Pharma&Biotech

Bacterial Endotoxins Test (BET):

Common Assay Issues
Erin Ross 24 September 2013
Saskia Ihle 25 September 2013

,© /Lonza
© Lonza
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
Sep-13
60-Minute Agenda

 45 minute presentation
 15 minute interactive Q&A
 Wrap-up

3
Sep-13
 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
Sep-13
 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
Sep-13
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 0.005 EU/ml)

6
Sep-13
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
Sep-13
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
Sep-13
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
Sep-13
 Variability in Endotoxin Testing –
Regulatory Requirements

Regulatory authorities have set some BET acceptance limits:

 ≥ │0.980│ 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
Sep-13
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
Sep-13
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
Sep-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
Sep-13
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
Sep-13
Product Independent Failures
 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
Sep-13
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
Sep-13
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
Sep-13
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 0.005 or 0.05 EU/ml
standard for Kinetic-QCL™ Testing)

19
Sep-13
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
Sep-13
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
Sep-13
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
Sep-13
“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
Sep-13
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
Sep-13
 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 non-
homogenous solution
25
Sep-13
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
Sep-13
 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
Sep-13
Product Dependent Failures
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
Sep-13
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
Sep-13
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
Sep-13
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 non-
homogenous)
 The product may have changed since validation
 Reassess and revalidate?
 More robust sample preparation procedure?

32
Sep-13
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
Sep-13
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
Sep-13
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
Sep-13
 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
Sep-13
Thank You for Your Attention