USP <85> Bacterial Endotoxin Validation

ARL Bio Pharma utilizes the kinetic turbidimetric method described in USP <85> Bacterial Endotoxins Test to provide results. An endotoxin test detects toxins that are released from the cell wall of disrupted gram-negative bacteria. This is a reaction over time test that quantitates the amount of endotoxin in a sample compared to a standard curve. This article discusses USP <85> challenges and why method validation is important to provide consistent results.

The most common challenge encountered while performing endotoxin testing is that many drug products cause the assay reaction to slow down (inhibition) or speed up (enhancement) during the testing process, which does not allow for an accurate result to be obtained. Those properties which cause the inhibition or enhancement must be overcome to obtain a reliable and repeatable test result. Microbiologists performing testing must determine the degree in which inhibition or enhancement is occurring. This is done by adding known amounts of endotoxin to test samples and comparing the results to those known values. If there is no inhibition or enhancement, results from the test samples spiked with a known amount of endotoxin will be the same as the expected results. Inhibition would artificially lower results for the test sample, and enhancement would increase results for the test sample, relative to the expected spike concentration. 

Once inhibition or enhancement is understood, microbiologists can develop a plan of action to overcome those sample properties.

Growth Promotion Testing

Growth Promotion demonstrates that media used in environmental monitoring programs, media fills, or personnel qualification are capable of supporting microorganism growth. 

Benefits of growth promotion testing:  

  • Reduces risk of false negative results if media does not support growth
  • Reduces risk of false positive results if media is contaminated (negative control sample required)
  • Ensures media used in environmental monitoring provides an accurate representation of environmental control in compounding areas
  • Meets FDA and USP microbiological growth media requirements
  • Verifies media vendor's certificate of analysis

ARL has a new submission form specific for "Growth Promotion" tests. This new form is a quick selection form for media types, organism tests, and incubation time and temperature.

Container-Closure Systems...

The container closure system for a drug product provides critical protection for stability and sterility. It consists of all components intended to seal and protect the package/delivery system and its contents. Container-closure integrity (CCI) is the ability of the system to keep contents in, and to keep harmful environmental contaminants out.

CCI testing (CCIT) detects leaks in the container closure system. A passing test result indicates the packaging system can maintain sterility and relevant physicochemical specifications. Three types of leaks that can be detected during CCIT include:

  • entry of microorganisms
  • escape of the product dosage form or entry of liquids or solids
  • escape of nitrogen gas or entry of oxygen, water vapor, or air gases

In February 2008, the Food and Drug Administration (FDA) introduced CCIT in lieu of sterility testing as a component of a stability written testing program for sterile products. A stability study measures the extent to which a product retains, within specified limits, and throughout its period of storage and use, the same properties and characteristics it possessed at the time of compounding. Sterility is a stability characteristic that should be conducted throughout a stability study to confirm a product's shelf life.

Drug Shortages

FDA has identified the following list of drugs for the purpose of the temporary enforcement policies described in 503B guidance and 503A guidance during the COVID-19 public health emergency. 

503B guidance establishes revised cGMP standards and allows outsourcing facilities to compound and distribute drugs needed to treat COVID-19 patients that hospitals cannot obtain from an FDA drug manufacturer due to drug shortage. 

503A guidance allows pharmacies to compound drugs and distribute to pharmacies that are unable to obtain an FDA approved version of the drug or one compounded by a 503B outsourcing facility. 

FDA recommends that State-licensed pharmacies consult with State authorities regarding local requirements.

USP <61> and USP <62> Microbial Tests for...

USP <795> states compounders are responsible for ensuring compounded drug products are of acceptable strength, quality, and purity. The chapter also directs compounders to USP <1163> for recommended quality control procedures.

USP <1163> specifies the appropriate microbiological tests of non-sterile products:

  • USP <61> Microbial Enumeration Tests


COVID-19 Industry News and Resources

  • FDA and USP
  • Pharmacy Associations
  • Pharmacy Suppliers

Importance of Sample Amounts...

This article outlines the importance of sample amounts submitted for potency testing and the impact it can have on potency results. Formulations require different sample amounts to allow for an accurate result representative of the compounded preparation. The table below can also be found in ARL's Sample Submission Guidelines

USP <51> Antimicrobial Effectiveness Testing

Antimicrobial Preservatives are excipients added to multi-dose containers for both sterile and nonsterile drug products. These preservatives are used to prevent growth of microbial contamination occurring during the use period. It's important to note that antimicrobial preservatives are not a substitute for good manufacturing practices. 

The USP <51> Antimicrobial Effectiveness Test is a challenge test for aqueous-based products. This test demonstrates the effectiveness of antimicrobial preservatives in preventing proliferation or limiting microbial contamination. It is performed on injections, topicals, oral products, and antacids packaged in multiple-dose containers. Click here to access a table of Common Pharmaceutical Preservatives.

An Update regarding COVID-19

ARL Bio Pharma is prepared and has taken steps to ensure our lab continues to provide testing services during this unprecedented time in the United States. We are taking every precaution to follow CDC guidelines and state and federal mandates regarding COVID-19.

Our laboratory is committed to our responsibility as a business partner in the drug supply chain to keep our clients up and running during this health crisis. To deliver the best technical service, we are available to support you Monday through Friday 7:30 a.m. to 6:00 p.m. CST at 800-393-1595 or 405-271-1144.

ARL is dedicated to assuring the health and safety of our employees, our customers, and the communities we serve. Our laboratory is working with our department and logistic partners to ensure supplies capacity in and out of our laboratory and are continuing to fulfill testing services. We will keep you informed of any impact on our ability to meet turnaround times and expectations.

Please follow the CDC website for the latest updates on COVID-19; and, ARL's website and online portal for service updates.

Thank you for your business and continued confidence in ARL to support your needs.

ARL Bio Pharma

Growth Media Differences

There are dozens of different types of microbial growth media available to Microbiologists, developed for a wide variety of purposes.  The USP references many of these to demonstrate the quality of both compounded drug products and the control of compounding environments.  The various types of media serve specific purposes based on the goal of the test.  This might include recovery of the broadest range possible of organisms in a sterility test, screening for a specific organism if it would be particularly dangerous in a certain dosage form, or capturing anything that might be in the compounding environment that might find its way into finished product.  Microbial growth media can be prepared in both liquid and solid forms, depending on the application.  Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB), for example, have the same nutrient profile and similar ingredients, but TSA is a solid and TSB is a liquid.  For conducting passive air sampling, for example, a solid TSA plate is easier to handle and leave out in the hood during compounding.  Liquid media, like TSB, is generally better at growing organisms, and should be used where possible for recovery of microorganism in drug products.  Liquid media is more conducive to growth, since nutrients, oxygen, and waste products move around more freely, and temperature is more uniform and constant in a fluid.