American Pharmaceutical Review – Endotoxin supplement http://www.gibraltarlabsinc.com/endotoxin-testing.html

Selma Tarcan & Alvan Chao

Proprietary and Confidential © Gibraltar Laboratories, Inc.

American Pharmaceutical Review

Endotoxin Supplement 2015

Low Endotoxin Recovery, Sterile Products,

Endotoxin Detection, Rapid Methods,

Contamination Control

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Lecture AGENDA Our course of discussion for today’s presentation

• General Information & History

• Definitions of endotoxin

• Detection Methods

• Rapid Methods

• Low Endotoxin Recovery

• Importance of Sterile Manufacturing

• Contamination Control

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Endotoxin Purpose and History of Endotoxin Testing

• Where do Endotoxins come from ? Endotoxin contamination can occur through water, raw materials, media, equipment, containers used

in the manufacturing.

• Manufacturing process must be microbiologically controlled to reduce and remove endotoxins by monitoring raw materials and in process

intermediates at critical steps.

• Why do we perform endotoxin testing? Code of Federal Regulations 21CFR 211.167(a) requires that any drug product claimed to be

sterile and non-pyrogenic be tested prior to release!

• The necessity to ensure that parenteral products are free from heat stable endotoxins and other pyrogens was recognized during World

War II when the need for intravenous fluids was huge.

• At the time the safety was ensured by conducting Rabbit Pyrogen Test (Florence Seribert) RPT became part of USP in 1942.

• LAL (Limulus amebocyte lysate) is an aqueous extract of blood cells from the horseshoe crab, developed by Frederick Bang and Jack

Levin found that amebocytes of horseshoe crab will clot in the presence of endotoxins.

• LAL did not completely replaced RPT because of interferences with the LAL assay.

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Endotoxin What are endotoxins?

• Components of the outer cell membranes of Gram-negative bacteria.

• Shed as part of the normal bacterial life cycle or by other processes that disrupt cells

• Contaminants in pharmaceutical raw materials, water systems, in process samples, and finished products.

• They are the major contributors to the pyrogenic response observed with contaminated pharmaceutical products

• Administration of parenteral products contaminated with pyrogens including LPS can lead to development of fever, induction of

inflammatory response, shock, organ failure and death in humans.

• Endotoxins contain lipopolysaccharide (LPS) molecules surrounded by surface proteins, lipoproteins and phospholipids.

• Lipopolysaccharides – Biologically active component of the endotoxin complex

• Located in the outer cell membrane of gram negative bacteria. • Composed of three regions

• Lipid A: Active toxic portion. This region important for maintaining structural integrity.

• Core oligosaccharide: less heterogeneous and conserved. Inner core and outer core.

• O-antigen: consists of repeating units of glycosyl residue and structure varies among different bacterial strands.

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Endotoxin Purpose, Test Method, and Definitions

• Naturally Occurring Endotoxin (NOE)

• NOEs are environmental, shedding from Gram negative bacteria.

• Control Standard Endotoxin (CSE)/Reference Standard Endotoxin (RSE)

• RSE preparation supplied by the FDA

• RSE and CSE are used as calibration and test standards in BET.

• RSE and CSE are purified LPS and are distinguished from Natural Endotoxins.

• CSE (Control Standard Endotoxin) is a commercially prepared lyophilized endotoxin and its potency is

determined against RSE.

• Contains stabilizers like human serum albumin, Polyethylene Glycol, and starch

• CSE/RSE VS Endotoxin

• CSE and Endotoxin not considered to be the same. CSE – purified LPS

• LPS and endotoxin are physically, chemically and structurally different.

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Endotoxin Test Methods - Bacterial Endotoxin Test (BET)

• Bacterial Endotoxin Test (BET) methods used:

• [USP <85> USP <151> USP <161>

• Gel-Clot method – qualitative method based on the formation of clotting of the test solution in presence of endotoxins

• Kinetic Chromogenic Method – quantitative method based on the color change of the reaction in the final test solution.

Measures the enzymatic reaction between bacterial endotoxin and the white blood cells of the horseshoe crab.

• Test compares sample or sample extract to standards prepared from Control Standard Endotoxin (CSE)

• Kinetic Chromogenic Test conducted by:

• Sample – test sample solution added to LAL reagent

• Positive Control - sample solution spiked with known amounts of CSE and added to LAL reagent.

• PPC Spike must recover 50-200% of CSE added

• <50% = Inhibition

• >200% = Enhancement

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Endotoxin Test Methods - Bacterial Endotoxin Test (BET)

• As per USP <85> samples are prepared to “non interfering state” (dilution) where a nominal level of CSE activity can be quantitatively

recovered. (Inhibition/Enhancement or Validation)

• There is no requirement that CSE be recovered in undiluted products!

• Product interferences with LAL assay are known to occur as it as a highly sensitive test.

• Inhibition

• added known spike of CSE is “under estimated” activity is masked or diminished.

• Enhancement

• added known spike of CSE is “over estimated”

• Reagents used to overcome Inhibition/Enhancement: B-glucan blockers, cationic dispersing agents, divalent cations, heating.

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Endotoxin Purpose, Test Method, and Definitions

• Low Endotoxin Recovery (LER)

• LER: “masking effect” caused by decline in the LAL reactivity to CSE after its addition to the test material.

• Is temperature dependent

• Primarily focus on Undiluted products

• Can be distinguished from interference commonly seen with the LAL assay that is normally overcome by dilution or other sample

preparation • What causes LER?

• Precise mechanism of the LER matrix interference unknown but according to number of theories by industry researches there are

combination of factors that can cause LER.

• Polysorbate that is found in many protein formulations.

• Chelators

• Ionic nature of the protein

• Inhibition/Enhancement can be due to adsorption or aggregation, cation concentration, proteins.

FDA take on LER – Recommends hold time studies. FDA Q&A 2012 #3 “Firms should establish procedures for storing and

validating (which includes product mixing) samples of bacterial endotoxin analysis using laboratory data to demonstrate the

stability of assayable endotoxin content. Protocols should consider the source of endotoxin used in the study, bearing in mind that

endotoxins might react differently from native sources of endotoxins.

Why worry about LER? – contamination can occur during manufacturing process and finished product may contain endotoxins

(residual from the contamination) but if they cannot be detected by test methods, it can be a health and safety concern.

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Endotoxin Test Methods

• Rabbit Pyrogen Test (RPT)

• RPT commonly used to detect endotoxin in products that is unable to be detected by LAL

method.

• in vivo [USP <151>] used to detect endotoxin in pharmaceutical products by monitoring the

increase in temperature or a fever following the intravenous injection of a test solution and is

designed for products that can be tolerated by the test rabbit in a dose not to exceed 10mL /kg

injected intravenously within a period of NMT 10 minutes.

• Limitations with RPT due to large number of use of animals for testing, continue to encourage

new methods.

• Rabbits may develop tolerance to endotoxin

• RPT may be waived if any other method demonstrated to be equivalent.

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Endotoxin Alternative Methods – What other methods?

• Recombinant Factor C – delivers same reliability as LAL

• Activation of the first component in the LAL cascade

• No animal resources

• No false positive reactions from beta glucans

• Monocyte activation test (MAT).

• Alternative to rabbit pyrogen test

• Can test for

• Gram Negative

• Gram Positive

• Other biological pyrogens (eg yeast)

• Parasitic pyrogens

• Viral Pyrogens

• Uses cryopreserved human blood as source of monocytes (immune response cells)

• Monocytes recognize pyrogens and respond by releasing fever-inducing signal molecules

• Fever inducing molecules detected by ELISA.

• Eliminates interference from turbidity, color, or clotting

• Rapid Detection Methods

• Reduce cost, ease of use, time.

• Optical, Electrochemical, and mass based biosensors

• Still in Research and development

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Ph. Eur Sterile Products guidance Future Update

• European guidelines under review – to revise annex 1 on manufacturing of sterile products

• Sterile manufacturing of pharmaceutical medicines plays an important role for microbiological contamination.

• Main risk concerns:

• Viable microorganisms

• Particulate matter

• Pyrogens (including bacterial endotoxin)

• Minimize risks

• Sterility achieved through

• Protective controls

• Good manufacturing practice

• Skill, training, and attitudes of personnel important.

• Quality assurance – to make sure carefully established and validated methods are strictly followed.

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Water for Pharmaceutical Use US vs Ph. Eur – European guidelines under review

• USP vs Ph. Eur WFI water systems

• USP: WFI can be produced using either Distillation or Reverse Osmosis.

• Ph. Eur only permits Distillation to be used. May allow both after update.

• Distillation: process where component substances from a liquid mixture are separated

through the combination of selective evaporation and condensation

• Good for removing endotoxin

• Reverse Osmosis: uses a semi-permeable membrane to remove larger particles from

water through the application of an applied pressure.

• RO used to be less efficient

• Water for Pharmaceutical use typically Water for Injection

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Water Water quality and usage

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Bioburden Contamination Control Pharmaceuticals, medical devices and personal care product manufacturing

• To minimize product contamination and to monitor the environments within such

products are produced.

• All aspects of the operation should be designed to establish, implement and maintain

a quality system that ensures the delivery of pharmaceutical and health care products. • Facilities

• Equipment

• Personnel

• Monitoring incoming raw materials

• Water systems

• Sanitizers

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Matrix Overview of contamination control

Validation Control Monitoring

Facility Qualification of Clean

Room area and HVAC

system

Maintenance of facilities sanitization,

Revision of Barriers, Traffic Patterns, or

Air Balance

Environmental

Monitoring (EM)

HVAC Qualification of the Clean

Room area and HVAC

System

Certification and Preventative

Maintenance (PM) of System, Repair of

HEPA Filters

EM

Water Qualification of Water

System

Certification and PM Regular

Sanitization of System

Bioburden Monitoring

of Water System

Equipment Qualification of the

Equipment as Suitable for

its Intended Use

Certification and PM Regular

Sanitization

EM, Finished Product

Release Testing

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Matrix Overview of contamination control

Validation Control Monitoring

Sanitization Validation of Cleaning, Sanitization

and Sporicidal Treatments

Regular cleaning and

Sanitization of facilities and

equipment

EM

Personnel Proficiency criteria, participation in

media fills, trending data by

operator

Training Discipline

Personnel Monitoring

Trending Data by

Operator

Process

Process Validation Acceptance Testing of Raw

Materials and Containers

In-Process Bioburden

Monitoring

Finished Product

Release Testing

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Conclusion Overview of contamination control

• Some thoughts do not reflect those of FDA or other regulation

• LER is a concern of FDA and Biologics License Applications

• No cases associated with endotoxin contamination since 1987.

• Further research needed for LER, Rapid endotoxin, new technologies

• Guidance's under review

• Process controls for Endotoxin

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