Bacterial Endotoxins Test (BET): Common Assay Issues

Transcription

1 Pharma&Biotech Bacterial Endotoxins Test (BET): Common Assay Issues Erin Ross 24 September 2013 Saskia Ihle 25 September 2013, / Lonza Lonza

2 Presenters Erin Ross Scientific Support Specialist, Lonza Pharma&Biotech Bioscience Solutions Lonza Walkersville, Inc. Dr. Saskia Ihle Scientific Support Specialist, Lonza Pharma&Biotech Bioscience Solutions Lonza Cologne GmbH 2

3 60-Minute Agenda 45 minute presentation 15 minute interactive Q&A Wrap-up 3

4 Variability in Endotoxin Testing There are many sources of variability in any test system: Equipment Environment Consumables Reagents Documentation and interpretation of procedures Results analysis Technician 4

5 Variability in Endotoxin Testing Equipment and Environment Some can be controlled more easily than others: Equipment, i.e. readers, heating blocks, pipettes, timers, should be installed, validated and maintained appropriately The laboratory environment should be assessed during qualification to ensure factors such as humidity, temperature, light, cleanliness, air handling and cross contamination risk are managed within acceptable limits 5

6 Variability in Endotoxin Testing Consumables Consumables for use in BET should, according to the Pharmacopeia, be free of detectable endotoxin and do not interfere in the test The test is defined as the method in use. For example, endotoxin load should not exceed the sensitivity of the assay in use (a 0.25 EU/tip certification would not be appropriate for use in kinetic chromogenic testing to EU/ml) 6

7 Variability in Endotoxin Testing Consumables Consumables include sample vials, plates, tips, dilution tubes, reaction tubes, reagent reservoirs, water and any accessory buffers or solutions Lysate suppliers can provide consumables adequately certified for use in the sensitive BET methods Be wary of generic suppliers certification of apyrogenic and check the endotoxin limit used to test the product 7

8 Variability in Endotoxin Testing Consumables Due to lot to lot variability of endotoxin detection consumables, some labs also test each new lot of critical consumables such as 96-well plates or gel clot reaction tubes with the current in-use lot of reagents Take care to use the correct consumables compatible with the equipment in use (for example, the correct size of pipette tips for pipettes) 8

9 Variability in Endotoxin Testing Reagents/Consumables Limulus Amebocyte Lysate (LAL) reagents are biologically sourced, no two lots will react exactly the same Carefully controlled manufacture, formulation and Quality Control testing will help ensure reaction with known endotoxin standards is within an acceptable range For this reason, it is prudent to use the consumables recommended, tested and certified by the supplier of the reagents in use 9

10 Variability in Endotoxin Testing Regulatory Requirements Regulatory authorities have set some BET acceptance limits: correlation coefficient (R-value) %PPC (Positive Product Control) recovery should be 50% to 200% of the added spike value for photometric techniques and PPC must clot in gel clot methods Negative controls must not react 10

11 Variability in Endotoxin Testing Additional Limits BET reagent manufacturers also recommend limits for other kinetic test parameters based on what is achievable and acceptable: Slope and y-intercept limits %CV limits (<10% is usually recommended) Setting tighter limits than is recommended or regulated may be needlessly setting your test up for failure 11

12 Variability in Endotoxin Testing Procedures Ensure Standard Operating Procedures (SOP) are written clearly and are not open to misinterpretation. SOPs should also include procedures about resampling/retesting Results analysis for kinetic and recombinant Factor C testing should be easy provided the analyst is made familiar with the software Results analysis for gel clot testing is more open to analyst interpretation and manual calculations open to error. Ensure procedures and worksheets are clear, concise and easy to use 12

13 Variability in Endotoxin Testing Analyst Training Adequate training of analysts is imperative. Training should include SOP reading, observation, practice runs and validation runs Where appropriate, include re-qualification scheme for users who do not routinely run assays or those whose technique is called into question due to assay failures 13

14 First Indications of Possible Problems Product independent failures Negative control failure Poor %CV or single well reactions in standards or PPC or sample wells Standards/positive controls reacting atypically Failure of standard curve parameters in photometric technique testing Product dependent failures Change in PPC reaction profile Poor %CV or single well reaction in sample wells Failure to meet the assigned endotoxin limit 14

15 Product Independent Failures

16 Negative Control Failure There are many actions one can take to help limit the occurrence of this common cause of assay failure: Avoid environmental contamination such as dust, hair, skin cells, and other particulates by use of adequate personal protective equipment and cleaning routines Old air conditioning units and vortex mixers can shed particulates. Locate reader and preparation area away from these items Reduce the possibility of endotoxin contamination of the blanks/negative controls with good pipetting technique 16

17 Negative Control Failure Use of certified consumables further reduces the possibility of contamination Do not shorten the vortex mixing steps as this can compromise the standard curve as well as the difference between the blank and lowest standard endotoxin concentration 17

18 Negative Control Failure Take care to not contaminate the LRW used for negative controls Airborne or cumulative from repeated tip entry into bottle From tip used to reconstitute initial stock Control Standard Endotoxin (CSE) <0.005 EU/ml LRW endotoxin limit Water for Injection (WFI) endotoxin limit is 0.25 EU/ml so this water may not be appropriate for use in photometric test methods (unless tested to <0.005 EU/ml) Important for medical device washing. Other washing fluids may not be consistently low 18

19 Poor %CV Hot Wells This is a commonly used explanation when %CVs are out of specification, blanks react before the lowest standard or a single replicate reacts Hot wells are suspected to be an artifact of 96-well plate batch variability In practice, hot wells tend to show at very low levels of endotoxin. Most are seen in blanks, sample wells where endotoxin contamination is low and at or close to the limit of detection (for example in the wells of the or 0.05 EU/ml standard for Kinetic-QCL Testing) 19

20 Poor %CV Hot Wells This is a real problem seen globally However, repeated hot well type reactions should be investigated as they may indicate environmental contamination 20

21 Poor %CV Hot Wells The 96-well plates used for endotoxin testing are not produced specifically for endotoxin testing Tissue culture plates are treated to improve cell adhesion Endotoxin and/or some test articles may be affected by this treatment Technicians may see significant differences in reaction times with various plate manufacturers 21

22 Poor %CV Hot Wells Plastic microplates are gamma irradiated after packaging in a clean environment But sterility does not indicate depyrogenation Use plates which have been endotoxin tested and tested to be free from LAL interfering factors Contaminated plates may still be encountered not every plate can be tested There is one way around this: Quartz microplates Very expensive Need to be cleaned and depyrogenated between use 22

23 Hot Wells Misuse of Definition Hot wells are used frequently to describe large replicate variations which are more likely due to: Spot contamination An incubator failure if the reader uses well specific heaters (this is a rare occurrence) Pipetting error Hot wells should not be used as a coverall explanation for large replicate variation. Repeated Out of Specification/Out of Trend (OOS/OOT) results should be investigated Similarly but rarely there are also hot tips: Most commonly seen with tips > 5 ml volume Use adequately certified consumables 23

24 Poor %CV Air Bubbles Generally not a problem, unless they burst during an assay Mostly created during lysate addition By blowing out pipette with direct pipetting technique Use reverse pipetting technique instead 24

25 Poor %CV Air Bubbles Problem when testing viscous products such as Tween 20 or protein containing substances When the bubble bursts the optical density drops May go below the original base line The shaking step at assay initiation usually disperses any bubbles to the edge of the well as long as the well does not contain a viscous or nonhomogenous solution 25

26 Single Tube Control Reactions Gel Clot It is not as common to have a negative control single tube reaction (i.e. one replicate of the negative control is positive) in the gel clot test as the tests are simply not as sensitive Commonly due to: Mislabeling of tubes Contamination during pipetting into reaction tubes due to poor pipetting technique Contaminated tip Retesting usually gives expected result 26

27 Positive Control Failures Often a result of incorrect pipetting due to distraction or poor labeling of dilution tubes Gross system contamination so all kinetic standards react at the same time (a rare occurrence) Blocked or failed channel in the kinetic reader (alarm and system test failures) Failure of positive control reactions or standard curve parameters such as correlation coefficient usually due to: Poor standard preparation (pipetting errors, too short vortexing, contamination, use of plastic dilution tubes) Correlation coefficient, slope and y-intercept failures can indicate a kinetic reader incubator issue, however, most reader systems would alarm if the temperature was not constant or outside the settings 27

28 Product Dependent Failures

29 PPC Reaction Failures %PPC recovery should be 50% to 200% of the added spike value for photometric techniques and PPC must clot in gel clot methods Repeated PPC reaction failures should be investigated If found during routine testing of a validated product, this must be investigated It could indicate a change in the interference properties of the validated sample or during sample preparation It could indicate an analyst training issue It could indicate a laboratory contamination issue 29

30 PPC Reaction Failures If the product is validated and the PPC fails with %CV pass and no mismatched replicate reactions, resampling is required initially to try to isolate cause of failure A change in the interference profile of a product could be due to: Change of raw material or raw material supplier Change of chemical structure / formulation that has no clinical impact Recent cleaning procedure has left residues 10µl pipette out of calibration Sampling error A change in the sample storage vessel 30

31 PPC Reaction Failures If resample fails, this eliminates sampling error and the issue may be a change in the product being tested since validation was conducted 31

32 Sample Well Variance Sometimes poor %CV or mismatched replicate reactions are seen repeatedly for a particular product If this trend is seen during validation, or becomes a trend later in routine testing, it should not be ignored Issues with sample preparation? (i.e. the sample nonhomogenous) The product may have changed since validation Reassess and revalidate? More robust sample preparation procedure? 32

33 Sample Well Variance Temperature of sample prior to plating can affect mixing %CV limits are not a regulatory requirement so some labs may consider changing the limit to 15 or 20% in extreme cases (i.e. some biological or complex pharmaceuticals). 33

34 Endotoxin Result Failures Most sterile parenterals have no detectable or very low level endotoxin contamination results OOS results rarely seen, but more often OOT results For an OOS/OOT result or for mismatched replicate reactions, check the %CVs to rule out system contamination, pipetting error or poor technique 34

35 Endotoxin Result Failures If the variance and PPC reactions pass limits and are within trend, a full OOS/OOT investigation must take place to determine the source of the problem Sample contamination Batch contamination It is possible that the positive result could be LAL-RM (Reactive Material). This should be considered in the investigation, but also accept that it could be a real endotoxin contamination 35

36 Common Assay Issues Summary Variation in any endotoxin detection assay can be minimized by: Good product dependent and independent validation protocols Good technician training Adequate reporting and investigation of any unusual results Use of adequately tested and certified reagents and consumables GLP/GMP 36

37 Thank You for Your Attention