webinar: bacterial endotoxins test (bet) – common assay...
TRANSCRIPT
Pharma&Biotech
Bacterial Endotoxins Test (BET):
Common Assay Issues Webinar
Erin Ross 10 March 2015
Dr. Saskia Ihle 11 March 2015
Pharma&Biotech
© Copyright 2015, Lonza Walkersville, Inc. All rights reserved.
2
60-Minute Agenda
Sources of Variability in Endotoxin Testing
Product Independent Failures
Product Dependent Failures
Questions and answers
3
60-Minute Agenda
Sources of Variability in Endotoxin Testing
Product Independent Failures
Product Dependent Failures
Questions and answers
4
Mar-15
Sources of Variability in
Endotoxin Testing
Equipment
Environment
Consumables
Reagents
Documentation and interpretation of
procedures
Results analysis
Technician
5
Mar-15
Variability in Endotoxin Testing –
Equipment and Environment
Equipment should be installed, validated and maintained
appropriately
Laboratory environment should be assessed during qualification
Humidity, temperature, light
Cleanliness, air handling
Cross contamination risk
6
Mar-15
Variability in Endotoxin Testing –
Consumables
Consumables for use in BET should “be free of detectable
endotoxin and do not interfere in the test” (Pharmacopoeia)
“Test” = method in use
Example: a 0.25 EU/tip certification would not be appropriate for
use in kinetic chromogenic testing to 0.005 EU/ml
7
Mar-15
Variability in Endotoxin Testing –
Consumables
Lonza offers 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
Testing new batches of “critical” consumables might be
appropriate
Take care to use correct consumables compatible with the
equipment (e.g. correct tip size for pipettes)
8
Mar-15
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
Mar-15
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 (before the lowest standard)
10
Mar-15
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
Mar-15
Variability in Endotoxin Testing –
Procedures
Standard Operating Procedures (SOP)
Must be clear and not open to misinterpretation
Should also include procedures about resampling/retesting
Results analysis for kinetic assays and recombinant Factor C:
WinKQCL™ Software easy
Results analysis for gel clot testing:
More open to analyst interpretation
Manual calculations source of error
Ensure procedures and worksheets are clear, concise and easy to
use
12
Mar-15
Variability in Endotoxin Testing –
Analyst Training
Adequate training of analysts is imperative
Include SOP reading, observation, practice runs and validation runs
Where appropriate, include re-qualification scheme for
Users who do not routinely run assays
Users whose technique is called into question due to assay failures
13
Mar-15
First Indications of Possible Problems
Product independent failures
Negative control failure
Standards/positive controls reacting atypically
Failure of standard curve parameters in photometric
technique testing
Poor %CV or single well reactions in standards or PPC or
sample wells
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
Mar-15
60-Minute Agenda
Sources of Variability in Endotoxin Testing
Product Independent Failures
Product Dependent Failures
Questions and answers
15
Mar-15
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 (e.g. dust, hair, skin cells,
and other particulates) adequate personal protective
equipment, cleaning routines
Locate reader and preparation area away from old air
conditioning units and vortex mixers – can shed particulates
Good pipetting technique reduces possibility of endotoxin
contamination of blanks/negative controls
16
Mar-15
Negative Control Failure
Use certified consumables
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
Mar-15
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 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
Mar-15
Poor %CV – “Hot Wells”
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 (not produced specifically for endotoxin detection)
In practice, “hot wells” tend to show at very low levels of
endotoxin (blanks, sample wells where endotoxin contamination
is at or close to the limit of detection)
There is one way around this: Quartz microplates
Very expensive
Need to be cleaned and depyrogenated between use
19
Mar-15
“Hot Wells” – Misuse of Definition
Frequently used to describe large replicate variations which are
more likely due to:
Spot contamination
Pipetting error
An incubator failure if the reader uses well specific heaters (rare)
Repeated Out of Specification/Out of Trend (OOS/OOT) results
should be investigated – don’t use “hot wells” as coverall
explanation for large replicate variation
Similar, but very rare: “hot” tips
Most commonly seen with tips > 5 ml volume
Use adequately certified consumables
20
Mar-15
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
21
Mar-15
Single Tube Control Reactions – Gel Clot
Negative control single tube reaction is not as
common (gel clot test 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
22
Mar-15
Positive Control Failures
Often a result of incorrect pipetting due to distraction or
poor labeling of dilution tubes
Gross system contamination all kinetic standards react
at the same time (rare)
Blocked / failed channel in reader (alarm, system test failures)
Failure of positive control reactions or standard curve parameters
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 incubator
issue (however: usually alarm if temperature not constant / out of range)
23
Mar-15
60-Minute Agenda
Sources of Variability in Endotoxin Testing
Product Independent Failures
Product Dependent Failures
Questions and answers
24
Mar-15
PPC Reaction Failures
Requirement
Photometric techniques: %PPC recovery should be 50-200% of
added spike value
Gel clot methods: PPC must clot
Repeated PPC reaction failures should be investigated
If found during routine testing of a validated product: must be
investigated, could indicate
Change in the interference properties of the validated sample or
during sample preparation
Analyst training issue
Laboratory contamination issue
25
Mar-15
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
If resample fails, this eliminates sampling error and the issue
may be a change in the product being tested since validation
was conducted
26
Mar-15
PPC Reaction Failures
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
A change in the sample storage vessel
Recent cleaning procedure has left residues
Sampling error
10µl pipette out of calibration
27
Mar-15
Sample Well Variance
Repeatedly poor %CV or mismatched replicate reactions for a
particular product? Trend should not be ignored!
Issues with sample preparation? (i.e. sample non-homogenous)
Temperature of sample prior to plating can affect mixing
Product may have changed since validation
Reassess and revalidate?
More robust sample preparation procedure?
%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)
28
Mar-15
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
29
Mar-15
Endotoxin Result Failures
If %CV and %PPC recovery pass limits and are within trend: full
OOS/OOT investigation to determine source of the problem
Sample contamination
Batch contamination
Possible that positive result could be LAL-RM (Reactive
Material) should be considered in the investigation, but also
accept that it could be a real endotoxin contamination
30
Mar-15
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
31
Mar-15
60-Minute Agenda
Sources of Variability in Endotoxin Testing
Product Independent Failures
Product Dependent Failures
Questions and answers
32
Mar-15
Do You Have More Questions?
Contact our Scientific Support Team:
Team EU: +32 87 321 611
Team US: +1 800 521 0390 (toll free)
Secure your seat at our Global Endotoxin Testing Summit to discuss hot
topics in endotoxin testing including:
Low Endotoxin Recovery (LER) and Hold-time Studies
Increasing Demand for LAL and the need for Horseshoe Crab Conservation
Validation and Regulatory Acceptance of Alternative Endotoxin Detection
Methodologies
Learn more and register: http://www.lonza.com/endosummit
33
Mar-15
Interested in Learning More?
Follow us on social to receive first-hand technical tips and QC know-how:
https://www.linkedin.com/company/lonza-qc-testing-solutions
https://www.facebook.com/lonzaqctesting
https://twitter.com/Lonza_LAL
Sign up for our QC eNews
www.lonza.com/enews
Thank You for Your Attention