Endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), found in the outer membrane of various Gram-negative bacteria. When injected into the body, they may cause anything from mild to severe inflammation, to in the most severe cases, septic shock.
Presence of endotoxins in a compound is evidence of a current or previous contamination. Removal of endotoxins requires a specialized filtration system, and they are not removed by 0.2um filters used to sterilize compounded medications. Some typical sources of endotoxins are raw materials, water, instruments and reagents that are stored and used to compound, for example solutions used to adjust the pH of solution.
DYNALABS employs the kinetic chromogenic and photometric quantitative testing methodologies, the kinetic-chromogenic, and the kinetic-turbidimetric methodologies. Both technologies are sensitive down to 0.005 EU/mL. The test protocols are based on guidelines delineated in USP <85> Bacterial Endotoxins Test.
Samples are assayed in quadruplicate against a five standard curve run in duplicate. Two of the quadruplicate samples are spiked with a known amount of endotoxin that must recover within an acceptable range to verify that the sample is not inhibiting the test.
- Oil Injections
- Aqueous Injections
- Sterile Solutions
Microbial identification becomes critically important when samples are found to be contaminated with a microorganism and/or if routine monitoring of the clean room or flow hoods results in organisms growing in or on test media. Identification of contaminating organisms assists in understanding probable sources of the contamination, which will help in preventing or at least minimizing future contaminations. Identification is also important in determining the “usual or resident” microorganisms in the facility, so if new organisms are identified as part of an investigation, the source of the contamination may be new and/or not associated with the facility, people and/or processes.
DYNALABS employs a number of methodologies from gram staining and microscopic inspection, to amplification of the DNA from contaminating organism(s). Results are reported based on the tests performed.
FORMS TYPICALLY TESTED
- Positive Media Fill Tests
- Bench and/or Hood Swabs
- Settling Plates
- Contaminated Samples
Particles measured by this test are defined as insoluble, free floating substances that cannot be measured by chemical means. The presence of particles in solutions and parenteral injections is an indication that the active(s) and/or excipient(s) may have precipitated or crystallized. It is critically important to determine the number and size of particles, because if present they may cause blockages that could be detrimental to the health of the patient.
DYNALABS employs two method of measuring particulate matter:
- USP <788> Light Obscuration Particle Count Test – Instrumental analysis using a laser to count and size each particle. Specifications relate to the size of the particle counted and the volume of the solution/injection being tested (see table below).
|Less than 100mL||25/mL||3/mL|
|Greater than 100mL||6000/container||600/container|
- USP <797> Physical Inspection – Visual inspection of the sample held up against a lighted white and black background as specified in Finished Preparation Release Checks and Tests Section. The specification for this test is absolute (i.e., None Detected).
- Aqueous Injections and Solutions
- Oil Injections (Physical Inspection only)
- Medical Devices
The test for pH is to measure the acidity or basicity of an aqueous solution. The pH of water at 25°C is approximately 7.0. Solutions with a pH of greater than 7.0 are said to be basic and solutions with a pH of less than 7.0 are acidic.
DYNALABS tests pH using a standardized potentiometric instrument according to USP <791>. For samples with a monograph in the USP, DYNALABS will use the specification defined in the monograph, unless otherwise instructed by the customer. For samples that do not have monographs DYNALABS uses the customer-defined specification, and in the absence of a customer-defined specification will use a specification of “Report Result”.
- Aqueous Injections and Solutions
Potency: A measure of the concentration, strength or activity of a medication. Potency is usually expressed as the amount of active pharmaceutical ingredient (API) within some unit measure of the medication, such as, but not limited to mg/mL, mg/gm, IU/mL, mg/capsule, mg/pellet etc., where the numerator is the amount API, and the denominator is the unit measure of the medication. Specifications for potency are typically expressed as a range, such as but not limited to 90.0 – 110.0% of label value.
Purity: A measure of the pureness of a raw material. Purity is typically expressed as a percent of activity, and is most often used to determine the factor used to adjust for the amount of impurities within an API raw material. Specifications for Purity are also typically expressed as a range, albeit a narrower one, such as 98.5 – 101.5%
DYNALABS employs a number of technologies for testing potency. However, purity measures and stability studies are performed using technologies where the methods can be proven to be stability indicating (e.g., HPLC). Our list of potency/purity testing technologies, include but are not limited to, HPLC, LC-MS, GC, UV/VIS, NIR, and Titrations.
When testing for potency or purity, the concentration of the API is assigned a value based on prequalified, tightly controlled reference standards. Testing protocols are developed based on guidelines delineated in USP <621> Chromatography and USP <851> Spectrophotometry and Light-Scattering, and are validated according to USP <1225> Validation of Compendial Procedures and ICH Guidelines. The customer is notified immediately of any sample that does not test within acceptable limits.
Specific gravity is the ratio of the weight of a liquid, to that of an equal volume of water at the same temperature and barometric pressure.
Specific gravity is commonly used in industry as a simple means of obtaining information about the concentration of solutions. It is also used when using weight to control volume. A specific gravity of greater than 1 means the solution is heavier than water, and conversely a specific gravity of less than 1 means the solution is lighter than water. A solution with a specific gravity of 1.0234 would mean that 100mL of the solution would weigh 102.34gms.
DYNALABS employs digital density meter technology for testing specific gravity. Test protocols are developed in accordance to USP <841> Specific Gravity. Samples are assayed in triplicate and results are reported to 4 decimal places.
FORMS TYPICALLY TESTED
Sterility is a state of being free of microorganisms. Results are expressed as “No Turbidity Observed – Interim” for samples that are found to be free of microorganisms, or “Positive – XX” for samples that are found to contain microorganisms, where the “XX” represents the media in which the sample was found to be positive.
DYNALABS employs the Membrane Filtration methodology for testing sterility of compounded medications. The test protocol is based on guidelines delineated in USP <71> Sterility Tests.
The sample is split into two equal portions and each portion is transferred into one of two 0.2um filter canisters. A vacuum is applied to the canisters and the aqueous portion of the medication is filtered away. The canisters are then washed 3 times to remove any residual drug and preservative. One canister is filled with Tryptocase Soy Broth (TSB) and incubated at 22.5 ± 2.5⁰C. This media is designed to grow aerobic microorganisms and fungi. The second canister is filled with Fluid Thioglycollate Medium (FTM) and incubated at 32.5 ± 2.5⁰C. This media is designed to facilitate the growth of anaerobic and facultative anaerobes. Both canisters are incubated for 14 days. For turbid samples, a portion of the incubated media is transferred to fresh media on the 14th day and then incubated an additional 4 days.
Negative controls are run at the beginning and end of each batch of samples tested. All control and sample canisters are inspected every day, and the results updated according to the inspection findings. If any sample canister shows signs of growth the customer is notified immediately.
All Sterile Forms
Beyond Use Dating (BUD)
DEFINITION OF DRUG STABILITY
Beyond Use Dating (BUD) is the date determined beyond which medications that have been manipulated and/or repackaged and stored or dispensed in a container other than the original manufacturer’s storage container may not be used. (Example – A refrigerated drug that has a dating of nine days moves to a room temperature drug that has a dating of 90 days.) This is different than expiration date which is the date beyond which ideally stored medications in the unopened manufacturer’s storage container may not be used. Stability dating can be demonstrated on specific formulas which include drug, concentration, diluent, container, and storage conditions. Each product (sterile or non-sterile), should be tested from the compounded date and last point. Typically, it is good to track any trends for a particular product throughout the study.
DYNALABS has developed a “safe harbor” for hospitals and compounding pharmacies as it pertains to beyond use dating of selective drugs. We perform stability indicating tests (three lots to demonstrate consistency of stability) within the USP guidelines via specific storage condition of the drug. Dating is based on historical data that we have gathered on selective drugs with different variables. Our approach not only addresses the dating but also the implementation of the dating within the pharmacy workflow.
DYNALABS has a department dedicated to performing method development and method validation services. Our scientists have years of experience working on stability indicating methods, residual solvent methods, and dissolution methods. We have our own protocols that comply with FDA guidelines or we can follow client-supplied protocols. Our protocols are flexible enough to be custom designed to meet client specific requirements. We offer method development and validation services using a wide range of technologies including: HPLC, GC, TLC, ELISA, FTIR, UV-VIS Spectrophotometry. We have extensive experience in HPLC method development, HPLC validation, GC method development and GC validation. We use these analytical techniques to develop and validate methods for API’s, intermediates, raw materials and finished products.
We provide a report at the conclusion of each project. Included within this report is the analytical method itself which is written in a step-by-step format.
Our protocol includes the following parameters:
- Limit of detection (LOD)
- Limit of quantitation (LOQ)
- System Suitability
- Standard & Custom Forged Degradation
- Stability of Standard and Sample Solutions
DYNALABS brings years of experience performing all types of analytical testing on many drug substances and drugs. We bring the highest level of quality and attention to detail to our clients to assure that their analytical method development and validation projects are successfully executed. We are FDA and DEA registered, and cGMP compliant.
BACKGROUND When preservatives are used for the purpose of minimizing growth of microorganisms, the effectiveness of the preservative within the context of the compound’s formulation will need to be validated. There are standard organisms used for this test. Testing to show the preservative is effective should include out to and including the beyond use date. In addition to the standard organisms, it is always good practice (but not required) to test the effectiveness of the preservative against organisms isolated from the facility in which the product is compounded.
DYNALABS method for antimicrobial effectiveness testing is based on the standards delineated in USP <51>. DYNALABS will perform this test using customer supplied product. The standard organisms called out for in USP <51> will be supplied by DYNALABS. DYNALABS can work with the customer to help determine if testing of resident organisms found within the compounding facility is feasible and beneficial to them.
FORMS TYPICALLY TESTED
- All forms of medications with preservatives
- Multi-dose medications
When a claim of stability is made that is outside the standards delineated in the USP <797> for low, medium, and high risk compounds, the claim must be validated with testing. The criteria that can directly impact stability are:
- Compound Formula
- Compounding Process
- Container Closure System
- Handling and Storage Conditions
Once a claim has been validated, any significant changes in these criteria may require some level of revalidation. This may not mean all the original validation testing be conducted, however there may need to be some smaller subset of the testing that needs to be repeated.
In additions to claims made on end use doses, there may also be claims made on intermediate materials that are maintained within the pharmacy, that are used in the compounding of end use doses. These must also be included in the design of the stability testing program.
DYNALABS will work with the customer to create individual stability programs for specific compounds. However, we prefer to work with the customer to first create families of compounds based on formula, compounding processes, container closure system, and handling and storage conditions. We then apply our considerable experience and knowledge to design a testing program that minimizes the costs of implementing a stability program, while maximizing the value gleaned from the data created.
FORMS TYPICALLY TESTED
- All sterile and non sterile forms for which DYNALABS has testing methods.
Sterility Test Validation
For customers that perform in house sterility testing, it is important that for each compound and/or family of compounds tested, there be validation testing that proves the compound does not inhibit the sterility test. This is important for compounds containing preservatives and/or that are inherently antimicrobial (e.g., Vancomycin), and especially if direct inoculation testing methodology is employed.
DYNALABS method suitability test is performed in accordance to USP <71> using the customer supplied formula and compound, and organisms supplied by DYNALABS. If the test fails the method suitability test, DYNALABS will work with the customer to design a sterility method that will pass.
FORMS TYPICALLY TESTED
- All forms of sterile medications
Uniformity of Dosage
The degree of uniformity of drug substance between dosage units, where dosage units are defined as dosage forms containing a single or part of a single dose. Uniformity may be demonstrated using two methods, content uniformity or weight variation. Weight variation may be used in certain cases (see USP <905> Table 1), whereas the content uniformity method may be used in all cases, and if the compound being tested fails weight variation.
DYNALABS conducts content uniformity and weight variation in accordance with the standards delineated in USP <905>.
The number of samples is established during the experimental design phase of the project. Samples are analyzed using appropriate potency testing methodology to determine uniformity.
Specifications for uniformity are expressed as Acceptance Values which are calculated according to the instructions in USP <905> Calculation of Acceptance Value sections.
FORMS TYPICALLY TESTED
- All solid (e.g., capsules, tablets, pellets, suppositories) and liquid (e.g., injections, suspensions, gels, ointments, creams) dosage forms.