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Sterilty Testing

The Sterility Test specified in USP General Chapter <71>  is understood to mean that pathogenic bacteria, yeast and/or fungi are absent from the article labeled “sterile”. In the United States, Europe, Japan, Australia and Canada the word “sterile” has that legal meaning. We don’t often think that we live in an environment surrounded by millions and billions of invisible microorganisms. The vast majority of them do not make us sick and in fact we could not live on earth today without them. However, pathogens like E.coli or Pseudomonas or Salmonella or Mycoplasma can and do make us sick, especially if we are older, infirmed or immunocompromised.  Recently the methodology used to determine if an article labeled sterile is sterile (sterility testing) was  harmonized amongst the United States, European and Japanese Pharmacopoeias.  In our blog about USP Monographs, we mentioned that a USP  monograph defines specific quality attributes. USP monographs which contain the “sterile” attribute must meet the requirements of sterility as defined in Chapter 71. For example,  Sterile Water for Injection is prepared from Water for Injection that is sterilized and suitably packaged. It contains no antimicrobial agent or other added substance. Thus, sterility Testing is the official referee test to determine legally if an article is sterile or not. An article labeled sterile is manufactured by a process that itself is validated to be effective with a very high degree of probability. This concept is known as the sterilization assurance level [SAL]. The current state of the art is a SAL of one in one million meaning that not more than one article of one million is likely to be non-sterile if a successfully validated process was followed.

Gibraltar Laboratories conducts the membrane filtration test using the Millipore Steritest system and the direct inoculation method in an ISO Class 5 sterility suite. In both methods the samples are placed in Soybean Casein Digest Medium (SCDM) and Fluid Thioglycollate Medium (FTM) and incubated for 14 days.  The samples are observed for evidence of microbial contamination daily. Gibraltar Laboratories also is expert in special sterility applications such as defined in ST 79 involving challenge with relatively resistant spores to steam sterilization. Gibraltar is expert in defining the protocol to validate the steam sterilization process including Installation Qualification, Operational Qualification and Performance Qualification.

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USP Monographs

The United States Pharmacopeia (USP) is a non–governmental, official public standards–setting authority for prescription and over–the–counter medicines and other healthcare products manufactured or sold in the United States. USP also sets widely recognized standards for food ingredients and dietary supplements. USP sets standards for the quality, purity, strength, and consistency of these products–critical to the public health. USP’s standards are recognized and used in more than 130 countries around the globe. These standards have helped to ensure public health throughout the world for close to 200 years. USP Monographs are standards that specify the quality attributes of thousands of ingredients, raw materials excipients and finished products sold in the United States. These standards are recognized as official by the United States Food and Drug Administration. An example of a USP Monograph is Aspirin.

The nine quality attributes that must be certified by qualified laboratories such as Gibraltar Laboratories are listed below

USP Aspirin

C9H8O4 180.16

  1. Identification—
  2. Loss on drying 731
  3. Readily carbonizable substances 271
  4. Residue on ignition 281:
  5. Chloride 221—
  6. Sulfate
  7. Heavy metals
  8. Limit of free salicylic acid
  9. Assay
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Bacterial Identification

Gibraltar Laboratories helps its sponsors control microbial contamination in
the manufacturing environment. Raw material and finished products are
usually subject to a compendia and/or sponsor specification with respect to
what organisms, if any, can be present. Microbial identification is the
science of correctly identifying by name the organism[s] present. By knowing
what organisms are present it is possible to design control measures to
control them. These microbial control measures can then be verified for
effectiveness by monitoring [e.g. Bioburden testing, environmental air
sampling, cleaning validations, etc.] Clinically, precise bacterial
identification and pathogen detection is necessary for correct disease
diagnosis, treatment of infection and trace-back of disease outbreaks
associated with microbial infections. Bacterial identification is used in a
wide assortment of applications including microbial forensics, criminal
investigations, bio-terrorism threats and environmental studies. At
Gibraltar Laboratories microbial identification is based on assortment of
characteristics including, phenotypic, biochemical and genetic.

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Microbial Limits Testing

At Gibraltar Laboratories one of the most frequent questions we are asked about is what is the proper way to examine non-sterile products?  It is easy to test a sterile product because it is understood that the absence of pathogenic bacteria, yeasts and molds is expected. However, for non-sterile products it is understood that microorganisms are likely to be present. For ingredients or products specifically found in the USP/NF chapter 61 and 62 describe what needs to be done to perform and validate the test. Chapter 61 , Microbial Examination of Nonsterile Products: Microbial Enumerations tests describes how to determine how many organisms are present or the sample’s Bioburden. Chapter 62,  Microbial Examination of Nonsterile Products: Tests For Specified Organisms describes how to determine by microbial identification if specified organisms are present in the sample. Sometimes sponsors choose a similar test for the same purpose. If so, it cannot be called by the official USP name. In this case the test name may be Microbial Limits or Microbial Content. All of these tests share the common purpose of determining in a sample the number of organisms [colony forming units, CFU] present per gram. In many cases, regardless of the cfu/gram the sponsor applies a quality attribute to not accept certain organisms. The kinds of organisms that are not allowed are recognized to be pathogenic especially to the young, infirmed or otherwise immunocompromised patient. For example E.coli, Pseudomonas aeruginosa, Burkholderia , Staph aureus and Bacillus cereus are widely thought of as specified organisms to not allow to be present in the final product. However, there are many other organisms that are similar that may also pose a serious safety risk. For this reason, the sponsor must evaluate the kinds of organisms that are discovered during the enumeration testing. This is done by skilled microbiologists using sophisticated technologies such as the  Vitek biochemical identification and sometimes also by Genetic analysis. Gibraltar Laboratories is certified to help you with these important tests and performs them routinely with a  very fast turnaround time. In fact, our laboratory was the 2010-2011 winner of the ACIL Turn Around Time Award.

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Antibiotic Testing

Gibraltar Laboratories performs certification of label claim testing regarding the potency of antibiotics (antibiotic testing). Certification is necessary to conform to US FDA current good manufacturing practice regulations. Potency means that the labeled strength of the antibiotic is accurate and therefore an infection caused, for example, by gram positive bacteria can be treated successfully by bacitracin. The potency of antibiotics may be demonstrated under suitable conditions by their inhibitory effect on microorganisms.

A reduction in antimicrobial activity also will reveal subtle changes not demonstrable by chemical methods. Accordingly, microbial or biological assays remain gnerally the standard for resolving doubt with respect to possible loss of activity. The antibiotic testing performed at Gibraltar Laboratories is described in the chapter <81> ANTIBIOTICS—MICROBIAL ASSAYS of the USP. This chapter summarizes procedures for the antibiotics recognized in the USP for which microbiological assay remains the definitive method. The cylinder-plate or “plate” assay depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a circular area or “zone” around the cylinder containing a solution of the antibiotic.

The antibiotic test is a very demanding test that requires expert care in the preparation of the media and organisms used in the assay. The fundamental principle is that the potency of the antibiotic is directly proportional to area of death it caused. If the antibiotic is not effective all of the bacteria added to the agar will form a confluent lawn. However, if the antibiotic is potent it will diffuse throughout the test system causing a clear zone where bacterial growth was prevented . This zone can be precisely measured with a sophisticated analytical instrument. This clear area of growth is also referred to as a zone of inhibition. Standards are always included in the assay so that one knows that a given zone area corresponds to a known potency. A regression curve is obtained from several concentrations of the standard and the unknown’s potency is obtained from its zone area.

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Endotoxin Testing

Gibraltar Labs Inc. is an ISO 17025 certified testing laboratory to perform Limulus Amebocyte Lysate (LAL) Test.  LAL, also known as Bacterial Endotoxin test, is a highly sensitive test used to detect or quantify bacterial endotoxins. Bacterial endotoxins, also known as pyrogens, are fever causing byproducts of gram negative bacteria. It is performed as an end-product endotoxin test for human and animal injectable drugs, biological products, and medical devices.

History: Rabbit pyrogen test was the first test used to detect endotoxins. LAL test officially replaced the rabbit pyrogen test due to its greater sensitivity because in rabbit pyrogen test, endotoxin amount wasn’t quantifiable. Amebocyte lysate from the horseshoe crab (Limulus poylphemus) was discovered to be the agent responsible for the blood clot in the amebocytes of the crab when came in contact with endotoxins. Amebocyte extract is now commercially prepared to be used as the major reagent used to detect and measure endotoxins. LAL test is a valuable test because it prevents the administration and or use of products and drugs which may cause fever, shock and death if highly endotoxic.

There are two methods of LAL endotoxin testing performed daily by Gibraltar’s highly experienced technicians. Gel-Clot method is based on the formation of clotting of the lysate in the presence of bacterial endotoxins in the test material. Kinetic Chromogenic method utilizes a microplate reader to automatically detect the amount of endotoxin concentration present in the sample when tested against a known amount of standards. Sensitivity of each test is based on the manufacturer’s label claim of the Lysate. Each new lot of reagents, a confirmation of label claim is performed by our certified technicians before a test can be considered valid and be performed on a routine basis. Gel-Clot has 0.03 EU/mL labeled lysate sensitivity and Kinetic Chromogenic has a sensitivity of 0.005 EU/mL.

Analyst qualification: Each one of our technicians who are certified to perform endotoxin testing also perform a proficiency test yearly to show competence in LAL Gel-Clot and/or Kinetic Chormogenic Test.

There are many other  qualifications, calibrations and proficiencies that we routinely perform and document so as to be certified to offer this critical service to you our customers.

All methods used in Gibraltar Laboratories follow Current USP <85> and <161>, EP, JP, ANSI/AAMI ST72 and FDA guidelines and are performed based on Standard Operating Procedures under GMP/GLP environment.

Any questions and/or concerns about the LAL bacterial endotoxin test, Gibraltar’s team of LAL experts will be happy to assist you.

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Steam Sterilization

Steam sterilization is a process of thermal sterilization employing highly pressurized saturated steam in a chamber [autoclave]. Steam sterilization is a widely employed sterilization process. The basic principle of operation is that the air in the sterilizing chamber is displaced by the saturated steam, achieved by employing vents or traps in gravity-displacement cycles or by active removal in dynamic-air-removal cycles. In order to displace air more effectively from the chamber and from within articles, the sterilization cycle may include air and steam evacuation stages. The design or choice of a cycle for given products or components depends on a number of factors, including the heat lability of the material, knowledge of heat penetration into the articles, and other factors described under the validation program (see above). Apart from that description of steam sterilization cycle parameters, using a temperature of 121C, the F0 concept may be appropriate. The F0 , at a particular temperature other than 121C, is the time (in minutes) required to provide the lethality equivalent to that provided at 121C for a stated time. Medical instruments and their sterilization trays need to be validated both with respect to cleaning effectiveness and sterilization. Most commonly companies submit validation data performed in accordance with ST79. The key component of a persuasive validation is a thorough assessment of worst case conditions. Gibraltar Laboratories recommends that worse case be defined not only with respect to the article subject to the validation but also with respect to the steam sterilizer under maximal and minimal load configurations. This is necessary for each sterilization cycle type, namely gravity-displacement and dynamic-air-removal [pre-vacuum]. Accordingly one selects the instrument in the case that is judged to be the most difficult to sterilize. This instrument is then challenged with artificial soil and highly resistant biological spores. The tray is wrapped in accordance with ST79 and placed in the steam sterilizer at its cold spot. The cold spot is determined by thermal mapping with NIST traceable thermocouples. Three half-cycles are performed at 134C. In order to pass the validation all of the biological spores must be killed and the instrument or its tray must be completely dry.

A related and additional validation involves cleaning instruments that have been challenged. For example one can challenge with oils, protein, blood, endotoxin, particulates and cleaning agent. Cleaning is performed using one or both of a defined automated cleaning process or defined manual cleaning process. Successful cleaning is demonstrated by the removal of the challenge and absence of particulates and cleaning agent.

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Total Organic Carbon

Total Organic Carbon (TOC) is a highly powerful analytical chemistry method that is valuable for many types of determinations. Two of the most common uses of Total Organic Carbon testing are for Purified Water testing and Cleaning Validations.  Gibraltar Laboratories excels in these areas because we use state of the art equipment manufactured by Sievers and our scientists are extensively trained and proficient.

Purified Water is water obtained by a suitable process. It is prepared from water complying with the U. S. Environmental Protection Agency National Primary Drinking Water Regulations or with the drinking water regulations of the European Union or of Japan, or with the World Health Organization’s Guidelines for Drinking Water Quality. It contains no added substance. The purity is judged by the absence of trace amounts of organic matter which is measured by the Total Organic Carbon test.

Total Organic Carbon is an indirect measure of organic molecules present in pharmaceutical waters measured as carbon. Organic molecules are introduced into the water from the source water, from purification and distribution system materials, and from biofilm growing in the system. Total Organic Carbon  can also be used as a process control attribute to monitor the performance of unit operations comprising the purification and distribution system. A Total Organic Carbon measurement is not a replacement test for endotoxin or microbiological control. While there can be a qualitative relationship between a food source (TOC) and microbiological activity, there is no direct numerical correlation. In addition to TOC, Purified Water must also meet the requirements of being relatively non-conductive as well as not harboring microbial coliforms such as E. coli. Customers typically rely upon Gibraltar Laboratories to quickly determine if the water they rely upon meets the requirements of purity such as low level of TOC, low conductivity and the absence of coliforms. Because Total Organic Carbon testing is so sensitive special care is needed to sample water. For example, the glassware used to collect the water must be specifically certified for use in TOC analysis or the determination will not be accurate. In addition, glassware used to collect samples must not be exposed to alcohol. Alcohol, such as ethanol, is a very useful step to control microorganisms but its use is detected and produces high TOC determinations leading to unnecessary investigations and/or OOS documentation.

Cleaning validation is performed to demonstrate that a new manufacturing campaign will not be contaminated by ingredients or cleaning agents previously used on common equipment. Since Total Organic Carbon is a non-specific measurement of carbon, it has wide spread application for companies that must comply with regulatory requirements. Typically, the manufacturer must assure that the active ingredients, excipients and cleaning agents used in a previous process are effectively removed so as to not be present as contaminants in a subsequent process. Since TOC can detect levels in the part per billion range it is possible to demonstrate that the equipment is not just visually clean but that it is clean scrupulously clean.

Gibraltar Laboratories is available to help and assist you to write validation protocols and reports to meet your needs. Please contact us for more information on Total Organic Carbon and other testing laboratory services.

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Tracking Your Information-Gibraltar Report Portal

Dear Gibraltar Friend,

As you have noticed we have improved our appearance recently. We have updated our logo to emphasize our widely known  company name. At  the same time we have updated our web page which makes it easier than ever before to do business with us. For example, now you can login to see a historical record of all of the reports that have been completed over the last six months. This makes it easier for you to verify that reports from us have been delivered and therefore the corresponding invoice should be approved for payment. In addition, if a report was misplaced you can conveniently login to review and download it. If you have not set up your account please contact us at 973.227.6882 or write me at DanielPrince@gibraltarlabsinc.com.

Sincerely,

Daniel Prince, Ph.D.

President

 

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Giving Back to Charity

Festival of Hope November 30, 2010

12-18-10

Dear GBL Reader,

When I am not busy leading Gibraltar Laboratories I spend a lot of my free time helping the LIONS organization do charitable work in the areas of Vision, Hunger, Youth and Environment. I strongly believe that we should all reach out and help those less fortunate than ourselves. We should give thanks for all of our blessings and abundance. Even now, as hard as it for many of us, we are better off than so many of the people alive today. As LIONS International we are part of the largest service organization in the world with 1.4 million members in 204 countries. If you can make a donation please consider the work of the Bridgewater Lions http://on.fb.me/fnKpkF. As a not for profit organization your gift is tax deductible. Recommended support levels for gifts are $1,250 Corporate/ $125 personal.

In 2010 we served our community by providing eye glasses, eye screening and food. Most noteworthy was our Festival of Hope event in Honor of the Bridgewater NJ Senior Citizens. Over 200 people attended our multi-faceted program which included eye screenings, presentation of a major gift to the senior citizens center, a choral concert, and birthday cakes.
The event started at 11 AM with the eye screening in the Eye Mobile with two professional ophthalmologists, Dr. Duan and Dr. Sun, and their staff. The doctors provided follow-up consultation for seniors. For the next few hours, about 30 senior citizens received free eye exams.
We also wanted to give a lasting and practical gift to the Senior Citizen center. In a moment of fun-filled suspense the Bridgewater Lions unveiled a 50” high-definition TV that the club donated to Bridgewater Senior Center. Adding an emotional component to this event, members of Harmonium, a Classical Choral Society, performed a concert with many familiar holiday tunes. The performance was of professional caliber and their beautiful voices were greatly appreciated by the audience. Sometimes, the audience even joined Harmonium in sing-along.
It was a wonderful day that meant so much to the seniors as well Lions and others in attendance. Several VIPs attended from local government. Namely, Somerset County Freeholder Patrick Scaglione and Bridgewater Council President Matt Moench.

Giving Value to the Community: The community was moved and touched by our efforts. Many seniors came up to us and shared that “…this was wonderful.”

In summary the value to the community was:

1. All who wanted eye screenings were provided for.
2. All seniors were valued as important and remembered
3. A permanent gift was made to the Senior Citizen Center [Large television]
4. An emotional connection was made through the performance by Harmonium Choral Singers

The BridgewaterPatch captured this sentiment in their issue of December 4, 2010 [http://bit.ly/eovv8h].
“Senior center member Beatrice Este, of Bridgewater, said she really enjoyed the event, especially because it fell on her 94th birthday.”Today is nice because it’s my birthday and I’m very entertained,” she said. Este said she is excited to watch some of her favorite musicals and comedies on the big screen, so long as there is “nothing sad,” she said.”
The article concluded,
“Overall, participants were impressed that so many people took time out of their busy work days to come and serve the seniors. From doctors to singers to volunteers, the day was about donating talents to help others.”

For more details see
ICEPN television http://bit.ly/dPgzxD.
http://www.facebook.com/pages/Bridgewater-NJ-Lions-District-16D/155295417824944?v=wall
BridgewaterPatch http://bit.ly/eovv8h
World Journal http://bit.ly/fXTW0q

PS: Please give generously so we may continue our great work when so many are in need. Please make your check payable to Bridgewater NJ Lions 16D.
Sincerely,
Daniel Prince, Ph.D.
President Gibraltar Laboratories and Bridgewater Lions NJ 16D