Fundamentals of Bacterial Endotoxin Testing Validation

1

CRYSTAL BOOTH, REGIONAL MANAGER, EAST COAST, MASTERS OF MICROBIOLOGY, NORTH CAROLINA STATE UNIVERSITY

Fundamentals of Bacterial Endotoxin Testing Validation

biotech.com | [email protected] | 700 Corporate Center Drive Pomona, California 91768

• Overview of the Assay– What is the test used for– The BET (bacterial endotoxin testing) reaction– CSE (Controlled Standard Endotoxin)– LAL (Limulus Amoebocyte Lysate)

• Regulatory Guidance– Guidance Documents and Guidelines– How is the test harmonized with the various compendia– Regulatory Observations

• Validation Strategies– BET definitions and calculations– Setting appropriate specifications for your application– Overview of Various Assays– Method development and validation tips– Common causes of invalid endotoxin assays

Agenda


• Any views, opinions, or recommendations made here are that of the author and are made for educational purposes.

Disclaimer


Overview of the Assay


http://www.horseshoecrab.org/med/testing-methods.html



• USP <85>: the bacterial endotoxins test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab.17

• Three Common Techniques:

– Gel-clot technique

– Turbidimetric technique (KTA)

– Chromogenic technique (KCA)



• Pyrogens are defined as any substance circulating in the body that produces a fever.13

– Chemical or Endotoxins

• Endotoxins are the lipopolysaccharide (LPS) portion of Gram-negative outer membranes.6

– Endotoxins are pyrogenic.6

– Heat-stable toxins present in the intact bacterial cell.

– Mainly released when the cell wall of the bacterium is destroyed.




https://askmicrobiology.com/gram-negative-bacterial-cell-wall/


• A product can be sterile, but endotoxin positive.

• Endotoxin’s may cause:– Chills

– Fever

– Leukopenia (decrease in number of circulating white blood cells)

– Shock

– Death

– A variety of other symptoms




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https://www.frontiersin.org/files/Articles/523904/fmars-07-00153-HTML/image_m/fmars-07-00153-g001.jpg


Controlled Standard Endotoxin (CSE)• Environmental (Unpurified or Native) endotoxins are a complex mix

of proteins, carbohydrates, and lipids.16

• RSE (Reference Standard Endotoxin)- are purified endotoxins that are highly characterized and understood preparations that standardize the LAL test.3

– Expensive to purchase– Limited supply

• CSE (Controlled Standard Endotoxin)- are secondary standards.3

– CSE are purified endotoxins– CSE are a molecule of polysaccharides and Lipid A without proteins. 16

– Prepared from Escherichia coli. – Does not contain live cultures – Safe to handle. – CSE clumps more readily in solution

• Endotoxin standards are weighed in nanograms (ng) and assayed to obtain the value of endotoxin units (EU) in the preparation.3




Limulus polyphemus is the Atlantic Horseshoe Crab, it is found on the Atlantic Coast from Maine to the Yucatan Peninsula in Mexico.1

http://qualityalchemist.blogspot.com/2008_03_01_archive.html




https://www.chesapeakelimulabs.com/horseshoe-crab-season-underway



https://www.theatlantic.com/technology/archive/2014/02/the-blood-harvest/284078/

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https://www.criver.com/products-services/qc-microbial-solutions/endotoxin-testing/3Rs-achieving-sustainability-endotoxin-testing


Regulatory Guidance

Regulatory Guidance

https://www.everteam.com/en/4-regulations-that-can-influence-your-governance-program/


• Code of Federal Regulations (CFR) Title 21: Food and Drugs.

• Food and Drug Administration (FDA) (2012) FDA Guidance for the Industry: Pyrogens and Endotoxins Testing: Question and Answers

• European Pharmacopeia (EP) 2.6.14 Bacterial Endotoxins

• Japanese Pharmacopeia (JP) 4.01 Bacterial Endotoxins Test

• United States Pharmacopeia (USP) <85> Bacterial Endotoxins Test

• PDA Technical Report 82- Low Endotoxin Recovery

Regulatory Guidance


• USP <85>, EP 2.6.14, and JP 4.01 are harmonized with one another and the assays described in the documents are equivalent. Portions that are not harmonized within the compendia are marked as such.

• Even though the tests are harmonized with one another, individual monographs for specific raw materials may not be harmonized with one another in the various regions.

Regulatory Guidance

https://www.advisoryexcellence.com/zte-tests-chinas-commitment-to-international-law/

https://www.advisoryexcellence.com/zte-tests-chinas-commitment-to-international-law/


• Regulatory Observations

Regulatory Guidance

https://www.webinarcompliance.com/product/good-preparation-practices-for-fda-audits-and-responding-to-fda-483s-meena-chettiar-fdb1698/


FDA 483 Dated 15Oct2019 18

• “Acceptance criteria for the sampling and testing conducted by the quality control unit is not adequate to assure that batches of drug products meet each appropriate specification and appropriate statistical quality control criteria as a condition for their approval and release.

• Specifically, Your firm failed to adequately test for endotoxin present you in your drug product, [REDACTED], in a manner that is statistically significant. Your firm tests [REDACTED] units out of [REDACTED] units produced per lot. This sample size accounts for [REDACTED] of each lot of this drug product produced. Your firm could not provide any scientific rationale for choosing this sample size. Your firm obtained out of trend results for endotoxin levels in lots [REDACTED] of your drug product [REDACTED]. These lots were retested by sampling an [REDACTED] units [REDACTED] passed release testing and were distributed to the United States. Your firm has manufactured and distributed [REDACTED] lots of this drug product to the United States.” 18

Regulatory Guidance


FDA 483 Dated 03Dec2019 18

• “Your firm does not test any lots of intrathecal drug products for endotoxin prior to release. All lots of intrathecal drug products are produced using non-sterile bulk drugs which are then [REACTED] and stored under refrigeration in syringes until use.

• In addition, your firm assigns a Beyond Use Date of nine days after production for all intrathecal drug products. However, your firm has no data to substantiate the nine day Beyond Use Date.

• In the last 12 months, your firm produced approximate [REDACTED] lots of intrathecal drug products which were not tested for endotoxin prior to being dispensed to patients. For example, Hydromorphone 5mg/ml, lot [REDACTED] was produced on l 0/30/ 19 and used in Rx [REDACTED] for patient [REDACTED] on the same date.” 18

Regulatory Guidance


FDA 483 Dated 03Aug2018 18

• “Failure to ensure [REDACTED] used in the final manufacturing steps of a non-sterile API intended for a sterile drug product is monitored and controlled for total microbial counts, objectionable organisms, and endotoxins.

• During the observation of the Bacterial Endotoxin Testing, the operator:

– Did not wear gloves while performing Gel Clot bacterial endotoxin testing

– Failed to maintain sterile bacterial endotoxin [REDACTED] ,inside Laminar Flow while pipetting

– Failed to handle sterile glass pipets to maintain aseptic conditions

– Failed to mix tube solution thoroughly before placing it in the test tube incubator

– Failed demonstrate a 180° inversion of tubes as required when reading results

• In addition, worksheet packet lacks documentation to identify the sample numbers of each of the samples tested, glass pipettes, pipettor and pipette tips used during the testing.” 18

Regulatory Guidance


FDA Warning Letter Dated 06Mar2019 9

• “Failure to validate with a high degree of assurance, a process whose results cannot be fully verified by subsequent inspection and test, as required by 21 CFR 820.75(a). During the inspection we observed:

• Your firm did not have any data to demonstrate bacterial endotoxin testing of the EBM medical devices, including [REDACTED] Soft Tissue Repair Matrix, has been validated adequately. For example, comparison of your current procedure, QCP-055 - Performance of the [REDACTED], Quality Control Procedure with your previous [REDACTED] for EBM Process Validation revealed the following discrepancies:– Your firm failed to use the medical device with the largest surface area as “worst

case” sampling for the [REDACTED] bacterial endotoxin verification study. [REDACTED] Soft Tissue Repair Matrix, with product code 830-247, measures 8 cm x 12 cm with a surface area of 96 cm2. This exceeds the sampling size of “[REDACTED]” with a surface area of [REDACTED] as documented in section 8.8, Endotoxin Sample Preparation, in the firm’s procedure, [REDACTED].

– The revision history of QCP-055, Rev: 05 shows that in March 2017, you modified the [REACTED] instructions for running routine samples. Specifically, “Sections 8.10.2.1 and 8.10.2.2 removed instructions to [REDACTED]. Your firm did not have any data to demonstrate this step did not affect the finished test results.” 9

Regulatory Guidance


FDA Warning Letter Dated 06Mar2019 Continued…9

• “Failure to establish and maintain procedures to prevent contamination of equipment or product by substances that could reasonably be expected to have an adverse effect on product quality, as required by 21 CFR 820.70(e). For example:

– Your firm did not have any data to demonstrate your [REDACTED] Water System, used to manufacture [REDACTED] medical devices, including [REDACTED], included bacterial endotoxin testing.

• As you are aware, finished product testing is not a substitute for a thorough validation of your manufacturing processes.” 9

Regulatory Guidance


FDA Warning Letter Dated 24Sept2020 9

• “Your firm failed to establish laboratory controls that include scientifically sound and appropriate specifications, standards, sampling plans, and test procedures designed to assure that components, drug product containers, closures, in-process materials, labeling, and drug products conform to appropriate standards of identity, strength, quality, and purity (21 CFR 211.160(b)).

– During the inspection, we observed the analysis of water samples and [REDACTED] samples for endotoxin using the gel clot method. We observed that the analyst failed to perform the critical steps for vortexing samples. Additionally, the analyst failed to use a calibrated timer for timing the steps in the method and instead used a wall clock that had not been calibrated. Vortexing and timing are critical steps for this method to ensure that testing does not yield false negative results. Your gel clot method also failed to include sufficient instructions.

– Your response acknowledged that your method was inadequate and that you would revise your procedure to include vortexing steps, as well as use a calibrated external thermometer and a calibrated external timer. You also stated that, as a part of your investigation to assess impact on the product, you analyzed reserve samples of all commercial batches of [REDACTED] injection [REDACTED] and all batches complied with the acceptance criteria.

– Your response is inadequate. You failed to perform a retrospective review of your laboratory practices including but not limited to the adequacy of your test methods. You also did not provide an evaluation of analyst competencies and training sufficiency.” 9

Regulatory Guidance


Validation Strategies

http://www.t-ec.com.tw/chuansheng/ezfiles/t-ec/download/attach/3/gel-clot.html



BET Definitions• CSE: Control Standard Endotoxin; a purified extract of E. coli that is standardized

against the Reference Standard Endotoxin (RSE) as indicated in the CoA. 3

• RSE: Reference Standard Endotoxin; a purified extract of E. coli that is well characterized and understood. 3

• CSE/RSE Ratio: The potency of the CSE determined relative to the RSE3

• EL: Endotoxin Limit3

• Endotoxin: A heat-stable toxin associated with the outer membrane of Gram-negative bacteria6

• EU: Endotoxin Unit is an expression of the activity of the endotoxin in a LAL assay 3

• LAL: Limulus Amebocyte Lysate• Lambda (λ): The LAL sensitivity used in the test for the gel-clot assay or the

lowest point on the curve in kinetic assays• LRW: LAL Reagent Water• MVC: Minimum Valid Concentration• MVD: Maximum Valid Dilution



BET Calculations

• The EL calculation is used to determine if endotoxin levels are safe.

• EL=K/M 17

– K=5 EU/kg for parenteral drugs or 0.2 EU/kg for intrathecal drugs17

– M=Maximum Human Dose/kg of body weight that would be administered in a single one-hour period. 17 70 kg is typically used as the average human weight. However, Japan is using 60 kg.



BET Calculations

• Maximum Valid Dilution 17

• Minimum Valid Concentration 1

• Geometric Mean 17


MVD=Endotoxin Limit X Sample Concentration

λ (EU/mL)

MVC=λ

Endotoxin Limit

Geometric Mean = antilog(Σ e)

f


BET Calculations

• Results: Endotoxin in Test Concentration (Weight)1

• Results: Endotoxin in Test Volume1

• Standard Series Preparation to get to 20λ1


λ= EU/mg

Test Sample Concentration

Labeled Endotoxin Value= Required Dilution to reach 20λ

Desired Endotoxin Amount

λ X Dilution Factor = EU/mL


Setting Appropriate Specifications• Is there a Monograph?• EL= K/M 17

• Using the calculation, 350 EU are allowed for a 70 kg human per hour. – Many companies add an additional safety

factor when calculating endotoxin limits to that ensure consumers are safe.

• Consider all of the contributing sources of endotoxin when calculating endotoxin limits (e.g. water, components, raw materials)1



Overview of the Various AssaysGel-Clot1

• Performed on a heat block at 37°C (+ 1°C).• Standard Curve Series are made with Controlled

Standard Endotoxin (CSE), LRW (LAL Reagent Water) and LAL. The curve covers the range of 2 λ, λ, ½ λ, and ¼ λ. – Lambda is the labeled sensitivity of the LAL reagent.

The curve is used to make sure the reagents are working properly at the desired sensitivity. The standard curve must be positive within a 2-fold dilution of lambda.

– 20λ is used to positive controls.

• Product (or product dilution) is assayed in equal parts with LAL reagent.

• At the completion of a 1 hr (+ 2 minute) incubation at 37°C (+ 1°C), the tubes are inverted 180°. Anything other than a solid clot at the 180° inversion is a negative result. Thus, giving the assay the nickname, “the wet hand test”.





Overview of the Various AssaysKinetic Turbidimetric (KTA)1

• The standard curve typically used is 5 EU/mL, 0.5 EU/mL, and 0.05 EU/mL. Can vary depending on assay and reagents used.

• Lambda is the lowest point on the curve (e.g., 0.5 EU/mL)• 5 EU/mL (highest) dilution is used to create positive

controls• Assay is typically performed at 340 nm wavelength at

~37°C. – The software can be programmed to stop the assay when

lambda has a positive reaction (reaction curve crosses the predetermined optical density).

• The microplate is incubated for a preselected amount of time.

• Readings take place at preselected intervals (such as 30 or 60 seconds) with spectrophotometric light absorbance at suitable wavelengths. Reaction times are determined from these readings. These are often called onset times or optical density.

• The endotoxin result is determined by comparing the reaction time to that of the standard curve.


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Overview of the Various AssaysKinetic Chromogenic (KCA)1

• KCA is similar to KTA in almost every aspect. • A standard curve is prepared and used to determine the results

of the assay, like KTA.• KCA uses a different wavelength to measure the results. • A color cleaving substrate is added to the LAL. When

endotoxin is detected, the substrate reacts and produces a color (typically yellow).

• Results: In order for a KTA or KCA reaction to be valid, the plate must meet the following criteria:

– Linear Correlation Coefficient: r is between –0.98 and –1. The slope of the curve must fall within the range of –0.12 to –0.35. The onset OD value is between 0.03 and 0.2.

– Contamination in the negative control invalidates the entire plate. The negative control onset time is greater than the onset time for the lowest standard.

– The coefficient of variation (C.V.) must be 10% or less between standard replicates for the study to be valid, or <20% between replicates and matched positive product controls (PPC) for the assay to be valid.

– Recovery of the interference control for each unknown (PPC or “hot spike”) must be within 50% to 200% of the specified control level. Failure to achieve recovery invalidates this test for the product, which recover outside of this range.





Overview of the Various Assays

Portable Test System (PTS) or Multi-Cartridge Endotoxin Testing System (MCS)1

• Runs on KCA technology.

• Reagents and controls are lyophilized on the cartridges.

• Only uses 25 μL of product per channel.

• Results in 15 minutes.


https://www.criver.com/sites/default/files/resource-files/Endosafe_Kinetic_Testing_Solutions_Technical_Sheet.pdf

https://www.criver.com/sites/default/files/resource-files/Endosafe_Kinetic_Testing_Solutions_Technical_Sheet.pdf


Method Develop and Validation Tips• Method Development begins with overcoming possible interference

issues.– Inhibition and Enhancement Screens are performed by diluting down to the

MVD or MVC.1

– Inhibition occurs when positive controls that should be positive, give negative results or when the endotoxin amount of the dilution can not be determined. 1

– Enhancement occurs when a positive control is a lot higher than it should be (>200%), or negative products are giving positive results. 1

• Enhancement is difficult to detect in Gel-clot and one must assume the product is positive unless it can be proven otherwise.1

• Inhibition and Enhancement issues are often overcome by dilution, balancing a pH, or eliminating possible Glucan Interference.1

• Recommend to perform experimentation trials in a notebook.



• Method Validation includes choosing an appropriate dilution from the method development.1

– For gel-clot one would ideally choose the 3rd highest positive dilution in the interference screen.1

– For the kinetic assay, one is ideally looking 100 + 25% in the positive controls. 1

– It is also desirable to be a 4-fold dilution away from the endotoxin limit. 1

• 3 lot validations are required for method validation. • At good internal rule is to validate 1 lot for clinical products and 3 lots for

commercial products. Only produce what is needed for the clinic and it may be a long time in between fills. In addition, during the developmental stages of a product, the formulation may change multiple times.

• The compendia requirements must be met. pH must be documented to be between 6-8. If the pH was adjusted during validation, it must be adjusted during routine testing.1



• For Gel-clot 17

– A product standard series is made in the product dilution (2λ, λ, ½ λ, ¼ λ) 17

– There are 4 replicates for each product dilution17

– There are a minimum of 2 replicates for the standard series. 17


Product Standard Series

Standard Series in LRW


• For KTA and KCA 17

– A normal standard series is made (e.g., 5 EU/mL, 0.5 EU/mL, and 0.05 EU/mL)

– Product dilutions are inoculated to the equal the middle point of the curve. 17



Common causes of invalid endotoxin assays

• Invalid assays are different than OOS results14

• Invalid results may require an investigation into the cause of invalidity. 14

• Common causes of invalidity include:14

– Pipetting errors14

– Incorrect well selection14

– Subpotent endotoxin standards (particularly the lowest endotoxin concentration.) 14

– Dilution errors14



Summary

Review Time

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• “The Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).”1

• Manufacturer’s try to take measures to preserve the future of the horseshoe crabs.

• There are regulations governing BET and warning letters and 483 observations have been written.

Summary


• The test is carried out in a manner that avoids endotoxin contamination. All materials for the test must be pyrogen free. 1

• There are three main techniques for this test: – Gel-clot technique– Kinetic turbidimetric (KTA)– Kinetic chromogenic technique (KCA)

• Any method can be used for the test. But, in the event of doubt or dispute, the final decision is made based upon the gel-clot limit test unless otherwise indicated in the monograph for the product being tested. 17

• There are three primary reagents required for this test– Endotoxin free water (also known as limulus amoebocyte reagent water or LRW)– Limulus Amoebocyte Lysate (LAL)– Controlled Standard Endotoxin (CSE)

• Follow the instructions on the manufacturer’s CoA• Endotoxin limits can be calculated with the equation K/M where K is 5 USP-

EU/kg of body weight for any route of administration other than intrathecal route and M is the maximum dose per kg of body weight per hour. 17

Summary


• λ: the labeled sensitivity in the Gel-Clot Technique (EU/mL) or the lowest concentration used in the standard curve for the Turbidimetric Technique or Chromogenic Technique. 1

• MVD= the maximum allowable dilution of a specimen at which the endotoxin limit can be determined. 1

• MVC= the minimum concentration at which a product can be tested and the endotoxin limit be determined. 1

• To ensure the test works as intended:1

– The reagents must be qualified

– The analyst must be trained

– The methods must be validated.

Summary


• Validation testing is typically performed in two parts: Interference Screening (Inhibition and Enhancement) and the Validation Screening. 1

• Validations are performed on 3 separate lots of product.

• For clinical products, one lot of product tested 3 times may be sufficient temporarily.

• The pH of the testing dilution with LAL should be in a neutral range of 6-8. 17

Summary


Common causes of invalid endotoxin assays

• Invalid assays are different than OOS results

• Invalid results may require an investigation into the cause of invalidity.

• Common causes of invalidity include:14

– Pipetting errors14

– Incorrect well selection14

– Subpotent endotoxin standards (particularly the lowest endotoxin concentration.) 14

– Dilution errors14

Summary


References1. Charles River Endosafe Workshop, August 2008

2. Code of Federal Regulations (CFR) Title 21: Food and Drugs. Accessed on 13Mar2021 at

https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm

3. Dawson, M. (1993) LAL Update. Endotoxin Standards and CSE Potency. Accessed on 13Mar2021.

4. Dawson, M. (1995) “Endotoxin Limits” LAL Update, Volume 13, No. 2. Associates of Cape Cod, Inc. Woods Hole, Massachusetts.

5. Dubczak, J. (2008) "From Horseshoe Crabs to LAL Testing" Charles River LAL Workshop, Charleston, SC. 19Aug2008. Lecture.

6. Endotoxin. Medical Dictionary. Accessed on 08Feb2021 at https://medical-dictionary.thefreedictionary.com/Endotoxins

7. European Pharmacopeia (EP) 2.6.14 Bacterial Endotoxins

8. Food and Drug Administration (FDA) (2012) FDA Guidance for the Industry: Pyrogens and Endotoxins Testing: Question and Answers, June 2012, Food and Drug Administration, Rockville, MD, USA.

9. Food and Drug Administration (FDA) Warning Letters. Accessed on 13Mar2021 at https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/compliance-actions-and-activities/warning-letters

10. Fujifilm (2014) The Impact of Endotoxin on the Human Body. Accessed on 13Mar2021 at https://www.wakopyrostar.com/blog/post/the-impact-of-endotoxin-on-the-human-body/

11. Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic

Cell Therapy Investigational New Drug Applications (INDs) (2008). Accessed on 13Mar2021 at https://www.fda.gov/media/73624/download

12. Japanese Pharmacopeia (JP) 4.01 Bacterial Endotoxins Test

13. Pyrogen. Medical Dictionary. Accessed on 08Feb2021 at https://medical-dictionary.thefreedictionary.com/Pyrogens

14. Schultz, J. (2008) "Testing Invalidities and Stage One OOS Laboratory Investigation" Charles River LAL Workshop, Charleston, SC. 19Aug2008. Lecture.

15. The Horseshoe Crab (2013). Accessed on 13Mar2021 at https://horseshoecrab.org/

16. Tirumalai, Radhakrishna Ph.D (2016) Naturally Occurring Endotoxin: A New Reference Material Proposed By the US Pharmacopeia | American Pharmaceutical Review. Accessed on 13Mar2021 at https://www.americanpharmaceuticalreview.com/Featured-Articles/190860-Naturally-Occurring-Endotoxin-A-New-Reference-Material-Proposed-By-the-US-Pharmacopeia/

17. United States Pharmacopeia (USP) <85> Bacterial Endotoxins Test

18. WCG FDA News. Form 483s Database. Accessed on 13Mar2021 at https://www.fdanews.com/form483


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