A s s o c i a t e s o f C a p e C o d , I n c .

Current Regulatory Considerations for Endotoxin Testing

Michael E. Dawson, Ph.D.

Director of Regulatory Affairs

7th June, 2011

PN002356 rev.3

-CONFIDENTIAL-

• The information in this presentation is given for the purposes of education and discussion.

• It is not intended to be, and it should not be used as, a substitute for regulations or regulatory guidance.

• Decisions and actions should be based on the relevant regulations, guidance documents and pharmacopeial chapters, not on this presentation.

GO TO THE SOURCE DOCUMENTS

Disclaimer

-CONFIDENTIAL-

• This presentation includes:

– Recent changes in the pharmacopeial Bacterial Endotoxins Test (BET) chapters

– Requirements for the Pharmacopeial BET chapters -implications for the marketplace

– Pyrosate

– The status of the US Food and Drug Administration (FDA) guidance documents on endotoxin testing

– The AAMI/ANSI standard, ST72

– Draft ICH guideline Q4B Annex 14

Introduction

-CONFIDENTIAL-

• United States Pharmacopeia (USP)– USP 33 became official October 1, 2010 – delayed by withdrawal

– BET chapter completely rewritten for USP 33

– Interim revision announcement (IRA, February 25, 2011) for the BET–became official on April 1, 2011 - supersedes USP 34, which became official on May 1, 2011

• European Pharmacopoeia (EP)– BET essentially unchanged in EP 7.0, effective Jan. 1, 2011

• Japanese Pharmacopoeia (JP)

– JP 16 issued on March 24, 2011; became effective on April 1, 2011

– Includes minor changes to the BET

Recent Changes to BET Chapters

-CONFIDENTIAL-

• Impact of Changes

– The changes do not alter the fundamental principles of the BET chapter.

– These principles were established when the BET chapters in the USP, EP and Japanese Pharmacopoeia (JP) were harmonized in 2001.

Recent Changes to BET Chapters (cont.)

-CONFIDENTIAL-

1. Description of Techniques (methods)– USP chapter now describes three techniques:

• Gel-clot, turbidimetric and chromogenic technique, not two (gel-clot and photometric).

– The EP also describes three techniques but continues to list 6 methods:

A. Gel-clot limit testB. Gel-clot quantitative testC. Turbidimetric kinetic methodD. Chromogenic kinetic methodE. Chromogenic end-point methodF. Turbidimetric end-point method

– The JP lists two techniques:• Gel-clot and photometric

Summary of Changes to the BET - USP 33 Forwards

-CONFIDENTIAL-

2. LAL Reagent– USP now specifies reagent “manufactured in accordance

with the regulations of the competent authority” - like the EP chapter (but not the JP).

– Potential implication for Lonza’s recombinant Factor C (rFC) reagent, which is unlicensed.

Note: The 1987 FDA Guideline states that reagent licensed by CBER should be used for validation, release and in-process testing.

Summary of Changes to the BET - USP 33 Forwards

-CONFIDENTIAL-

3. β-Glucans – USP

– The note on β-glucans has been moved from a footnote into the main text and refers to lysates with factor G removed or added glucan blockers - like the EP.

4. Reconstituted Lysate Reagent – USP

– USP now refers to reconstituted lysate as lysate TS, like the JP, but not the EP, which refers to lysate solution.

5. LAL Reagent Water – USP

– The footnote “LAL Reagent Water ” has been replaced with “Water for BET” in the text, like the EP.

– The JP also refers to water for BET.

Summary of Changes (cont.)

-CONFIDENTIAL-

6. Endotoxin Reference Standard

– USP: Details of reconstitution, use and storage of USP (Endotoxin RS) are replaced with reference to instructions in the “package leaflet.”

– JP: Added that the Reference Standard Endotoxin has been calibrated to the current World Health Organization (WHO) International Standard for Endotoxin.

– EP: No changes to this section.

7. Use of Dilutions of Standard Endotoxin – USP

– The caution: “Use dilutions as soon as possible to avoid loss of activity by adsorption” (like the EP and JP) replaces:

“Do not store dilutions … in the absence of supporting data …”

Summary of Changes (cont.)

-CONFIDENTIAL-

8. pH – USP and EP

– The statement that the reaction mixture pH is usually within range if the sample pH is between 6.0 – 8.0 has been removed, but not from the JP.

– Now states that the pH of the reaction mixture should be within the range specified by the lysate manufacturer, usually 6.0 to 8.0.

9. Endotoxin limit (K/M) – USP, EP and JP

– M now defined as the maximum bolus dose (no longer generally defined as the maximum dose per hour).

– Added text: “When the product is to be injected at frequent intervals or infused continuously, M is the maximum total dose administered in a single hour period.”

– No real change.

Summary of Changes (cont.)

-CONFIDENTIAL-

10. Endotoxin Limit Tightened - USP Interim Revision Announcement (IRA) - not in EP or JP.

– The generic endotoxin limit for drugs administered on a per square meter basis (primarily anti-cancer drugs) has been halved – in a footnote on p. 1. Not applicable to most drugs (dose per kg or person).

– The value of K for such drugs has been reduced from 5 to 2.5 EU/kg.

– Addresses FDA concerns due to pediatric doses of these products.

11. Repeating Test for Interfering Factors, Gel-clot Technique – USP IRA

– Added requirement to repeat the test for interfering factors when any condition likely to influence the result changes.

– Gives consistency with photometric methods and the EP and JP.

Summary of Changes (cont.)

-CONFIDENTIAL-

12. PhotometricLysate Qualification – USP and EP

– Added the requirement to perform the test for the Assurance of Criteria for the Standard Curve for each lysate lot.

– This was/is specified for the gel-clot technique and in the JP.

13. Validation of Treatments to Overcome Interference: JP– Demonstrate that a sample treatment eliminates interference

without loss of endotoxins – was/is in USP and EP.

14. EP Guidelines

– The section titled “Test for bacterial endotoxins: guidelines” has been removed from the BET chapter (2.6.14)

– Is now a separate new chapter (5.1.10) under General texts.

Summary of Changes (cont.)

-CONFIDENTIAL-

• Interim Revision Announcement (IRA) for the BET chapter was published on February 25, 2011.

– First announced in USP’s Pharmacopeial Forum (PF) in November 2010.

– Became effective on April 1, 2011.

– See: http://www.usp.org/pdf/EN/USPNF/2011-02-2585BACTERIALENDOTOXINS.pdf

– Only on the website – not published in PF.

• Included two changes (discussed above), one of which is significant to some drug manufacturers.

USP Interim Revision Announcement

-CONFIDENTIAL-

• The IRA supersedes the BET chapter in USP 34.

• This despite the fact that the official date of USP 34 (May 1, 2011) is after that of the IRA.

– There is potential for confusion here.

USP Interim Revision Announcement (cont.)

-CONFIDENTIAL-

• General Information

• Followed by two sections: Gel-clot technique and Photometric techniques - each includes:1. Preparatory Testing:

• Confirmation of test performance

– Gel-clot method: Test for confirmation of labeled sensitivity

– Photometric methods: Assurance of standard curve criteria (|r| ≥0.980)

• Test for interfering factors

– Show sample does not interfere with detection of endotoxin

– Detect known amounts of endotoxin added to the sample

2. Test Procedure:

• Gel-clot: limits test and quantitative test described

• Photometric methods: same as the test for interference

Elements of BET Chapters

-CONFIDENTIAL-

• A minimum of 9 wells are required to run a test as specified in (i.e. in compliance with) the BET. If negative controls are included in triplicate, 12 wells are needed.

• The test described in the USP cannot be performed in the PTS.

Test Requirements for BET Compliance

USP/EP/JP BET Requirements For Photometric Tests:Confirmation of Test Performance - Assurance of Criteria

for the Standard Curve

At least three standard endotoxin concentrations (typically λ, x.λand x2.λ, where x is the dilution factor).

Include at least three replicates of each standard concentration.

Negative controls are not specified – they are required for the test for interfering factors and for all gel-clot tests.

-CONFIDENTIAL-

• At least two replicates of each required. Therefore, a minimum of 12 wells are required to run a test as specified in (i.e. in compliance with) the BET.

• The test described in the USP cannot be performed in the PTS.

Test Requirements for BET Compliance (cont.)

USP/EP/JP BET Requirements For Photometric Tests:Test for Interfering Factors and the Test Procedure

A. Sample solution (without added endotoxin)

B. Sample solution with endotoxin added to give aconcentration from the middle of the standard curve

C. At least three standard endotoxin concentrations

D. Water for BET (negative controls)

-CONFIDENTIAL-

Note: There is no provision in the BET for use of an archived (stored) standard curve, which the Charles River Endosafe PTS reader depends upon.

Implications of BET Requirements for Photometric Test Systems

USP/EP/JP BET Requirements For Photometric Tests:Assurance Criteria for the Standard Curve

Pyros Kinetix®

Tube Readersand

Microplate Readers

PortableTest

System

At least three standard endotoxin concentrations

Include at least three replicates of each standard concentration.

√ X

-CONFIDENTIAL-

Again, there is no provision in the BET for use of an archived (stored) standard curve.

Implications of BET Requirements for Photometric Test Systems (cont.)

USP/EP/JP BET Requirements For Photometric Tests:Test for Interfering Factors and the Test Procedure

Pyros Kinetix®

Tube Readersand

Microplate Readers

PortableTest

System

A. Sample solution (without added endotoxin) √ √B. Sample solution with endotoxin added to give

a concentration from mid standard curve √ √

C. At least three standard endotoxin concentrations √ X

D. Water for BET (negative controls) √ X

-CONFIDENTIAL-

• Pyrosate® is unlicensed.• The instructions for use state:

“Pyrosate is a rapid endotoxin test specially designed for water, dialysate or other testing in applications that do not require use of a Limulus Amebocyte Lysate (LAL) reagent licensed by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration.”

• The Pyrosate page in the ACC Catalog states:

“May be used of all endotoxin testing applications not requiring FDA-licensed LAL”

Pyrosate®

-CONFIDENTIAL-

Pyrosate® and the USP BET, <85>

USP Requirement Pyrosate®

“… product manufactured in accordance with the regulations of the competent authority.”

Unlicensed – but is manufactured to the same standards as licensedproduct.

Test for Confirmation of Labeled Lysate Sensitivity

Not in IFU – but can be done.

Test for Interfering factors Not in IFU – but can be done.

Limit Test IFU covers half of this. Does not include the negative control and positive control of endotoxin at 2λ that is required in the BET. Can be done with Pyrosate by using water for BET as the sample.

Quantitative Test Not in IFU – but can be done.

-CONFIDENTIAL-

• The 1987 Guidelines and the 1991 Interim Guidance are reportedly slated for withdrawal.

– This would remove the only documents that mention archived standard curves.

– Implications for the CRE PTS reader.

• FDA planning to publish a Question and Answer paper in place of these documents.

– Drafts of this Q & A document have not been published or circulated outside of FDA.

FDA Guidance Documents

-CONFIDENTIAL-

• The AAMI/ANSI ST72:2002/(R)2010 “Bacterial endotoxins—Test methodologies, routine monitoring, and alternatives to batch testing”

– Reaffirmed by ANSI on March 17, 2010

– A recognized consensus standard by FDA Center for Devices and Radiological Heath (CDRH)

• Not by the Centers for Drug and for Biologics Evaluation and Research (CDER and CBER)

AAMI Association for the Advancement of Medical Instrumentation.

ANSI American National Standards Institute

AAMI/ANSI Standard, ST72

-CONFIDENTIAL-

• Scope: determination of bacterial endotoxins on or in medical devices, components, or raw materials

– But it also states:“Although the scope of this standard is limited to medical devices, it specifies requirements and provides guidance that may be applicable to other health care products.”

• A final draft revision of this standard has been published for comment: AAMI/FDS-1 ST72.– It contains minor changes and adds an annex on OOS

investigations.

AAMI/ANSI ST72 (cont.)

-CONFIDENTIAL-

• The standard is consistent with the FDA Guidelines/Guidance and the USP BET.

– Specifies the use of licensed reagent.

• Lonza’s rFC reagent does not meet this requirement.

• Adds provisions for alternatives to batch testing.

– The new draft specifies that this should be considered in the context of a process risk management plan.

• Annexes provide background and guidance.

AAMI/ANSI ST72 (cont.)

-CONFIDENTIAL-

• Draft ICH guideline Q4B Annex 14: Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions on Bacterial Endotoxins Tests – General Chapter states:– BET in the EP, JP and USP can be used interchangeably in the

ICH regions subject to the following conditions:

• 2.1.1 Any of the three techniques can be used. In the event of dispute, the gel-clot limit test should be used to make the final decision.

• 2.1.2 The Endotoxin Reference Standard should be calibrated to the current WHO International Standard for Endotoxin.

– FDA added that they might request demonstration that the chosen method is acceptable.

– Issued as an FDA Guidance document September 2010.

– To be accepted by Europe, Japan and Canada.

ICH Draft Consensus Guideline

-CONFIDENTIAL-

• Recent revisions to the USP, EP and JP BET chapter do not change any fundamental principles– Except for the endotoxin limit for drugs administered per m2

– The current USP BET is the IRA

• Implication of BET Requirements - compliant tests cannot be performed in all systems

• Pyrosate is currently unlicensed - but can perform compliant tests

• FDA guidances on endotoxin testing are expected to be withdrawn and replaced by a Q & A document

• AAMI/ANSI ST72 includes many of the points in the FDA Guidance documents

• ICH guideline Q4B, Annex 14 on the BET– States the equivalence of the chapters– Specifies standardization to the WHO International Standard for Endotoxin.

Conclusion

T h a n k Yo u .

Q u e s t i o n s & D i s c u s s i o n

Current Regulatory Considerations for Endotoxin Testing

Michael E. Dawson, Ph.D.

Director of Regulatory Affairs

7th June, 2011

PN002356 rev.3

-CONFIDENTIAL-

AbbreviationsAAMI Association for the Advancement of Medical Instrumentation

ANSI American National Standards Institute

BET Bacterial Endotoxins Test

CBER Center for Biologics Evaluation and Research (agency of USFDA)

CDRH Center for Devices and Radiological Heath (agency of USFDA)

CDER Center for Drug Evaluation and Research (agency of USFDA)

EP European Pharmacopoeia

ICH International Conference on Harmonisation

IFU Instructions for use (package insert)

IRA Interim Revision Announcement

JP Japanese Pharmacopoeia

LAL Limulus Amebocyte Lysate

PF Pharmacopeial Forum

rFC recombinant Factor C

RS Reference Standard (as in the USP endotoxin RS)

USFDA United States Food and Drug Administration (often just FDA)

USP United States Pharmacopeia