusivity of each of the media used by the system was extensively studied.  The data shows good inclusivity and exclusivity.
  • Limit of detection (LOD):  are the lowest microbial counts in a sample that can be detected under the conditions used.  The limit of detection was evaluated for Total Aerobic count, Yeast and Mold count and Gram negative Bile Tolerant bacteria.  The data shows that for all 3 media the limit of detection was close to 1-3 cfu/vial.
  • Precision and Repeatability:  is the degree of agreement among individual test results when repeatedly testing multiple samples of suspensions of microorganisms across the range of the test.  It is usually expressed as standard deviation (SD) or coefficient of variation (CV) of a series of measurements. Generally, a %CV of 15%-35% is acceptable. All the results obtained were well within the acceptable range.
  • Robustness: indicates the capacity of the device or technique to absorb small variations such as: incubation conditions, incubation temperature, media and sample volumes, etc.   The data demonstrates the robustness of the system with various media.
  • Ruggedness: is related to the reproducibility of the results considering different equipment (different instrument units), personnel, different lots of reagents, etc.  The BioLumix system has been demonstrated to be very rugged with high precision of test results obtained by analyzing the same microorganisms under a variety of different operating conditions including different analysts, different instrument units, and various  lots of media.
  • False negative rate: A false negative is a test in which samples that are inoculated or naturally contaminated generate a positive result in the reference method (plate count) but are negative in the test method (BioLumix). Naturally contaminated samples were tested at various specification levels, the BioLumix procedure detected the samples as being contaminated in all cases while the plate count method had a false negative result.
  • False positive rate: A false positive is a test in which a sample that does not contain the target organism generates a positive result in the BioLumix system but not in the Plate Count method. A false positive rate of 2.5% was seen in total aerobic count and no false positives were seen in the yeast and mold assay.

Conclusion

There is a need 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.  New rapid automated methods in microbiology need to be validated, and the BioLumix system comes with a validation book.

  • Unique validation book created for each customer.
  • Single platform testing for all assays
  • Real-time communication – early warning of contamination
  • Automated Certificate of Analysis in 48 hours
  • No product interference with ready-to-use vials with organism specific enrichment medium
  • Technology based on traditional media
  • Stackable compact instruments take up little bench-top space
  • Technical service available 24/7

References

Food  and  Drug  Administration  (FDA)  (2007),  21  CFR  Part  11,  Guidance  for Industry; Electronic Records; Electronic Signatures – Scope and Application. The National Formulary, Rockville, MD

United States Pharmacopeia (USP) (2008), Chapter <1223>, Validation of Alternative Microbiological Methods. The National Formulary, Rockville, MD

United States Pharmacopeia (USP) (2008), Chapter <2021>, Microbial enumeration tests-Nutritional and dietary supplements. The National Formulary, Rockville, MD

United States Pharmacopeia (USP) (2008), Chapter <2022> Microbiological procedures for Absence of Specified Microorganisms- Nutritional and dietary supplements. The National Formulary, Rockville, MD

Detection of psuedomonads in dairy and water samples using a quantatative one-step testing protocol in just one day

By Roger Brideau*
*Presented in part at the International Association of Food Preservation
‘IAFP’ Conference in Charlotte NC USA

dairy microbiology detection of psuedomonads Introduction- Pseudomonad organisms are a major cause of bacterial spoilage of pasteurized milk and dairy products due to post process contamination.  Early detection of Pseudomonad’s can be a predictor of product shelf-life as they are the predominant psychotropic bacteria present.  BioLumix has developed a rapid method for the detection of Pseudomonad’s in dairy products and the method is also applicable to their detection in process water.

Purpose- To evaluate the ability of the BioLumix system to detect Pseudomonad’s in dairy products, determine the speed to results, sensitivity, selectivity and ability to predict shelf-life.

Methods- the BioLumix system is an optical system that detects growth of Pseudomonad’s using a CO2 sensor in selective growth media.  The BioLumix system was directly compared to the plate count methodology for milk samples stored at refrigerated temperatures and held overnight at room temperatures (enriched).  Testing of water was also accomplished in side by side studies to show the capability of the BioLumix system for quantitation of Pseudomonads.

Results:  Growth of Pseudomonad’s in the BioLumix vial
Table of quantitation of Pseudomonads
A growth comparison was made for detection of each Pseudomonad in the BioLumix system using PSE-B vials and on CFC (Pseudomonas agar) spread plates.  Table 1 summarizes the growth of freshly diluted samples of organisms that were enriched in TSB during the prior 18-24 hrs.  The PSE-B vials are selective, as shown by not allowing growth of unrelated gram positive and gram negative bacteria, yeast or mold.  Four different species of Pseudomonad’s grew in the PSE-B vial and on CFC plates.

Milk Sample Testing
Commercial milk samples were tested upon arrival in the laboratory.  Five of twenty were positive for the presence of Pseudomonad’s using both PSE-B vials and CFC spread agar plates.  After storage for 3-7 days, twelve of twenty samples were positive for Pseudomonad’s including after enrichment at RT for 16-18 hrs.  Thus, refrigerated milk samples have varying incidence of Pseudomonad flora.

Dairy Microbiology Calibration dataMilk Calibration Curve
Organisms from milk samples that grew in PSE-B vials and on CFC plates were used to generate the Calibration Curve shown in Figure 1. These data suggest that low numbers (~10) of Pseudomonad’s should detect within 24 hrs in the PSE-B vial. The Calibration Curve can be embedded into the BioLumix software on the instrument and used to generate a read-out of cfu per gram of milk.  This enables quantitation of the milk sample for the presence of Pseudomonad’s before 24 hr; a distinct advantage over plate methodology taking 48-72 hours.

Dairy Microbiology detection time distributionDistribution of Data
In the dairy settings the goal is to separate a “good” sample that has a potential to maintain quality over a product’s shelf-life from a “bad: sample that will have a shorter shelf life. Criteria for separation between a “good” and “bad” product based upon the Pseudomonad’s numbers can be established. If one selects a count of 1,000 cfu/ml as the separation point: the Histogram shown in Figure 4 indicated at 12.5 hrs all samples with higher counts (in red) detected, while all the samples below 1,000 cfu/ml did not.

Results: Testing of Process Water for the presence of Pseudomonads
Eight different types of process water samples were found to be free of Pseudomonad’s after testing using PSE-B vials and CFC spread plates (data not shown).  Clean process water samples were then inoculated with individual isolates of Pseudomonad’s were used to generate a calibration curves for water, similar to the milk calibration curve. Pseudomonad growth in inoculated process water was measured using PSE-B vials and PA spread plates and was used to generate the Calibration Curve. Detecting vials were confirmed to contain Pseudomonas by the Oxydase test.

Summary
The data presented show equivalency between the BioLumix PSE-B vial and CFC (Pseudomonas agar) plates for the detection of Pseudomonad’s found in commercial milk samples and in inoculated process water samples.  PSE-B vials detected as little as 1-3 organisms (data not shown).
The number of organisms in commercial milk was found to increase over time at refrigerated temperatures and this agreed with a previously published report (Burdova et al 2002) showing the affect of storage temperature on milk shelf-life.
The BioLumix assay is completed in 18 hours and offers an advantage over spread plate methods for time to results and ease of calculation of cfu per gram of milk or water.  A single vial is all that is needed and thus both time and material costs are reduced.  Calibration Curves were easily generated for both milk and water sampling and can be used to generate a cfu/ml of sample in less than 1 day to yield an estimate of cfu/gram.

REFERENCE:  Burdova, O. et al (2002).  Bulletin Vet Med. Poland. 46:325-329. Hygiene of Pasteurized Milk Depending on Psychrotrophic Microorganisms.

BioLumix Exhibits @ SupplySide West 2013 – Stop by Booth # 30038!

SupplySide WestSupplySide West brings together the suppliers and buyers that drive the dietary supplement, food, beverage, animal nutrition, personal care and cosmetic marketplace. Key professionals and thought leaders from executive management, R&D, QA/QC and marketing teams will meet in Las Vegas November 12-16.  The 2013 show floor is the largest yet, featuring over 1,700 booths, please make sure that you visit BioLumix at Booth #30038.

Rapid automated methods, such as the BioLumix System, can speed up time to results from 7-10 days to 1-2 days.  The time saved has a direct impact on many operational profits drivers. . . and is The reason over 450 BioLumix Systems are actively being used by many of the largest manufacturers in the industry.

Here are just a few of the many benefits of the BioLumix Partnership Program:

Ready-to-use vials  with specialized media which do not require QC on your end

Very Fast Results—C of A within 48 hours  for expedited product releases

Simplified, automated microbiology testing

    • Paperless laboratory
    • 60% savings in labor
    • Operated by non-microbiologist

ALL assays available within one automated system

Validated Method in compliance with USP <2021>, <2022>, <2023> and USP <61>   and <62>

Remote access/support for your system via internet and interactive phone calls to facilitate optimal instrument performance

Product reports tailored to specific applications

Dedicated microbiologist assigned to support your    facility

Real-time continuous support for reviewing microbiology assays. . .  and more

BioLumix takes pride in delivering the highest level of customer support for our BioLumix Partners.  Did you know that in a recent customer satisfaction survey, ALL of our customers would recommend us to their vendors, suppliers and customers?

With the BioLumix System, we can help your company internalize microbiology testing.  If you already have an internal laboratory, we can help you save time and money by automating your testing!  Many of our customers have gone through audits and we have received great feedback from them.

biolumix automation drives profitsWe welcome all of our customers to stop by and say hello!  If you are not yet a customer, we welcome the opportunity to work with you.  Our jobs center around satisfying our customers and providing unparalleled support.  We would be happy to give you a demonstration of the system including testing your samples for free.  After all, we wouldn’t want to sell you a product that doesn’t work for you.  Still not convinced?  We would be more than happy to provide you a long list of customer referrals.  Allow us the opportunity to demonstrate the many benefits of the BioLumix Partnership Program.

Dealing with difficult to read plates or manual data transfer is a thing of the past. With automated monitoring of ready-to-use assay vials and automated data processing and archiving (paperless), the microbiologist‘s job got a lot easier with the same accurate results in less than half the time.  Real-time data communication: Early warning of contaminated samples as well as sample release information could be automatically communicated through your intranet, significantly improving your company’s efficiencies.  BioLumix system can be used to test powders, oils, enzymes, botanical material, tablets, capsules, powders and liquids easily and without product interference.

BioLumix trainers will provide each customer with a full and customized Validation book, during the two and a half days of on-site training and installation of the system, including installation qualification, Operation Qualification and Performance qualification.

With this internal automated microbiological system, customers have control over their testing, leading to accurate results and accelerated product release while reducing costs.

The BioLumix Rapid Microbiology System can save multiple days versus current USP testing methods, and dramatically impact key profit drivers, such as elimination of idle time with raw materials, work in progress, and finished goods.

Microbiology of Dietary Supplements

Official Methods for Dietary Supplements

The USP methods for dietary supplements include:

USP <2750> Manufacturing Practices for Dietary Supplements: Contains the official FDA ruling on dietary supplement cGMPs (21 CFR Part 111).

USP <2021> Microbial Enumeration Tests-Nutritional and Dietary Supplements: Enumeration of the total number of aerobic bacteria, enumeration of the total number of combined yeast and molds, and the presence of bile-tolerant gram negative bacteria present in all nutritional supplements, from raw materials to the finished product.

USP <2022> Microbiological Procedures for Absence of Specified Microorganisms-Nutritional and Dietary Supplements: Procedure for determining the absence of specified objectionable organisms. Good manufacturing practices require that objectionable organisms be absent from non-sterile nutritional and dietary products. Objectionable organisms are defined as microorganisms that may cause potential health hazards to the user of the product and would adversely affect the product safety.

USP <2023> Microbiological Attributes of Non-sterile Nutritional and Dietary Supplements: contains information about frequency of microbiological sampling and testing, recommended microbial limits for botanical ingredients and products, and finally, recommended microbial limits for dietary supplement ingredients and products. However, specifications are typically are developed by the manufacturer with guidance from USP <2023>.

USP <1223> Validation of Alternative Microbiological Methods:  Any alternative method must be validated against these UPS methods using the methodology described in USP <1223> including side-by-side testing, determination of the accuracy, specificity, limit of detection, robustness, ruggedness, etc.

Many methods validated for food products, are not necessarily validated or appropriate for dietary supplements. For example, many methods have been written for very specific products such as eggs, meat, milk, nuts, etc. These methods were not written with dietary supplements in mind. And “dietary supplements” encompass such a large scope and variety of products therefore, it is risky to simply assume that a method written for example for eggs will also work for supplements.  Therefore, a method having AOAC certification is not necessarily appropriate for dietary supplements.  AOAC had not certified any microbiological method for dietary supplements, all their methods are certified for food only.

Often, one method will work for one product but not for a different product. Petrifilm methodologies are excellent methodology for many food products. However, Petrifilm should not be used on dietary supplement samples without thorough validation. For instance, samples that are dark in color and many common dietary ingredients might make it difficult to see growth on the Petrifilm. Viscous products such as Psyllium begins to harden before an appropriate spread on a Petrifilm can be obtained.

Product Interference or Matrix Effect

Spirulina, turmeric, Psyllium, honey, Calcium carbonate, dyes, and oils: these are just a few of the many products that the Nutraceutical industry works with, and struggles with. Many botanicals, herbals, enzymes or protein powders create challenges for microbiological testing (Tackling those challenging Nutraceutical Products with Rapid Automated Microbiology Testing!, C. Ockerman 2013). Granules (which can often be mistaken for a bacterial colony), extreme pH, viscous products can all interfere with the plate count methodology.

Microbial Testing of Dietary Supplement

Microbiological testing in the dietary supplement industry consists according to the USP chapters, of monitoring levels of naturally occurring indicator organisms and spoilage organisms, as well as the absence of objectionable organisms (e.g., Salmonella, Staphylococcus aureus, and E. coli) in finished products.

The typical battery of microbiological tests for a dietary supplement includes:

• Total Plate Count

• Yeast and Molds

• Enterobacteriaceae (gram negative bile tolerant bacteria)
• E. coli
• S. aureus

• Salmonella

• Pseudomonas (for liquid products or products with high water activity, or topical/cosmetic products)

How to Test for Microbiology

Many companies outsource the microbiology testing to third party commercial testing labs instead of performing it in house.  Some companies fell that whenever products are sent out for testing quality results cannot be questioned, but time is sacrificed and a premium price is paid for the service. Therefore, companies tend to only bring their microbiological testing in-house when they reach a critical mass such that the cost of sending out is equal to or more than starting up a laboratory.

In house Laboratories can perform using the USP methodology.  In preparing its own media and reagents the lab needs to pay attention to USP <1117> (Good Microbiological Laboratory Practices).  Because in an internal laboratory the quality of the results depends on the quality of the culture media prepared.  This includes accurate weighing of dehydrated components, the use of high quality (USP Purified) water, completely dissolving the dehydrated media or individual ingredients, and the need to control the heating of the media to avoid damaging heat-labile components of the media. The quality control of each batch of the media is a critical concern.

It is important to take production timelines into consideration when determining what methods fit each company individual needs.  A major issue with microbiological testing is the hidden costs in labor and inventory hold time.  The one assay that often keeps a quality control manager up at night is their yeast and mold testing—which takes five days to obtain a result.

Why Automated Rapid Microbiology?

Internalizing the microbiology testing and especially adopting rapid microbiological methods (RMM) can speed up the time to results from 7-10 days to 24-48 hours. The time saved has a direct and REAL impact on many key operational profit drivers.  The Advantages of RMM can include: Greater accuracy, better sensitivity, and increased sample throughput, automated data capturing and allowing easier data handling, and reduced cost for product release.  Rapid methods allow test results to be obtained faster. More importantly, if a product is above a desired microbiological specification, the results are obtained a lot sooner, and corrective action can be taken days before traditional methodology would allow.

All analytical methods need to be validated prior to their introduction into routine use, and this is especially true for novel technology platforms, such as rapid microbiological methods (RMMs). Because many RMM technologies consist of a combination of instrumentation, software, consumables and reagents, in addition to specific detection, quantitative or identification methodologies, it is important to develop a comprehensive and holistic approach to the validation process to ensure that the entire RMM system is suitable for its intended use.

The BioLumix Advantage
The BioLumix System is a system that fits a need in the Dietary supplement industry in terms of speed, working with all product matrices, and automatically documenting the results in real-time, thereby reducing the labor requirement for microbiological testing.

The BioLumix system was extensively validated for dietary products and compared to the methodology described in USP <2021> and <2022>.  The validation methodology followed USP <1223> “Validation of Alternative Microbiological Methods”.  This included performance criteria such as: side-by-side comparison to the USP methodology, accuracy and precision, specificity,  limits of detection and quantification, along with ruggedness and robustness, reproducibility of the results, false positivity rate and false negative rate.

A full validation package including Installation Qualification (IQ), Operational qualifications (OQ) and Performance qualification (PQ) is included with the BioLumix system
The BioLumix Advantages include:

  • The only system with all assays on a single platform
  • Real-time communication – early warning of contamination
  • 48-hour Automated Certificate of Analysis
  • Simplified testing with ready-to-use vials
  • Each vial comes with a certificate of analysis, negating the need for QC of the vials
  • Validated for dietary supplement products – IQ, OQ, PQ
  • No product interference
  • 21 CFR Part 11–compliant software
  • Remote-access customer support 24/7/365

A Novel Rapid Automated Method for Suitability Testing

Suitability Testing by USP Methodology

Suitability testing is performed in order to verify that the method utilized eliminates the effect of any
antimicrobial properties of the product. Therefore, the media diluent combination does not inhibit the
recovery and growth of microorganisms, if present in the sample. The goal of the suitability testing is to
establish the ability of the test to detect microorganisms in the presence of product.

The suitability described in USP <61> verifies the
validity of the testing method by showing the
recovery of microorganisms in presence of the
product. Total Aerobic Microbial Count and Total
combined Yeast and Mold can be carried out by
membrane filtration, pour plating or spread plate
method.

Suitability using USP <62> can use selective media to
detect various organisms such as: Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli,
bile-tolerant gram-negative bacteria, Clostridia,
Salmonella and Candida albicans. The samples are
first enriched by incubating in Trypticase Soy Broth
(TSB) or another appropriate neutralizing media, and then streaked onto selective agars for the
determination of presence of specified or the objectionable microorganisms.

The new USP <61> and USP <62> tests also provide harmonization to existing European Pharmacopeia
methods for testing non-sterile pharmaceutical products. Additional in order to verify the testing
conditions, a negative control is performed using the chosen diluent that shows no growth of
the microorganisms. While conducting the suitability testing precautions must be taken to avoid
contamination so they do not affect the microorganisms that are being tested. The procedure involves
the inoculation of the neutralized sample with low (not more than 100 cfu) and detecting the organisms
by the prescribed method.

Even though USP uses traditional microbiology methods, from the USP <61> and <62> states
that “Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopeial method has been demonstrated” and “any validated method,
including, Rapid Methods can be used”.

The New BioLumix Method

A study was recently conducted to show the utilization of BioLumix system (see figure below) in
suitability tests using a variety of products from both the pharmaceutical and cosmetic industries.

Sampling was conducted by taking ten gram of the product
and placing it into 90ml of M Letheen broth (or another
appropriate neutralizing broth) for a final dilution of 1/10.
An overnight culture of the target organism was diluted to
not exceed 100 cfu and the inoculum not exceeds 1% of the
volume of the diluted product. Then 1.0 mL of the
neutralized sample containing organism was placed into the
appropriate vial and a side by side comparison was done
with the appropriate USP method.

The products tested included Aloe, Hand Sanitizer, Lip Balm, Flavored Lip Balm, Medicated Lip Balm,
Breath Spray, Medicated ointment, and Sun Screen. A variety of different types of each product were
tested.

32 product samples were tested for suitability. Four bacteria (Staphylococcus aureus ATCC 6538;
Pseudomonas aeruginosa ATCC 9027; Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739; a
yeast (Candida albicans ATCC 10231), and a mold (Aspergillus niger ATCC 16404) were used to show the
effectiveness of the neutralization step. Thirty one
products were properly neutralized by the M Letheen
Broth as evidenced by detection time in the vials and
colonies on the plates. Only one product tested contained
a high level of ethanol, which required a 1:100 dilution in
M Letheen Broth to obtain neutralization. There was 100%
correlation between the two methods.

Typical data obtained by the system is shown in the figure:

The product was inoculated with ~ 100 cfu/g of three organisms: Staphylococcus aureus ATCC 6538 (light blue); Bacillus subtilis ATCC 6633 (Dark blue) and Escherichia coli ATCC 8739 (green). The Detection times obtained (shown as triangles on the curves) are comparable to data obtained without product.

What are the advantages of the BioLumix system?

Time Saving: The results are available much faster, for example, the results of the Yeast and Mold vialsoccurred in less than 48 hours, while the Aspergillus took nearly five days for countable colonies. All
products tested with bacteria using the automated BioLumix assay yielded results typically 10-13 hours,
instead of 48 hours for the plate count methodology. The advantage is that you can see results an
entire day early if the product is exhibiting any sort of inhibition. Data generation is slower using classic
microbiology so it can also slow down production improvements as well.
Labor Saving: The setup of the assay can be done much faster using the BioLumix system as opposed
to traditional plating methods, saving significant hands-on labor due to its automation and simplicity of
use. BioLumix can make the microbiological testing simpler, faster, and automated- saving significant
time and labor. It is paperless, increasing efficiency and saving on disposables, time and space, and best
of all the system is unaffected by product interference. The System is extremely easy to operate, with
its straightforward, streamlined testing design it offers accurate results leading to reduced material-
holding time for faster product release.

High Correlation with USP: The BioLumix System showed a high correlation between the instrument
results and the USP methodology. The system is fully automated with automated data archiving,
data maintenance in databases, and automated report generation. Regulators encourage rapid
microbiology methods for improved process control and product release. The BioLumix system
is validated as being at least equivalent to the compendial method. Under general notices of the
USP states that “Automated procedures employing the same basic chemistry as those assay and
test procedures given in the monograph are recognized as being equivalent in their suitability for
determining compliance.”

References:

USP <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

USP <62>: MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TEST FOR SPECIFICIED
MICROORGANISMS

Rapid Microbiological Testing of Capsules, Softgels and Pills Encapsulation of Pharmaceuticals

Pharmaceutical products may be encapsulated in a relatively stable shell for oral consumption.  These shells are called capsules and can either be hard-shelled or soft-shelled.  Hard-shell capsules are commonly filled with dry powdered ingredients, pellets, or granules.  Soft-shell capsules (softgels) are made from gelatin and primarily contain oils or active ingredients that are dissolved or suspended in oil.  Both hard and soft-shell capsules may contain colorants, dyes, opaquing, dispersing, or hardening agents, and preservatives.  Tablets are solid doses of medicinal substances and may be soluble effervescent, chewable, molded, or compressed.

Traditional methods for testing

Plate count methodology as described in USP <61> is regularly used to test capsules and tablets.  Using this methodology, it takes two days for Aerobic Count results, and five days for Yeast and Mold results.  When testing for objectionable organisms, it may take several days using selective broths or agars to determine the absence or presence of these bacteria.   The colors of the shells and the products and the viscosity of the 1:10 dilution sometimes interfere with the reading of the plates.

Rapid Detection with the BioLumix System

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, and reduces the error rates produced by paper-based activity recording and batch data entry.  The BioLumix system also helps automate microbiological quality control processes.

To validate the equivalency of the BioLumix system to USP <61> or USP <2021>, over 100 types of capsules, softgels, and tablets were tested with the BioLumix system and plate count methodology at various specified levels.  The products were tested for total aerobic microbial count (TAMC), Total yeast and mold count (TYMC), and the absence of E. coli, P. aeruginosa, S. aureus, and Salmonella (objectionable organisms) in 10 grams of product.  For each assay, a 1:10 dilution was created by adding 10 grams of product to 90 mL of TSB and further dilutions were performed depending on the desired specified level.  Some of the samples were inoculated with various bacteria.


Figure 1 shows the BioLumix curves obtained for the Total Aerobic assay.  The purple curve represents an inoculated colored capsule.  All the curves along the baseline represent different kind of capsules that were not contaminated.


Figure 2 shows the BioLumix curves for the Yeast and Mold assay.  The dark blue curve represents an inoculated product. All the curves along the baseline represent different kind of capsules that were not contaminated.

 

As seen in Figure 3, the BioLumix vial design is separated into two zones: the incubation zone where the sample is present and the detection zone where the reading takes place.  Therefore, the system prevents any product interference.

Figure 3 shows two vials that contained dissolved blue capsules.  The sample on the right contained bacteria while the sample on the left was clean.

Final results are seen in the BioLumix system roughly 25-40 hours faster than the plate count method in the Total Aerobic Count assay and 40-72 hours faster in the Yeast and Mold assay.

When testing for objectionable organisms, results may be obtained several days sooner than the plate count method.  Following the BioLumix protocol, 0.1 mL of the incubated sample in TSB is added to a selective vial and data is collected in the BioLumix instrument for 18-24 hours, depending on the assay.  If detection occurs, the sample may be verified with a confirmation assay.  This procedure eliminates hours or days waiting for plate results.

The BioLumix system is faster, less labor-intensive, and more sensitive than the plate count method.  With an automated certificate of analysis generated within 48 hours, the time-to-results is reduced and allows for quick release of products.

Caron Ockerman

References:

USP <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

USP <2021> MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS

USP <2022> MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MICROORGANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS 

BioLumix DMF Submission for Rapid Microbiological System Accepted by FDA

Ann Arbor, MI, May 20, 2011: BioLumix announced today that the US Food and Drug Administration (FDA) have accepted a BioLumix Drug Master File (DMF) entitled “Microbiological Rapid Method for the Detection and Enumeration of Microorganisms in Pharmaceutical Products”.  The DMF provides specific technical and regulatory information to the FDA which allows companies planning to use the BioLumix Rapid Microbiological System to obtain regulatory approvals for prescription drugs. 

Dr. Ruth Eden, President of BioLumix said, “The DMF can be referenced by drug manufacturers, reducing FDA review times and accelerating the regulatory approval process for use of the BioLumix System.  In addition, Over the Counter (OTC) products can be tested in conjunction with the BioLumix Validation Package.  The BioLumix DMF includes the information the FDA requires to assess alternative microbiological detection systems; such as, information about the technology, its accuracy, specificity, limit of detection, robustness, ruggedness, and equivalence to USP methodology.”

“FDA actively encourages use of new technologies including rapid microbiology methods (RMM)” stated Dr. David Hussong, FDA’s Associate Director for New Drug Microbiology, Office of Pharmaceutical Science, CDER.  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. 

“The BioLumix System is extremely easy to operate,  with its straightforward, streamlined testing design it offers  rapid, accurate results leading to reduced material-holding time for faster product release. “ said Kevin LaBrecque, Director of sales and Marketing at BioLumix. He continued, “It offers simplified single-platform testing for all assays with a 48-hour Automated Certificate of Analysis, while avoiding any product interference.”

ABOUT THE BioLumix DMF: In the DMF BioLumix shares specific technical and regulatory information with the FDA, including details about reagent composition, mode of action of reagents and growth media, and performance characteristics of its system. The FDA will be able to evaluate the use of the BioLumix system in pharmaceutical applications for rapid detection of microorganisms, when referenced by the customer.

ABOUT BioLumix:  BioLumix provides microbial testing products for the rapid detection of microorganisms to the Pharmaceutical, Nutraceutical, Dietary Supplements, Cosmetics, Toiletry and the Food Industries. Using an internally developed proprietary advanced technology a wide variety of test kits are available covering all required microbiological assays. By reducing microbial testing time and consequently, time for product release, the BioLumix system increases testing efficiency, manufacturing productivity and improves supply chain management.

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.

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.