Industry Drivers in the Development of Recombinant Lysate

 

With recombinant lysate, a recombinant protein is an artificially produced (and often purified) protein. The recombinant process occurs, in general terms, when recombinant DNA encoding a protein is introduced into a host organism to express foreign proteins. This process requires the use of specialized expression vectors together with restructuring by foreign coding sequences.

 

Tim Sandle has written a review of recombinant lysate for use with the bacterial endotoxin test.

 

The idea for the use of recombinant lysate is to provide a reagent that reacts in the same way as the natural cascade within the four species of horseshoe crab: (Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda). The cloning technology serves an alternative to the fishing and bleeding of horseshoe crabs (where bleeding is performed through a large dorsal blood sinus called the pericardium). In addition, the use of recombinant technology to substitute the specific pathway seeks to address the concern with some lysates that also detect beta glucans through the activation of factor G. A further advantage with recombinant lysates stems from the production of the test reagent through the use of cell culture.  This may result in the supply of a more consistent product.

 

From around 2003 recombinant lysate has been commercially available, albeit not recognized by any compendia until 2016. However, it has only been in the past few years that manufacturing has become more consistent and subsequently a number of quality control testing laboratories have been looking into the use of recombinant lysate. This somewhat gradual process is attributable to a clash between drivers (seeking to conserve horseshoe crabs, albeit with the mortality rate an imprecise and often contested figure)4 and points of hesitancy (regulatory uncertainty, lack of standardization, imprecise validation requirements and so on). As of 2021, recombinant lysate will become an established alternative to the traditional LAL test within Europe but within the U.S., when a new chapter appears, a greater level of comparative validation will be required in order to show equivalence. While this disparity is understandable, and this article attempts to show this through a discussion of the historical perspective, the current situation is a disservice to laboratory users. Much of what has been written on rFC to date has looked at things from a U.S.-centric perspective (rather than U.S. and European) and from a supplier or regulatory perspective; this article embraces the laboratory user perspective.

 

The reference is:

 

Sandle, T. (2020) Historical Milestones and Industry Drivers in the Development of Recombinant Lysate for Bacterial Endotoxin Testing, American Pharmaceutical Review, at: https://www.americanpharmaceuticalreview.com/Featured-Articles/569887-Historical-Milestones-and-Industry-Drivers-in-the-Development-of-Recombinant-Lysate-for-Bacterial-Endotoxin-Testing/

 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Pharmacopeial changes in relation to pyrogens and endotoxin

The European Pharmacopeia (Ph. Eur.) is proposing a series of changes in relation to the testing of pyrogens and for bacterial endotoxin. The changes represent the most significant changes to the approach since the Ph. Eur harmonised the bacterial endotoxin test (BET) chapter with the United States and nJapanese pharmacopeia during the early 2000s.

A new article from Tim Sandle for the GMP Review outlines the key changes.

For a copy of the article, please contact Tim Sandle

The reference is:

Sandle, T. (2014) Pharmacopeial changes in relation to pyrogens and endotoxin, GMP Review, 13 (3): 10- 12

Posted by Tim Sandle

Variability and Test Error with the LAL Assay

The Limulus amebocyte lysate (LAL) assay is the compendial test for the examination of bacterial endotoxin in pharmaceutical products (as described in USP chapter <85>), in-process material, and pharmaceutical grade water.

With any biological tests, measurements are susceptible to variations in analytical conditions. Here the LAL assay has a relatively high level of variability even for a biological assay. This variation derives from 3 principle sources: reagents, product, and method. This paper examines some of the reasons for LAL test variation, focusing on photometric methods (chromogenic and turbidimetric), and considers how variation can be assessed through good laboratory quality control.

To review a new paper by Tim Sandle that looks at the variations and reasons for test error with the LAL assay, see American Pharmaceutical Review.

Reference:

Sandle, T. (2014) Variability and Test Error with the LAL Assay, American Pharmaceutical Review, October 2014, pp1-5

Posted by Tim Sandle

Endotoxin testing – European Pharmacopeia changes

A proposal has been made to amend the European Pharmacopeia chapter on guidance for the bacterial endotoxin test – chapter 5.1.10.

The proposed changes are:

In the context of the new bacterial endotoxins Ph. Eur. policy, a new section (2-4) includes aspects to be considered when establishing an endotoxin limit for a specific substance or product; also, reference is made to the fact that an endotoxin limit is not always provided in a specific monograph.

– Reference is made to general chapter 2.6.30. Monocyte-activation test as an alternative to the rabbit pyrogen test, and a recommendation is given to perform a risk assessment when using the bacterial endotoxin test as a pyrogenicity test, due to the potential for contamination by non-endotoxin pyrogens. In this respect, the previous section 11 concerning the replacement of the rabbit pyrogen test by a test for bacterial endotoxins is substituted with a new text in agreement with a strategy to be applied for testing of bacterial endotoxins or non-endotoxin pyrogens. A distinction is made between replacement methods already described in the Ph. Eur. and other alternative methods.

– In the context of the 3R’s, a test for bacterial endotoxins or a monocyte-activation test is preferred to the rabbit pyrogen test. Therefore, implementation of the rabbit pyrogen test has to be justified and authorised and is appropriate only when the bacterial endotoxin test or the monocyte-activation test cannot be validated.

– Reference is made to the use of alternative reagents to the Limulus amoebocyte lysate, such as recombinant factor C: this practice avoids the use of endangered animal species and can be considered in the context of the use of an alternative method as described in the General Notices.

A number of additional specific revisions have been made.

– In line with current knowledge, Method A is no longer declared as the reference method, and all methods A to F of general chapter 2.6.14. Bacterial endotoxins can be used. Where the method is stated in the monograph, the use of another method must be supported by evidence of validation.

–The expression ‘threshold endotoxin concentration’ has been replaced by the more appropriate expression ‘endotoxin limit concentration’ to harmonise with general chapter 2.6.30. Monocyte-activation test.

– A new entry has been included in Table 5.1.10.-1 for formulations administered per square metre of body surface.

Finally, the structure of the general chapter has been modified to improve its clarity.

The proposal has been added to Pharmeuropa 26.4.

Posted by Tim Sandle

Bacterial endotoxins Ph. Eur. policy for substances for pharmaceutical use

The European Commission has made an important announcement about endotoxin limits in relation to substances for pharmaceutical use. For new substances, pharmacopeia monographs will no longer specify endotoxin limits. It will be left to the user to determine if a substance requires testing and what the appropriate limit should be.

The issued text reads:

Bacterial endotoxins Ph. Eur. policy for substances for pharmaceutical use (Approved by the Ph. Eur. Commission at its 149th Session, June 2014)

1.Reasons for requirements for testing for bacterial endotoxins

Bacterial endotoxins are contaminants from gram-negative bacteria and are the most common cause of pyrogenicity in pharmaceutical products. Any preparation administered parenterally should be sterile and comply with the test for bacterial endotoxins (BET) as described in the Ph. Eur.

Substances to be used in parenteral preparations must comply with the BET, whatever their origin, since:

• Contamination by bacterial endotoxins can take place prior to or during the manufacturing process;
• Bacterial endotoxins cannot easily be removed by the manufacturing process;
• Bacterial endotoxins should be detected as early as possible in the manufacturing process.
• It is to be noted that the ICH Guideline Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products provides the following recommendation under section 4.1 Drug Substance Specification: “Pharmacopoeial tests (e.g., endotoxin detection) should be performed on the drug substance, where appropriate.”

2. Requirements for bacterial endotoxins in the Ph. Eur.

The Ph. Eur. provides requirements for testing for bacterial endotoxins as follows.

The general monograph Substances for pharmaceutical use (2034) and individual monographs on substances for pharmaceutical use require compliance with the BET when substances are used in the manufacture of parenteral preparations. The monographs refer to general chapters 2.6.14. Bacterial endotoxins and 5.1.10. Guidelines for using the test for bacterial endotoxins.

According to the general monograph Parenteral preparations (0520), pharmaceutical preparations to be used parenterally have to comply with the test for bacterial endotoxins or, where justified and authorised, the test for pyrogens.

3. Problem statement

During the elaboration of a monograph, it is not always known at the level of the manufacturing of the substance whether the substance is to be used for the production of a parenteral preparation and therefore it is not known whether compliance with the BET is needed or not.

With regard to the limits to be applied for the test, individual monographs provide limits whereas general chapter 5.1.10 provides a way to calculate the endotoxin limit:

Values of K to be used for the calculation of the endotoxin depend on the route of administration and are given in the general chapter (Table 5.1.10.-1). There are other ways to establish limits, for example based on process capability, patient population or specific requirements of the competent authority. These are not clearly stated in the Ph. Eur. This might result in apparent contradictions between the different BET requirements.

4. New policy on the way to prescribe the BET in the Ph. Eur.

With consideration to the above, the Ph. Eur. Commission recommends the following approach to bacterial endotoxins.

Elaboration of new individual monographs

A test for bacterial endotoxins is not included in new monographs for substances for pharmaceutical use. The requirements of the general monograph Substances for pharmaceutical use (2034) apply.

• A test is included only where a specific method has to be described, for example if a specific sample preparation has to be used or if a specific method has to be applied.
• If a test is included in the monograph, no limit is given for the test.

Existing monographs

BET specifications are kept in individual monographs for substances for pharmaceutical use. Existing limits remain in individual monographs to maintain the use of well-established limits.

In order for the policy to be applied, the following changes are proposed to existing Ph. Eur. texts.

• General chapter 5.1.10 is expanded with further considerations regarding the setting up of limits.
• General monograph Substances for pharmaceutical use (2034) is slightly reworded in order to take the above policy into consideration.

5. Consequences of the new BET policy for Ph. Eur. users

It is up to the user of the Ph. Eur. to determine whether compliance to BET is needed or not for a given substance. Where a test is included in the monograph with no specific limit, it is up to the user to set the limit for the substance, based on the following considerations: use of the substance (route of administration, patient population); calculation according to the formula given in general chapter 5.1.10; process capability; or any other considerations raised by the competent authority.

Source: Ph. Eur.

Posted by Tim Sandle

A Tale of Two Species - Blue Blood

Horseshoe crabs' blue blood, which contains copper, not iron, is prized by the biomedical community for its ability to detect bacteria in human medicines. It's just one of the amazing qualities of the 350-million-year-old evolutionary marvel detailed in "Crash: A Tale of Two Species."

"Crash: A Tale of Two Species" aired on PBS Sunday, March 20, 2011 at 8pm as part of the 28th season of the Peabody and Emmy award-winning series produced by Thirteen in association with WNET.ORG for PBS. Major support provided by Canon U.S.A. Inc.

Here is a fascinating excerpt:

Horseshoe crab amebocytes coagulate around as little as one part in a trillion of bacterial contamination. Even better, the reaction takes 45 minutes, not two days as with mammalian equivalents. Coagulan, the chemical that makes this possible, is used for testing medical equipment and vaccines prior to use, without which many more people would die from infections. Unfortunately, coagulan synthesis is in its infancy so a quarter of a million crabs are harvested each year for their blood, as shown in this video:

Posted by Tim Sandle

Performance Characteristics Of Automated LAL Tests 

By Tim Sandle, Ph.D, M.A., BSc (Hons), CBiol, MSBiol., MIScT

Introduction

The use of automated Limulus amebocyte lysate (LAL) methods to perform Bacterial Endotoxin Testing (BET) is now commonplace in most pharmaceutical laboratories. By automated I am referring to the use of software driven heated tube and plate readers for turbidimetric or chromogenic LAL testing for laboratories testing against Ph. Eur. 2.6.14 or USP <85>.

These ‘semi' - automated methods have a number of advantages over the gel-clot method in both testing terms, such as easier demonstration of inhibition / enhancement testing, and in aiding cGMP through the various reports that can be obtained. The Gel-clot test is still widely used and, by virtue of its relative simplicity, remains a robust method. However, laboratories facing ever more regulation are more often employing quantitative methods.

Testing using the automated methods requires validation, which any reputable laboratory demonstrates, in simple terms, by testing three different lots / batches at a sample within the maximum valid dilution and demonstrate freedom from interfering substances. The automated instruments themselves will have been calibrated by the supplier for factors like uniformity of temperature.

Not all laboratories, however, have examined these instruments for their performance characteristics in the way that a biochemist would look at the equipment used for conducting more classical assays. Yes – for some microbiologists it's news that the LAL test is an assay. Due to the fractions of endotoxin (in terms of weight of lipopolysaccharide) that the test detects it cannot fall within the tight scope that the biochemist would apply to an assay, for aspects like accuracy or precision or other parameter pulled out of the ICH guidelines.

This doesn't mean that microbiologists can ignore the performance of their instruments. This article sets out some of the performance characteristics which could be considered for automated LAL instruments. I am not suggesting that these all need to be looked at or that already busy microbiology lab have to grapple with yet another validation task. However, the microbiologist responsible for such systems should at least understand their strengths and limitations. Types of automated LAL instruments Automated LAL instruments fall into two broad categories depending upon the nature of the test: turbidimetric (where the rate of turbidity (or the ‘gelation') of the lysate is proportional to the amount of endotoxin present) and chromogenic (where a synthetic ‘yellow' chromogenic substrate is used in the clotting cascade and the intensity of this yellow is related to the amount of endotoxin). Turbidimetric tests are read using photometric instruments (such as spectrophotometers) and chromogenic tests are more often read using ELISA plate readers. This article doesn't attempted to weigh up the pros or cons of competing instrumentation (or step into the cut throat world of competing LAL reagent suppliers) except to acknowledge that different types of software affect the test and different manufacturer's lysates differ considerably, not least in the sensitivity to LAL Reactive Materials, such as, glucans.

Applying performance characteristics

I am probably taking it as read that some performance characteristics should be applied to automated LAL tests. Understanding the abilities of the instrumentation is important in examining and interpreting test results and in defending those test results against misunderstanding from Production and QA departments. Some characteristics will be included in annual or six-monthly calibrations of the instrument, such as, temperature distribution, and others are shown through demonstration of the suitability of the instrument over time, such as the results from the negative control samples (the ‘blanks' to assayist).

What assay characteristics can be applied to automated LAL testing so that the performance of the instruments and test can be judged? In this article the following will be examined:

Limit of Detection
Limit of Quantitation
Reproducibility
Repeatability
Precision
Accuracy
Robustness

I would suggest that these factors need to be performed, examined and filed as part of the instrument's equipment validation.

Tim Sandle, Ph.D, M.A., BSc (Hons), CBiol, MSBiol., MIScT – Dr. Sandle is the Head of Microbiology at the UK Bio Products Laboratory. Dr. Sandle is a chartered biologist and holds a first class honors degree in Applied Biology; a Masters degree in education; and obtained his doctorate from Keele University. His role involves overseeing a range of microbiological tests, batch review, microbiological investigation and policy development. In addition, he is an honorary consultant with the School of Pharmacy and Pharmaceutical Sciences, University of Manchester and is a tutor for the university's pharmaceutical microbiology M.Sc course. Dr. Sandle serves on several national and international committees relating to pharmaceutical microbiology and cleanroom contamination control (including the ISO cleanroom standards). He is currently chairman of the PharMIG LAL action group and serves on the NBS cleaning and disinfection committee. He has written over eighty book chapters, peer reviewed papers and technical articles relating to microbiology. He is currently the editor of the Pharmaceutical Microbiology Interest Group Journal and runs an on-line microbiology forum (www.pharmig.blogspot.com). Dr. Sandle is an experienced auditor and frequently acts as a consultant to the pharmaceutical and healthcare sectors.

Posted by Tim Sandle

WHO update endotoxin guidance

The World health Organization has updated its guidance document on the Bacterial Endotoxin Test. the document is available as a draft for public comment. The document is titled:

“IMPLEMENTATION OF THE 1 REVISED GENERAL MONOGRAPH ON PARENTERAL PREPARATIONS IN THE INTERNATIONAL PHARMACOPOEIA: LIMITS FOR THE TEST FOR BACTERIAL ENDOTOXINS (3.4)”.

With the document, individual monographs on injectable dosage forms in the

International Pharmacopoeia (Ph.Int.) were investigated with a view to add a limit for bacterial endotoxins to each monograph that currently does not include such a requirement. The result of the review is that a number of endotoxin limits have been proposed.

Furthermore, in the new general monograph on Parenteral preparations the following statement is made:

“For powders and concentrates for injections and intravenous infusions, the amount of the preparation to be tested and the nature and volume of the liquid in which it is to be dissolved, suspended or diluted is specified in the individual monograph.”

The new guidance proposes to delete this sentence since the preparation of the sample solution is described in Chapter 3.4 Test for bacterial endotoxins. The text requires that samples should be dissolved or diluted in aqueous solutions so that the final solutions do not exceed the maximum valid dilution (MVD).

The aim is for the document to be implemented in October 2014. The draft can be viewed via the WHO website.

Posted by Tim Sandle

Endotoxin detection: techniques and developments

American Pharmaceutical Review has issued a special supplement dedicated to endotoxin testing.

The supplement features interesting articles on the endotoxin concern of biologics (by Kevin Williams); regulatory issues pertaining to endotoxin testing (by Michael Dawson); a comparative study examining different methods of endotoxin destruction (by Tim Sandle); an examination of future developments in endotoxin testing (by Karen Zink McCullough); and preparation of a naturally occurring endotoxin challenge study (by Kim Bowers and Lynn Johnson).

The supplement also contains other information relating to endotoxin and LAL testing, and makes for a useful and interesting read in relation to more advanced aspects of the bacterial endotoxin test.

For further details, see APR.

Posted by Tim Sandle

Beta glucans as pharmaceutical product impurities

β-Glucans (beta-glucans) are polysaccharides of D-glucose monomers linked by β-glycosidic bonds. Glucans are important because they can react with certain lysates and cause interference with the LAL test. Furthermore, glucans can, in certain circumstances, cause physiological effects in humans (β-glucans are known as “biological response modifiers” because of their ability to activate the immune system).

To discuss the implications on both the LAL test and in relation to pharmaceutical products, Tim Sandle has written an article for the American Pharmaceutical Review. The article has been published as a special supplement ‘Further Pharmaceutical Microbiology’.

The reference is:

Sandle, T. (2013). Pharmaceutical Product Impurities: Considering Beta Glucans, American Pharmaceutical Review, 16 (5) Supplement S1: 16-19

Posted by Tim Sandle

Glucans and the Bacterial Endotoxin Test

The issue of beta-glucans and the relationship of these substances to the LAL test remains an important area not described in great detail in compendial or regulatory documents. β-Glucans (beta-glucans) are polysaccharides of D-glucose monomers linked by β-glycosidic bonds. Glucans are important because they can react with certain lysates and cause interference with the LAL test.

In relation to this area of pharmaceutical concern, Tim Sandle has written an introductory paper. It can be accessed here: glucans.

Posted by Tim Sandle