LexaMed is a FDA registered and ISO 13485 certified Contract Research Organization (CRO) with laboratories focused on providing the highest quality consulting services and microbiology and chemistry testing for compounding pharmacies. With years of industry experience, LexaMed offers a proven approach of combining integrated consulting and laboratory services that align our clients operations with the regulations like the FDA 21 CFR 201/211.
Since 2012, the United States Food & Drug Administration has continued to aggressively increase regulatory actions, pressure and scrutiny aimed at compounding pharmacies. LexaMed is uniquely positioned to guide compounding pharmacies through the challenging times of this heightened regulatory environment. Our industry expertise and decades of experience in the pharmaceutical industry includes, but is not limited to the following: Quality Systems, Laboratory testing both microbiology and analytical chemistry, validation of equipment and aseptic processes, environmental monitoring of both viable and non-viable particulates, stability programs and FDA interactions.
Container Closure Integrity Dye Immersion Test
The Antimicrobial Preservative Efficacy test is designed to verify the efficacy of antimicrobial preservatives added to non-sterile dosage forms or sterile multi-dose containers in order to inhibit the growth of microorganisms that may be introduced inadvertently, during the manufacturing process or product use. Antimicrobial effectiveness must be demonstrated on all injectables packaged in multiple-dose containers and other products such as ophthalmics containing antimicrobial preservatives.
The USP Chapter <51> Antimicrobial Effectiveness Test (AET) specifies the use of Candida albicans (ATCC 10231) Aspergillus brasiliensis (ATCC 16404), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538) as challenge organisms for evaluating the efficacy of the antimicrobial preservative.
Bacteria are grown on Soybean Casein Digest Agar plates (SCDA). Yeast and Mold are grown on Sabouraud Dextrose Agar (SDA). All organisms are adjusted to approximately 1.0 x 108 colony-forming units (CFU)/mL by diluting in sterile saline.
Individual test samples and a positive control are prepared for each organism. At Time 0, the test samples and positive controls are individually inoculated with each organism to reach a concentration of 1.0 x 105 – 1.0 x 106 CFU/mL. The positive controls and samples are assayed immediately after inoculation to determine the challenge level and to provide confirmation that the samples were inoculated.
The inoculated samples are stored in a 20 – 25°C incubator at 7, 14 and 28 days. After incubation the samples are removed from the incubator and assayed for surviving organisms. The organisms recovered from each time point are compared to those seen in the Time 0 positive control. Changes in the concentrations of organisms are converted to Log reduction values in order to assess the antimicrobial effectiveness of the samples. Acceptance criteria are established based on the type of product (ex: injectable, oral dose) and are specified in USP Chapter <51>.
Endotoxin contamination of an injectable product results from the presence of Gram negative bacteria or components of their cell wall and can occur as a result of inadequate manufacturing controls. Drug product components, containers, closures, manufacturing equipment and water systems are among the areas to address in establishing endotoxin control. Adequate cleaning, drying, and storage of equipment as well as routine monitoring of water systems and incoming materials can help to ensure that the manufacturing process does not contribute endotoxins to the final product.
USP Chapter <85> Bacterial Endotoxins Test (BET) is an in-vitro assay to detect and quantity Gram negative bacteria, the source of endotoxin. The BET is performed as part of lot release testing for injectable pharmaceutical products and medical devices with direct or indirect contact to the cardiovascular system, lymphatic system or cerebrospinal fluid. The assay is also used as a screening test for incoming components and as a Quality Control (QC) test for water used in the manufacturing process.
There are three methods used for this test: the semi-quantitative Gel-clot method, the quantitative Kinetic Turbidimetric method and the quantitative Kinetic Chromogenic method.
A liquid sample or sample extract prepared from a medical device or pharmaceutical product is assayed using Limulus Amebocyte Lysate (LAL). LAL is a reagent made from the blood of the horseshoe crab and in the presence of bacterial endotoxins, forms a clot in the Gel Clot method a change in turbidity in the Turbidimetric method and color change in the Chromogenic method. The test sample response is compared to a standard curve made from known endotoxin concentrations. All tests are performed in duplicate. A positive product control and negative control are included as part of each assay.
Prior to conducting the BET assay each product must be validated to demonstrate that the product does not interfere with the ability to detect endotoxins. This is accomplished with the positive product control (also called the spike recovery) for the kinetic test methods (Turbidimetric and Chromogenic), and with a separate inhibition and enhancement assay for the gel-clot test.
Products requiring a treatment such as reconstitution in a solvent other than water, heat denaturing, centrifugation or filtration should be validated to demonstrate that the treatment does not result in the loss of endotoxins. This is accomplished by inoculating the sample with endotoxin, subjecting the inoculated sample to the chosen treatment then testing for endotoxin recovery.
Endotoxin limits for pharmaceutical products are specified in individual USP monographs and are based on the maximum dose that can be administered in a 1-hour time period. The limit for medical devices is not more than 20.0 EU/device for devices with blood contact and not more than 2.15 EU/device for devices with cerebrospinal fluid contact.
USP Chapter <71> Sterility Test states that prior to conducting a sterility test on a sterilized product, its level of bacteriostatic and fungistatic activity should be determined, that is the degree of inhibition to microbial growth that may be caused by substances in or on the product. Bacteriostasis/Fungistasis (B/F) should be evaluated following the first sterilization or aseptic manufacturing of all new products prior to sterility testing or when significant product or manufacturing changes occur. The testing is designed to evaluate B/F activity and to validate the chosen sterility test method.
Based on the media type to be used in the Sterility Test, low numbers (<100 CFU) of selected bacteria and fungi challenge organisms as specified in the USP, are added to containers with the test article present to demonstrate that they can be detected in the presence of the test article. For samples sterility tested by the membrane filtration method, test samples are filtered through a 0.45 µm membrane filter and rinsed with a sterile diluent. The rinse is then inoculated with the chosen organisms and the membrane filters are aseptically added to the appropriate media. For samples sterility tested by the direct transfer method, test samples are aseptically transferred into a volume of media sufficient to cover the sample, the containers are then inoculated with the chosen organisms. The volume of media utilized in the B/F testing must be the same amount utilized for routine sterility testing procedures. The B/F validation will demonstrate reproducibility of the method to reliably recover representative microorganisms. If growth is inhibited, a documented investigation must be initiated and modifications such as increased dilution, additional membrane filter washes or addition of inactivating agents should be implemented to the test method to optimize recovery. [/av_textblock] [/av_two_third] [av_two_third first] [av_heading heading='Container Closure Integrity Dye Immersion Test ' tag='h3' style='' size='' subheading_active='' subheading_size='15' padding='10' color='' custom_font=''][/av_heading] [av_textblock size='10' font_color='' color=''][/av_textblock] [av_textblock size='' font_color='' color=''] A container closure system refers to the sum of packaging components that together, contain and protect the integrity of the contents of the container. The ability of the container closure system to maintain the integrity of its barrier, and hence, the sterility of a drug product throughout its shelf life must be demonstrated. The Container Closure Integrity (CCI) Dye Immersion test is used to assess the integrity of a sample container. It can be used to test vials, screw cap bottles, and syringes. Study Methodology:
Test samples are immersed in a solution of 0.1% Methylene Blue Dye suspension in a specifically designed test chamber and a pressure differential (vacuum and/or pressure) is applied. Following a specified exposure time, the vacuum/pressure is released and the containers are washed to remove dye from the outer surfaces. Validated breached positive controls ranging from 10-30 µm are included with each run to verify the performance of the vacuum chamber with the test vials/containers.
Test and positive control containers are analyzed after exposure for the presence of dye using an ultraviolet-visible (UV/Vis) technique. Products that are viscous or opaque are not appropriate for this test method.
Microbial identification is critically important when monitoring clean rooms, flow hoods or when samples are found to be contaminated with a microorganism resulting in growth in or on test media. Characterizing a microorganism can provide important information as to its origin and potential impact in relation to a product or in relation to the environment in which it was isolated. Identification of resident microorganisms in the facility helps in an investigation where a new source of contamination may have potentially been introduced through the facility, people, and/or processes.
There are a number of methodologies for microbial identification from microscopic inspection to gram staining to amplification of the DNA from contaminating organism(s).
Particulate Matter consists of randomly sourced extraneous substances coming from a manufacturing environment that cannot be quantitated by a chemical analysis because of their heterogeneous composition. Ophthalmic Solutions should be essentially free from visually observed particulates. USP Chapter <789> describes a two stage test approach for enumerating particles within specific size ranges for ophthalmic solutions. The solution is first tested by a light obscuration method (Phase 1) if it fails to meet prescribed limits it must then be tested by the microscopic method (Phase 2) which has its own limits. If a solution does not have the clarity and viscosity similar to water it may provide erroneous data when tested using the obscuration method therefore in those cases it is acceptable to use the microscopic method only.
The results obtained from testing a group of test articles cannot be extrapolated to other test units with certainty, therefore, it is important to develop a sound sampling plan. The plan should be based on operational factors, product volume, historical particulate numbers, particulate size distribution and variability of counts between units.
Phase 1 – Light Obscuration Method
For large-volume parenterals single units are tested, for small-volume parenterals, less than 25 mL, the contents of 10 or more are pooled for testing. The test solutions are analyzed using a HIAC liquid particle counting system. A minimum of three aliquots are withdrawn and particles ranging from 2.0 μm to 150 μm are counted and sized. The counter calculates the average cumulative counts, average differential counts, average cumulative counts per ml and average differential counts per ml. The ophthalmic solution meets the requirements of the test if the average number of particles does not exceed 50 per mL for ≥ 10 µm size particles or 5 per mL for ≥ 25 µm. If the average number of particles counted exceed these limits the test solution must be tested by the Microscopic Method.
Phase 2 – Microscopic method
The total pooled solution volume or a single unit volume are vacuum filtered through a 1.0 µm or finer pore size membrane filter. After filtration, the membrane filters are placed into petri slide dishes and dried. Dried filters are analyzed under a microscope, using a calibrated graticule, at 100X magnification. Using oblique illumination at an angle of 10˚ – 20˚, the particulates on the surface of the membrane are sized and counted using the graticule micrometer. The ophthalmic solution meets the requirements of the test if the average number of particles does not exceed 50 per mL for ≥ 10 µm size particles, 5 per mL for ≥ 25 µm or 2 per mL for ≥ 50 µm size particles.
Potency is a measure of drug activity expressed in terms of the amount required to produce an effect of a given intensity. Potency testing provides documentation that the concentration, strength or activity of the active ingredients are as labeled for the compounded preparation.
LexaMed has access to a number of technologies for potency testing including state-of-the-art HPLC technology, LCMS, GC, UV/VIS, NIR and titrations.
Stability programs are designed to determine a beyond-use-date (BUD) for a preparation. When a claim of stability is made that is outside the standards outlined in USP <797> for low, medium and high risk compounds, the claim of that stability must be validated with a documented testing program. Once validated, any significant changes to the documented validation may require some level of revalidation.
Sterility testing is necessary for pharmaceuticals and medical devices that claim to be sterile or free from viable microorganisms. Sterility testing methods are required to be accurate and reproducible, in accordance with 21CFR 211.194 and 211.165. USP Chapter <71> Sterility Test is the principal source used for sterility testing of sterile drug products produced by aseptic processing. The quantity and number of products to be tested is determined both by batch size and individual container fill volumes. It is important that the samples represent the entire batch and processing conditions. Samples should be taken at the beginning, middle, and end of the aseptic processing operation.
The test evaluates samples for sterility by transferring the solution into growth media, incubating for a minimum of 14 days and then checking for evidence of microbial contamination. To optimize aseptic transfer of samples into the test media, all testing is performed in a state-of-the-art ISO Class 5 cleanroom.
Samples may be tested using either a direct transfer or membrane filtration method using a soybean casein digest broth (SCDB) and fluid thioglycollate media (FTM). Samples tested by direct transfer are aseptically immersed in the test media. Samples tested by membrane filtration are passed through a 0.45µm filter, and the filter is immersed in the test media. SCDB is used for aerobic and fungal growth at incubation temperatures of 20 – 25°C and FTM is used for anaerobic growth at an incubation temperature of 30 – 35°C.
The successful completion of a USP sterility test does not assure that all products in a sterilization lot are sterile. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedure which, along with the use of routine controls and monitoring, forms the foundation of a sterility assurance program.