webinar: bacterial endotoxins test (bet) – common assay issues

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.

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60-Minute Agenda

Sources of Variability in Endotoxin Testing

Product Independent Failures

Product Dependent Failures

Questions and answers

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60-Minute Agenda





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Mar-15

Sources of Variability in

Endotoxin Testing

Equipment

Environment

Consumables

Reagents

Documentation and interpretation of

procedures

Results analysis

Technician

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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

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Mar-15


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

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Mar-15


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)

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Mar-15


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

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Mar-15


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)

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Mar-15


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

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Mar-15


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

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Mar-15


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

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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

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Mar-15

60-Minute Agenda





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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

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Mar-15


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

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Mar-15


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

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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

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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

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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

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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

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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)

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Mar-15

60-Minute Agenda





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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

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Mar-15


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

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Mar-15


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

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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)

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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

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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

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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

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Mar-15

60-Minute Agenda





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Mar-15

Do You Have More Questions?

Contact our Scientific Support Team:

Team EU: +32 87 321 611

[email protected]

Team US: +1 800 521 0390 (toll free)

[email protected]

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

mailto:[email protected]

mailto:[email protected]

http://www.lonza.com/endosummit

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Mar-15

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webinar: bacterial endotoxins test (bet) – common assay issues

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