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Biotech Contract Development Services
The following is a list of the extraction methods offered. The extraction method is the procedure for removing the microorganisms from the product being tested.
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Immersion
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Flush / Rinse
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Surface Sampling
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Dilution
The following is a list of the enumeration methods offered. The enumeration method is the procedure to determine the number of viable microorganisms that are extracted from the product being tested.
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Membrane Filtration
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Direct Plating
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Most Probable Number (MPN)
The microbial load test should be validated to ensure that the method is effective, accurate and reproducible in determining the material microbial load in a test article. The most suitable methodology should be determined as part of method development. Validation will be performed in the best method that shows higher recovery of organisms and no inhibition. Validation is performed per USP/EP for Pharmaceutical or Biotechnology products and AAMI for medical devices. Cosmetics are tested per FDA BAM manual. MQA can develop custom bioburden procedures for difficult to test samples.
MICROBIAL ENUMERATION TEST
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Note: Microbial load test is performed on undiluted sample; additional dilutions will be an additional charge. Microbial load liquid samples must be shipped cold. Liquid samples will be tested within 24 hours of receiving at the lab. Samples should be collected in sterile containers.
BACTERIAL ENDOTOXIN
The Limulus Amebocyte Lysate (LAL) test is in-vitro assay used for detection of pyrogenic substances (endotoxin) in sterile parenteral drugs, in-process manufacturing samples, cleaning validation rinse samples and medical devices. Limulus amebocyte lysate is an aqueous extract of blood cells (amebocytes) from the horseshoe crab, Limulus polyphemus. The LAL test can be performed by any of the following methods: Gel-Clot method, Kinetic Turbidimetric or Kinetic Chromogenic. Selection of the most suitable LAL test method is based on the assay results obtained during the assay development and validation.
The Gel-Clot LAL test is performed by adding the LAL lysate and the test specimen in a pyrogen-free reaction tube. The reaction solution is placed immediately in a dry block incubator or water bath at 37 ± 1°C for 60 ± 2 minutes. After 60 minutes the tube is removed from the incubator and inverted. A positive test is a firm gel (clot) that remains intact in the bottom of the reaction tube after inversion of 180°. The concentration of endotoxin in the tube is greater than or equal to the sensitivity of the LAL lysate. Lack of a gel indicates a negative test. The Gel-Clot LAL test can detect as little as 0.015 EU/per mL.
In the Kinetic Turbidimetric LAL method, either the rate of increase in turbidity or the time taken to reach a particular level of turbidity (the onset time) is determined. The assay requires a 96 well plate reader to incubate multiple samples at a controlled temperature (37°C) and take optical density readings at regular intervals. Standard curves are prepared by plotting the log onset time against the log concentration of standard endotoxin and are used to calculate endotoxin concentrations in specimens. The sensitivity of this test is 0.01 EU/mL.
In the Chromogenic LAL test, the LAL reagent is mixed with test sample in a microplate and incubated in a reader at 37 ± 1°C. Absorbance measurements are collected with time after addition of Chromo-LAL and analyzed by the K-QCL software. The time (onset time) taken for a sample to reach a specified absorbance (onset OD) is calculated; and a standard curve, showing the linear correlation between the log onset time and the log concentration of standard endotoxin, is generated. The sensitivity of this test is 0.005 EU/mL.
Water, liquid and powder samples are tested per USP <85> "Bacterial Endotoxin or EP 2.6.14 "Bacterial Endotoxin". Devices are tested per USP <161>, Transfusion and Infusion Assemblies and Similar Medical Devices and USP <85>. Other methods such as ANSI/AAMI ST72:2002, Bacterial endotoxins- Test methodologies, routine monitoring, and alternatives to batch testing and USP are available. Samples are stored at 2-8°C for 24 hrs or -20°C if not tested in 24 hours.
BACTERIAL ENDOTOXIN
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MICROBIAL LIMIT TEST
The Microbial Limit Test is designed to perform the qualitative and quantitative estimations of specific viable microorganisms present in samples. It includes tests for total viable count (bacteria and fungi) and screening for pathogenic organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Clostridium and Escherichia coli. The type of pathogen screening will depend on the product (e.g., oral products are typically tested for absence of E. coli and Salmonella). When test samples have antimicrobial activity or when they include antimicrobial substances, these antimicrobial properties are eliminated by dilution, filtration, neutralization, inactivation, or other appropriate means.
Samples should be submitted at a temperature similar to that in which the sample is stored. Avoid extreme temperatures.
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BIOLOGICAL INDICATORS (BI's)
MQA can provide biological indicators (BI) designed to meet the ANSI/AAMI/ISO/EN 11138 standards and USP guidelines. We purchase our BI's from selected vendors. BI's provided by MQA are tested for spore population and identity by MQA. D value verification can be arranged per customer request.
Biological Indicators are:
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Used to evaluate the effectiveness of sterilization processes. |
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Essential in performing sterilization cycle validation. |
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Used for ethylene oxide gas, dry heat, steam and radiation sterilization processes. |
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BI's are available in a wide range of organisms, populations and packaging. |
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BI's are available as strips (standard size or mini), discs, suspensions, self-contained, ampoules nd custom configurations. |
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BACTERIA AND MOLD IDENTIFICATION
Bacterial isolate characterization services include two methods: Phenotypic - bioMerieux API ID system and Genotypic - Dupont RiboPrinter system.
The RiboPrinter® microbial characterization system provides the speed, accuracy and resolution needed to identify bacteria and then compare them at the strain level for efficient and consistent characterization. Using powerful DNA-based information, the RiboPrinter® system provides an automated genetic snapshot (RiboPrint® pattern) of any bacterial strain in less than eight hours. RiboPrint® patterns identify and characterize environmental isolates, pathogens, spoilage organisms, control strains, beneficial organisms or any bacterium that is important to the pharmaceutical, personal care and food safety industries.
The bioMerieux API system is a phenotypic system that uses an index profile database. The API bacteria identification includes organism isolation: Gram stain, Oxidase, or Catalase and the ID.
Mold identification is performed by macroscopic and microscopic examination of the isolate. Additional confirmatory testing services are as follows:
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BACTERIOSTASIS AND FUNGISTASIS TEST
Bacteriostasis and fungistasis testing is required by all major pharmacopeias as a means of validating the Sterility Test. This essential step should be conducted on all new products or materials initially submitted for testing and should be repeated periodically to assure continued compatibility with established testing procedures.
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CONTAINER CLOSURE STUDIES
A container closure system refers to the sum of packaging components that together contain and protect the drug dosage form. Regulatory agencies require that companies must demonstrate that the container closure system used to package a drug or medical device will protect the product from microbial contamination or preserve the sterility for the life of the product. The product container closure system can be tested by physical (Dye Leak test) or microbiological means (microbial immersion test).
The Dye leak test is performed by immersing the samples in a vacuum chamber filled with methylene blue dye. After exposure to the dye and test conditions, the samples are inspected for the presence of the dye.
The microbial challenge test is performed by immersing the simulated samples filled with Tryptic Soy broth in a media broth inoculated with B. diminuta. The samples are incubated for 14 days at 30-35°C and then inspected for contamination.
Typically, the test is performed using the same number of samples as the sterility test plus positive and negative controls.
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USP and EP ANTIMICROBIAL EFFECTIVENESS TEST
The antimicrobial effectiveness test (AET) is designed to provide a laboratory test that gauges the level of biological activity possessed by the preservative system in a pharmaceutical or cosmetic product. The USP <51> and the BP/EP have specific requirements and different acceptance criteria for each type of pharmaceutical products. When performed according to USP <51>, five indicator organisms are utilized for the purpose of challenging the preservative system in a product. Three of the five USP indicator organisms, Escherichia Coli, Pseudomonas aeruginosa, and Staphylococcus aureus, address the growth of bacteria. Candida albicans is the representative yeast, while Aspergillus
The test is performed by inoculating the product aliquot with a number of organisms between 1 x 105 (100,000) to 1 x 106 (1,000,000) colony forming units (CFU) per mL of product. At various intervals, depending on the category, the product is tested to determine its ability to control reproduction or destroy the microorganisms. A logarithmic reduction is evaluated at each test interval required for the category.
For testing purposes, the USP has divided test articles into four separate categories:
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Category 1 - |
Injections, other parenterals including emulsions, otic, sterile nasal products made with aqueous bases or vehicles. |
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Category 2 - |
Topically used products made with aqueous bases or vehicles, non-sterile nasal products, and emulsions, including those applied to mucous membranes. |
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Category 3 - |
Oral products other than antacids made with aqueous bases or vehicles. |
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Category 4 - |
Antacids made with an aqueous base. |
The AET test is typically performed during product development and as part of a stability study, to determine if a preservative system will stand up to the product's shelf life. The first time a product is tested for Antimicrobial Effectiveness, a validation is necessary to show the microorganisms are able to withstand the formulation. A full validation is performed in three independent studies with each of the studies recovering not less than 70% of the growth inoculum versus the control.
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DISINFECTANT EFFICACY STUDIES
All disinfectant, sanitizer or sporicidal solutions must pass testing requirements by the EPA, FDA and EU regulatory bodies in order to be license. Testing requirements for licensing are very limited and specific and do not represent actual use. FDA requires that drug and medical devices manufacturers validate the disinfectant to demonstrate the efficacy under use conditions and microbial flora specific to the manufacturing site. European disinfectant testing requirements are indicated in CEN/TC 216 (European Committee for Standardization Technical Committee 216 - Chemical Disinfectants and Antiseptics).
Disinfectant Testing Standards
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USA
AOAC
Carrier test
USP <1072> Disinfectants and Antiseptics
Kill time
Surface coupons
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EU
CEN/TC 216 (European Committee for Standardization Technical Committee 216 - Chemical Disinfectants and Antiseptics)
Phase I:
Basic Suspension test to examine bacterial and fungicidal activity.
Phase 2:
Step 1:
suspension test to examine bactericidal, fungicidal, or sporicidal activity under laboratory conditions appropriate to its intended use (under conditions representative of practical use).
Step 2: Handwash, Handrub and Surface tests
Handwash, Handrub and Surface tests
Phase 3:
Field tests under practical conditions
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ANTIMICROBIAL PROPERTIES EVALUATION
MQA provides a comprehensive program for evaluation of antimicrobial properties of compounds, liquid or solid materials. Depending on the compound testing may consist of method development, Kill Time studies, MIC and MBC.
Method development
MQA will develop an evaluation method that provides accurate and reproducible results.
Kill Time Studies
Kill Time studies are performed by spiking challenge organisms on aliquots of the liquid compound at different concentrations and test intervals. The purpose of the test is to demonstrate log reduction of the challenge organisms under the study test conditions.
MIC and MBC
The basic quantitative measures of the in vitro activity of antibiotics are the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The MIC is the lowest concentration of the antibiotic that results in inhibition of visible growth (i.e. colonies on a plate or turbidity in broth culture) under standard conditions. The MBC is the lowest concentration of the antibiotic that kills 99.9% of the original inoculum in a given time.
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MYCOPLASMA DETECTION BY PCR TEST
Mycoplasma can contaminate cell cultures and cost biotechnology companies lost time and reduce profits. The organism can severely reduce cell growth and metabololism, which will affect the field and possibly the the activity of the recombinant protein. Mycoplasma is a unique group of microorganisms that fall in the category between the bacteria and viruses.
Currently, European and
MQA developed a new method based on Polymerase Chain Reaction (PCR) that reduces the mycoplasma screening test to 1-2 days. MQA method is GMP validated using 6 different Mycoplasma species. In extracts of mycoplasma DNA and in the presence of CHO cell with antibiotic, the assay limit of detection for six different mycoplasma species is 10 genomic copies. The assay is very specific to detect Mycoplasma in the presence of other genomic DNA.
Cell line samples should be pre-cultured in the absence of mycoplasma active antibiotics for several days to maximize test sensitivity. Samples should be derived from cultures that are at 90-100% confluence (1.0 x 106 to 1.0 x 107cells). PCR inhibiting substances may accumulate in the medium of older cultures.
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MEDIA INCUBATION AND GROWTH PROMOTION
MQA provides media, growth promotion and sterility check studies, samples pick-up, incubation, plate inspection and reporting seven (7) days a week.
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Other Tests:
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