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Endotoxin vs microbial testing

Endotoxin testing is the measurement of bacterial endotoxin, the lipid A component of the lipopolysaccharide of Gram-negative bacteria, whereas microbial testing counts microorganisms or tests for their presence. ICH Q6B lists endotoxins, sterility and microbial limits as separate pharmacopoeial tests.[1],[2],[3],[4],[5]

Which compendial tests fall under each heading?

For endotoxin, FDA guidance names USP chapter <85>, Bacterial Endotoxins Test, and describes gel clot, photometric and kinetic test methods based on Limulus amebocyte lysate (LAL).[6],[7]

For microbial quality of nonsterile products, ICH Q4B annexes published by FDA name USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests, USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms, and USP <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products.[2],[3],[8],[9],[10],[11]

For sterility, ICH Q4B Annex 8 names USP <71> Sterility Tests. Each annex recommends that the named USP, European Pharmacopoeia and Japanese Pharmacopoeia texts can be used as interchangeable in the ICH regions, subject to its stated conditions.[4],[12]

Why can a material that passes a sterility test still contain endotoxin?

Endotoxin is part of the bacterial outer membrane: Raetz and Whitfield describe lipopolysaccharide as consisting of lipid A, or endotoxin, a core oligosaccharide and an O-antigen polysaccharide, and an FDA inspection technical guide places bacterial endotoxins in the outer membrane of Gram-negative bacteria.[1],[13]

An FDA inspection technical guide states that lipopolysaccharides are released from bacteria after death and lysis of the cell, and that endotoxins are not removed by sterilizing or microbiological filters.[13]

The same guide states that devices intended to be free of viable microorganisms have occasionally been shown to be contaminated with endotoxins, and that it is difficult to remove endotoxins from products once present.[13]

Why does a low microbial count not predict a low endotoxin result?

The FDA guide states that the growth of some types of microorganisms contributes to increased levels of endotoxin, and that the subsequent reduction of a high microbiological level will not be associated with a similar reduction of a high endotoxin level.[13]

It gives the example of microbiological testing of bioburden after filtration: that result provides some information about the adequacy of a sterilization process, but little, if any, information about whether endotoxin contamination was minimized.[13]

How do pyrogens relate to endotoxin?

The FDA guide states that pyrogenic substances can be produced by some Gram-positive bacteria, mycobacteria, fungi and viruses, but that the pyrogens produced by Gram-negative bacteria, the endotoxins, are of significance to the pharmaceutical industry.[13]

Current FDA guidance states that FDA has accepted an LAL test for endotoxins in lieu of the rabbit pyrogen test for more than 30 years. It also states that firms should consider the risk of non-endotoxin pyrogens, and that where they may be present the rabbit pyrogen test, USP chapter <151>, may be more appropriate.[6],[14]

How are the results reported and compared?

Endotoxin limits in the FDA guidance and in USP chapter <161> are expressed in endotoxin units, for example EU/mL.[6]

ICH Q4B Annex 8 states that the sterility acceptance criteria are harmonized between the three pharmacopoeias. Annex 4C states that, for each regulatory region, the pharmacopoeial text on acceptance criteria for nonsterile products is nonmandatory and provided for informational purposes only.[4],[8]

In each of these annexes, FDA notes that it might request that a company demonstrate that the chosen method is acceptable and suitable for a specific material or product, irrespective of the origin of the method.[2],[3],[4]

Limitations

An endotoxin result says nothing about living microorganisms, and a microbial or sterility result says nothing about endotoxin; each applies only to the samples tested by the method used.[5],[13]

The FDA inspection technical guide cited here is dated March 1985 and is quoted only for its descriptions of endotoxin; current test methods and limits are those of the FDA guidance and the compendial chapters it names.[13],[6]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Research peptide profiles in this library. What this page describes applies to peptide lots in general; none of these profiles reports a result of it for any lot.

References

  1. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635-700. PMID 12045108 · DOI 10.1146/annurev.biochem.71.110601.135414 · PMC2569852
  2. ICH Q4B Annex 4A(R1) Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests General Chapter. U.S. FDA guidance for industry, September 2010. FDA docket FDA-2008-D-0396
  3. ICH Q4B Annex 4B(R1) Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms General Chapter. U.S. FDA guidance for industry, September 2017. FDA docket FDA-2008-D-0398
  4. ICH Q4B Annex 8(R1) Sterility Test General Chapter. U.S. FDA guidance for industry, September 2017. FDA docket FDA-2009-D-0013
  5. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
  6. U.S. FDA. Pyrogen and Endotoxins Testing: Questions and Answers. Guidance for industry. FDA docket FDA-2013-S-0610
  7. United States Pharmacopeia, General Chapter <85> Bacterial Endotoxins Test (as named in FDA, Pyrogen and Endotoxins Testing: Questions and Answers). USP <85>
  8. ICH Q4B Annex 4C(R1) Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use General Chapter. U.S. FDA guidance for industry, September 2017. FDA docket FDA-2008-D-0400
  9. United States Pharmacopeia, General Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests (as named in ICH Q4B Annex 4A(R1), FDA guidance). USP <61>
  10. United States Pharmacopeia, General Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms (as named in ICH Q4B Annex 4B(R1), FDA guidance). USP <62>
  11. United States Pharmacopeia, General Chapter <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products (as named in ICH Q4B Annex 4C(R1), FDA guidance). USP <1111>
  12. United States Pharmacopeia, General Chapter <71> Sterility Tests (as named in ICH Q4B Annex 8(R1), FDA guidance). USP <71>
  13. U.S. FDA. Inspection Technical Guide No. 40: Bacterial Endotoxins/Pyrogens (March 20, 1985). FDA ITG No. 40
  14. United States Pharmacopeia, General Chapter <151> Pyrogen Test (as named in FDA, Pyrogen and Endotoxins Testing: Questions and Answers). USP <151>