James Zellner, Technical Sales Representative

USP 797 states “CSPs administered epidurally should have the same endotoxin limit as that of intrathecally administered CSPs.” This new requirement lowers the endotoxin limit for epidural products from parenteral limit calculations to intrathecal limit calculations.

The difference between parenteral calculated limits and intrathecal/epidural limits is a factor of 25, i.e. a calculated endotoxin limit of 350 EU/mL for a parenteral preparation becomes 14 EU/mL when calculated for intrathecal/epidural administration.

Common Epidural medications include:

• Bupivacaine
• Ropivacaine
• Lidocaine
• Fentanyl
• Sufentanil
• Morphine
• Hydromorphone

For more information about endotoxin testing or the changes coming in the new USP <797>, please contact ARL at 1-800-393-1595 or email info@arlok.com.

Andrew Taylor, ARL Bio Pharma Microbiology Lab Supervisor

Pyrogens are a group of fever causing substances which can be found in compounded sterile preparations (CSPs) if appropriate actions are not taken to reduce or remove them. One of the most widely known groups of pyrogens are bacterial endotoxins. 

Bacterial endotoxins are:

• Remnants of bacterial cells that can cause fever, diarrhea, or septic shock in patients  
• Not removed during filter sterilization
• Not removed during steam sterilization
• Not detected by a sterility test

USP <797> requires a Bacterial Endotoxin Test (BET) for each preparation of:

• Category 2 and Category 3 CSPs compounded from one or more nonsterile component(s)
• Multiple-dose CSPs

USP <797> also requires a description of the depyrogenation process employed, including the temperature, pressure (if applicable), duration, permissible load conditions for each cycle; and the use of endotoxin challenge vials (ECVs) must be included in the facility’s SOPs. The chapter also states that if a CSP is dispensed or administered before Endotoxin testing results are known, a facility must have procedures in place to Immediately notify the prescriber of a test failure with the potential to cause patient harm.

The Food and Drug Administration (FDA) requires a BET for all 503B outsourcing facility drug products reported to be non-pyrogenic.

Even if it is not mentioned specifically in the regulatory documents, it is important to check for the presence of endotoxins in raw materials, at various points in the compounding process, and in finished products before administering a drug to a patient.  

The limit for the maximum amount of endotoxin allowed to be present in a CSP can be found in the applicable USP monograph, or calculated using the route of administration, patient weight, and maximum bolus dose. Once the limit has been established, the primary test method used to determine the amount of bacterial endotoxins in a CSP is described in USP <85> Bacterial Endotoxins Test.   

ARL Bio Pharma’s Bacterial Endotoxin Testing Process

ARL Bio Pharma follows the procedures described in USP <85>. The test is performed using depyrogenated glassware and supplies and the product to be tested is prepared using reagents specifically designed for BET. Each sample is tested in duplicate, using a standard curve, negative control, and positive control. At the conclusion of the test, the amount of endotoxin is calculated for each sample analyzed. If the endotoxin concentration is found to be below the limit, and all quality control parameters of the test are met controls were acceptable, the sample complies with USP <85> requirements. 

Therapeutic Peptides: ARL Bio Pharma’s Testing Capabilities

Jessica Munson, M.S., ARL Bio Pharma Analytical Supervisor

Peptide therapies have become increasingly popular due to the ability to help build lean muscle, shed weight, increase energy, improve skin appearance, amongst other benefits. With the increasing popularity and improvements in peptide production this has allowed compounded preparations to become more readily available. Wider availability has allowed ARL to test a variety of natural and modified peptides that cover multiple therapeutic areas. 

Peptides available for testing

If you want to test a peptide not on this list, please contact ARL at 800-393-1595 or info@arlok.com.

References:

• O’Brian, Sarah Ashley. “The Next Fountain-of-Youth Craze? Peptide Injections.” The Wall Street Journal, Mar. 2023, https://www.wsj.com/articles/peptide-injections-home-shot-2040a709
• Wang, L., Wang, N., Zhang, W. et al. Therapeutic peptides: current applications and future directions. Sig Transduct Target Ther 7, 48 (2022). https://doi.org/10.1038/s41392-022-00904-4

United States Pharmacopeia published the revised <795> and <797> chapters November 1, 2022. These chapters become official November 1, 2023.

Revisions to the chapters can be accessed through a subscription to the USP Compounding Compendium. The subscription provides access to:

• USP <795> and USP <797> and 40+ compounding related General Chapters
• 70+ compounding monographs from the United States Pharmacopeia and the National Formulary (USPNF)
•  Continuous online access to the most up-to-date published version of the compounding compendium
• Compendial notifications to alert subscribers of newly released and/or updated content

On November 8, 2022, USP Compounding Executive Committee held an Open Forum on the 2022 revisions to General Chapters <795> and <797>:

• USP <795> Open Forum Recording
• USP <797> Open Forum Recording

In addition, the USP Compounding Executive Committee put together an extensive list of Frequently Asked Questions for the chapters:

• USP <795> FAQ (62 questions and answers)
• USP <797> FAQ (195 questions and answers)

When the revisions to USP <795> and <797>, which contain references to USP <800>, become official on November 1, 2023, <800> will be required and compendially applicable only to the extent to which USP General Chapters <795> and <797> apply.

Compounders are encouraged to check with state agencies (state boards of pharmacy), other regulators (Occupational Safety and Health Administration), and oversight organizations as these agencies may make their own determination regarding the applicability and enforceability of USP <800>.

The time to prepare is now for the chapter revisions. ARL published newsletters in June-September 2022 highlighting proposed chapter updates:

• Personnel Qualification and Environmental Monitoring Requirements for Compounded Sterile Preparations (CSPs) Categories 1 and 2
• Personnel Qualification and Environmental Monitoring Requirements for Compounded Sterile Preparations (CSPs) for Category 3
• Release Inspections and Testing for Categories 2 and 3
• Beyond Use Dating

Save the Date: USP will host a compounding workshop on February 7 – 8, 2023. The workshop will have options for both virtual and in-person attendance at the USP headquarters in Rockville, MD.  

The USP <797> BUD limits are based on the risk of microbial contamination or not achieving and maintaining sterility. The CSP formulation must remain chemically and physically stable, and its packaging must maintain its integrity for the duration of the BUD. A shorter BUD must be assigned when the stability of the CSP or its components is less than the hours or days stated in the applicable tables below. Additionally, the BUD must not exceed the shortest remaining expiration date or BUD of any of the starting components.

Beyond Use Dating Stability Factors:

• Chemical and physical stability properties of the drug and/or its formulation
• Compatibility of the container closure system with the finished preparation (e.g., leachables, interactions, adsorption, and storage conditions)

Beyond Use Dating Sterility Factors:

• Conditions of the environment in which the CSP is prepared (Cleanroom suite or SCA)
• Aseptic processing and sterilization method
• Starting components (Sterile or nonsterile starting ingredients)
• Whether or not sterility testing is performed
• Storage conditions (Packaging and temperature)

Category 1 CSP Longest Permitted Beyond Use Dates

Category 2 CSP Longest Permitted Beyond Use Dates

Multiple-Dose CSPs must be prepared as a Category 2 or Category 3 CSP.

Preserved CSPs

• BUD is assigned according to Category 2 or Category 3
• Must pass testing per USP <51>
• After container is entered or punctured, must not be used for longer than assigned BUD or 28 days if supported by USP <51>, whichever is shorter.

Nonpreserved CSPs (aqueous ophthalmic)

• BUD is assigned according to Category 2 or Category 3
• Must pass testing per USP <51>
• USP <51> is not required only if the CSP is for use by a single patient and the container label includes the following statement “Discard 24 hours after first opening when stored at controlled room temperature or after 72 hours when stored under refrigeration.”

Stability Data Requirements for Category 3 CSPs: 

The BUD assigned to a Category 3 CSP must be supported by stability data obtained using a stability indicating analytical method that is able to distinguish the active ingredient from its degradants and impurities (e.g., by forced degradation studies) and quantify the amount of the active ingredient.

• The Category 3 CSP must be prepared according to the exact formulation (API and other ingredients of identical grade and procedures) from which the stability data are derived. 
• The Category 3 CSP must be packaged and stored in a container closure of the same materials of composition as that used in the study. 
• The analytical method must be validated based on characteristics such as those described in <1225> Validation of Compendial Procedures. 
• The compounding facility must have documentation of the stability study, including a description of the methodology (e.g., number of samples taken, storage conditions), validation of the method, the stability-indicating analytical method, and all of the results of the study. 

If the Category 3 CSP is an injection (<788> Particulate Matter in Injections) or if it is an ophthalmic solution (<789> Particulate Matter in Ophthalmic Solutions), particulate-matter testing is conducted once per formulation with acceptable results. 

Once for each formulation and for each container closure system in which it will be packaged, the container closure system used is evaluated for and conforms to container closure integrity (see <1207> Package Integrity Evaluation—Sterile Products). 

Category 3 CSP Longest Permitted Beyond Use Dates

Release Inspections and Testing for Categories 2 and 3. 

Category 2:

• Must be prepared in a cleanroom suite
• May be assigned a BUD of >12 hours at controlled room temperature or >24 hours if refrigerated

Category 3:

• Have additional requirements that must be met at all times
• May be assigned a BUD longer than established for Category 2 CSPs, up to 180 days

Visual Inspection: 

• This determines whether the physical appearance of the CSP is as expected (e.g. free of inappropriate visible particulates or other foreign matter, discoloration, or other defects).
• This confirms the CSP and its labeling match the prescription or medication order. 
• This also includes visual inspection of container closure integrity (e.g. checking for leakage, cracks in the container, or improper seals).

Sterility Testing:

• This qualitative test is applied to substances, preparations, or articles required to be sterile. A satisfactory result indicates that no contaminating microorganism has been found in the sample examined under the conditions of the test.
• Method Suitability is required for USP <71> citation.
• An appropriate sample volume of a product lot is filtered or directly inoculated into two microbial growth medias.
• After an appropriate incubation period, the sample is observed for turbidity.
• After the sample has completed testing, a Certificate of Analysis (COA) is issued stating a Pass or Fail result.

Rapid Sterility Testing:

• If an alternative method is used for sterility testing, the method must be validated (see USP <1223>) and demonstrated to be suitable for that CSP formulation.
• Where USP <71> requires between 14 and 18 days of incubation before a final test result, a rapid sterility test result can be generated after only 6 days of incubation.
• This Celsis method can test all sample types including solutions, oil, and suspensions. 
• Method Suitability is required. 
• An appropriate sample volume of a product lot is filtered or directly inoculated into two microbial growth medias.
• After an appropriate incubation period, an analysis on the media is performed to detect microbial growth.
• After the sample has completed testing, a Certificate of Analysis (COA) is issued stating a Pass or Fail result. 

Bacterial Endotoxin Testing:

• The USP <85> Bacterial Endotoxins Test (BET) is a quantitative test to detect or quantify endotoxins from Gram-negative bacteria using lysate derived from horseshoe crabs.
• With a kinetic turbidimetric method, a small amount of sample is prepared via serial dilution, along with any necessary additional reagents to overcome enhancement or inhibition effects.
• The prepared sample is transferred to a 96-well plate, and placed into a plate reader.
• Over the course of approximately an hour, the reader examines the wells for a reaction between the lysate and any endotoxin in the product samples.
• Based on either a lack of reaction, or the time taken to observe a reaction, the system calculates a quantitative result based on the sample preparation.
• At the completion of the test, a numerical value is given and reported on a Certificate of Analysis (COA).
• A comparison between the test result and the endotoxin limit for a sample determines whether the test is a Pass or Fail result.

Antimicrobial Effectiveness Testing: 

• USP <51> Antimicrobial Effectiveness must be demonstrated for aqueous-based, multiple-dose topical, and oral dosage forms, and for other dosage forms such as ophthalmic, otic, nasal, irrigation, and dialysis fluids. 
• In addition, whether inherent in the product or produced because of adding an antimicrobial preservative, antimicrobial effectiveness must be demonstrated for all injections packaged in multiple-dose containers or for other products containing antimicrobial preservatives.
• For this test, aqueous is defined as a water activity of more than 0.6. 
• Method Suitability is required for USP <51> citation.
• Separate volumes of product sample are inoculated with high CFU counts of 5 different representative challenge organisms.
• Depending on which of the four categories in the chapter a product falls into, petri plates are prepared with growth media and a sample of the inoculated product at specific intervals over a 28-day period.
• At each of the plating intervals, a CFU count is obtained and compared against the criteria in the chapter.
• At the conclusion of the 28-day incubation and final plating, a Pass or Fail result is reported on a Certificate of Analysis (COA), depending on whether the product met the criteria in the chapter at each plating interval.

Personnel Qualification and Environmental Monitoring Requirements for Compounded Sterile Preparations (CSPs) for Category 3. 

Category 3:

• Have additional requirements that must be met at all times
• May be assigned a BUD longer than established for Category 2 CSPs, up to 180 day

Visual Observation of hand hygiene and garbing: This is a competency evaluation of hand hygiene, garbing procedures, and gloved fingertip and thumb sampling of both hands.

Gloved fingertip and thumb sampling:

• This test provides data on any microorganisms present on the technician’s gloves during compounding. 
• Press gloved fingertips onto one agar plate per hand after compounding. Label plate with all relevant information including, but not limited to, staff members name, date, and time of sampling. 
• Send plates to ARL for incubating, counting of colony forming units (CFUs), and reporting of a Certificate of Analysis (COA).
• Document test results.
• Determine if any additional actions are required including growth promotion of the media to prove the media was capable of detecting growth, identification of any organisms recovered, retraining of staff, etc.
• Repeat as necessary. 

Media Fill Testing:

• This test assesses the technician’s ability to compound aseptically.
• Personnel should perform the media fill test under the most stressful and challenging conditions at the pharmacy. A simulated compounding operation is conducted with microbial growth media in place of drug components and/or products.
• Send media fill containers to ARL for incubation and observation, and reporting of a Certificate of Analysis (COA).
• Document test results.
• Determine if any additional actions are required including growth promotion of the media to prove the media was capable of detecting growth, identification of any organisms recovered, retraining of staff, etc.
• Repeat as necessary. 

Post media-fill surface sampling test: 

• This test assesses the technician’s ability to compound aseptically. 
• At the completion of the media fill qualification procedure, the surfaces of the compounding area are sampled using agar press plates.
• Send plates to ARL for incubating, counting of colony forming units (CFUs), and reporting of a Certificate of Analysis (COA).
• Document test results.
• Determine if any additional actions are required including growth promotion of the media to prove the media was capable of detecting growth, identification of any organisms recovered, retraining of staff, etc.
• Repeat as necessary.

Surface sampling:

• This test monitors surfaces for viable particles.
• At the conclusion of a compounding session, agar press plates are used to sample the surfaces of the compounding space.  
• Send plates to ARL for incubating, counting of colony forming units (CFUs), and reporting of a Certificate of Analysis (COA).
• Document test results. 
• Determine if any additional actions are required including growth promotion of the media to prove the media was capable of detecting growth, identification of any organisms recovered, retraining of staff, etc.
• Repeat as necessary. 

Viable air sampling:

• This test monitors air quality for viable airborne particles. 
• Follow manufacturer’s instructions for operation of the active air sampling device, including placement of media. Test at least 1 cubic meter or 1000 L of air from each location sampled. 
• Send plates to ARL for incubating, counting of colony forming units (CFUs), and reporting of a Certificate of Analysis (COA).
• Document test results. 
• Determine if any additional actions are required including growth promotion of the media to prove the media was capable of detecting growth, identification of any organisms recovered, retraining of staff, etc.
• Repeat as necessary.

USP <795> states that pharmacists performing non-sterile compounding must ensure that the finished preparation has its accepted potency, purity, quality, and characteristics.  This article discusses the tests described in USP <61> and USP <62> that are used to determine if non-sterile products meet quality requirements.  These tests can also be used by sterile compounders for qualifying raw materials and performing in-process quality control testing.  

Testing Conditions

The general considerations for testing non-sterile products are much like USP <71> Sterility testing.  Growth promotion is required to demonstrate that every lot of media will grow the microorganisms required for each test.  Method suitability is also performed prior to product testing to establish the ability of the test to detect microorganisms in the presence of the specific formulation to be tested.  All testing must be completed under aseptic conditions to avoid extrinsic microbial contamination of the product tested and must include proper positive and negative controls.   

USP<61> Microbial Enumeration Tests

USP <61> is often called “Bioburden” or “Microbial Limits” because the test determines how many microorganisms are present in the sample.  To perform the test, the sample is plated onto two types of growth media (Soybean-Casein Digest Agar and Sabouraud Dextrose Agar).  The plates are incubated at a defined temperature and duration.  At the conclusion of the test, the number of colonies present on the plates are counted and the results calculated.   The results can then be compared to the acceptance criteria.  USP <1111> can be referenced for acceptable amounts of microorganisms present based on whether the sample is a raw material or finished drug product.  If the sample is a finished drug product, the acceptance criteria depends on the route of administration.  

USP<62> Tests for Specified Organisms

USP <62> provides instructions on how to perform tests that demonstrate particular microorganisms are not present in non-sterile substances and products.  These tests are performed similarly to USP <61> tests, but with microorganism specific growth media.  At the conclusion of incubation, a result of “Pass” or “Fail” is generated.  A passing result indicates the absence of the tested specified microorganism.  USP monographs and <1111> define which organisms to test based on the route of administration of the raw material or finished drug product.  For example, USP <1111> recommends that cutaneous (topical) products be tested for the absence of both Pseudomonas aeruginosa and Staphylococcus aureus, versus an oral preparation where the recommendation is to test for the presence of Escherichia coli. 

Please contact ARL (800) 393-1595 or info@arlok.com with questions.

Andrew Taylor, Microbiology Supervisor

Reference Documents:
United States Pharmacopeia <61>

United States Pharmacopeia <62>

United States Pharmacopeia <1111>

Liothyronine Sodium (T3) and Levothyroxine Sodium (T4) are iodinated amino acids and are typically formulated in microgram concentrations. These active pharmaceutical ingredients (APIs) contain water: up to 4% in the T3 powder and up to 11% in the T4 powder. The primary reasons for the difficulty in preparing formulations containing T3 and/or T4 arise from 3 common issues: microgram concentrations, iodination of the compound, and water.

Three Common Compounding Issues

1. Microgram concentrations

In the preparation of a powder blend of T3/T4 capsules, typically the active ingredients are mixed with microcrystalline cellulose and, potentially, other excipients like methyl-cellulose, to provide the correct dilution and desired slow-release properties. Sometimes additional materials are added, such as a coloring agent, to provide a visual indication of completeness of mixing. Normally, these ingredients are blended through the technique of geometric addition and mixed or stirred in the same fashion as other capsule formulations. This becomes an issue is treated the same as other capsule formulations as blending microgram quantities is significantly more difficult than blending milligrams, due to a microgram being 1/1000th of a milligram. A few particles of the API powder may equate to a microgram, thus these few particles must be evenly distributed throughout the powder blend for a homogenous preparation. To achieve this desired microgram distribution it often takes longer to blend in comparison to a milligram distribution preparation and may require different handling techniques. In T3/T4 blends the particle size of the API is significantly different from that of the other ingredients, so even when the particles are well-mixed, they may not stay mixed due to this difference. During handling, the vibrations generated in the process will cause the smaller particles to move toward the bottom of the container while the larger ones will migrate to the top.

2. Static charge

T3 and T4 both contain iodine atoms which are members of the halogen group. Halogens are electronegative because of their electron rich outer shell orbital. If the powder blend is mixed in a plastic container, the agitation of the cellulose along with the T3/T4 actives will develop a strong static charge on the walls of the container which will attract these microgram amounts of ingredients out of the blend and onto the surface of the container. The faster the powder blend is mixed, the stronger the static charge will be, and the more the API will migrate out of the blend. ARL has observed, through several rinse studies, T3T4 API migrating and staying attached to the walls of containers.

3. Water Content

Since a portion of the weight of the T3 and T4 powder is water, adjustments in the desired amounts of these ingredients must be considered to offset this content. An appropriate extra amount of the API powder should be included in the formulation so the final product will not be sub-potent.

Compounding Suggestions:

• Make sure your formulation appropriately accounts for the water content in the raw, starting material API powders.
• Both the active and inactive ingredients should be micronized to approximately the same particle size to help prevent stratification.
• Blends containing microgram amounts of T3 and or T4 actives must be blended longer and more completely than usual.
• Avoid using plastic containers for mixing and storage. The blending should be accomplished in either glass or metal containers and should have a tumbling action of side over side and end over end to ensure the actives are well mixed. Mortar and pestle mixing may not be enough or as effective by comparison.
• The time required for thorough mixing will vary, depending upon batch size and mixing speed, but a 2-hr. mixing time would not be unusual to achieve proper dispersion.

USP <797> states compounders must ensure products maintain labeled strength within monograph limits. The difficulty with determining limits (specifications) for compounded preparations is that there may not be a monograph to reference. In these cases, ARL assigns the specification by choosing them from a monograph that is closest to the product being tested. When there is not a monograph for a similar product, ARL defaults to 90%-110% per <797>. Clients may also provide their specifications at the time of sample submission. Contact ARL info@arlok.com with additional questions.