What is the role of LAL, LRW and CSE in Bactiral Endotoxin Testing?

LAL :

• Limulus Amoebocyte Lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the Atlantic horseshoe crab, Limulus Polyphemus.
• Amoebocyte acts coagulation mechanism.
• Anti aggregation agent must be added to inhibit the aggregation N-Ethylmaleimade.
• LAL mainly used for detection and quantification of bacterial endotoxins.
• LAL reacts with bacterial endotoxin lipopolysaccharide(LPS), which is a membrane component of Gram negative bacteria.
• Frederick Bang reported in 1956 that Gram negative bacteria, even if killed will cause the blood of the horseshoe crab to turn in to a semi-solid mass.
• Amoebocytes contains granules with a clotting factor known as coagulogen.
• In 1970 the U.S. Food and Drug Administration (FDA) approved LAL for testing drugs, product and devices that come in contact with the blood. prior to that date, much slower and more expensive tests on rabbit test  had been used for this purpose.
• LAL test useful of identification and detection of endotoxins.

How to get the LAL?

• Collect big size of healthy horseshoe crabs.
• Remove  the blood by using of Syringe.
• Separate the white blood cells (serum) consist of Amoebocyte by using centrifugation.
• Amoebocytes is then freeze- dried for further process.
• Horseshoe crab (only blood removed) blood doner.
• 500 mL blood (serum) = 10 ng/vial 
• LAL made of Amoebocyte/Gametocyte of L-polyphemus 1.5 % v/v of 25 % human serum albumin (stabilizer), 3 % Nacl and other appropriate ions.
• LAL have ( sodium ions, magnesium ions) buffer for automatic pH adjustment purpose.

LAL Reagent:

• different sensitivity of the  Limulus Amoebocyte Lysate (LAL) available in market.
• Choose one of the following LAL reagent (0.25, 0.125, 0.0625, 0.03125, 0.015625 EU/mL).
• Always LAL reagent detect on endotoxins above on  LAL sensitivity. (Example 0.25 EU/mL Lysate TS (Limulus Amoebocyte Lysate Test Solution) sensitivity detect on above 0.25 EU/mL only, not detected on 0.03 EU/mL) 
• Re hydrate immediately prior to testing ( 5 minutes to allow to come to room temperature and dissolve).
  
• Do not vortex Limulus Amoebocyte Lysate, because Lysate have sensitivity protein that protein is break down.
• Cover with an endotoxin -free surface if not in use.
• Stored the LAL recommended as per manufactures.

LAL Reagent Water (LRW):

• LAL Reagent Water (LRW) is sterile purified water prepared by distillation or reverse osmosis.
• LAL Reagent Water (LRW) is equivalent to water for bacterial endotoxin test.
• Its contains <0.005 EU/ml Endotoxin(Charles river),<0.001 EU/ml Endotoxin(cape cod).

Controlled Standard Endotoxin (CSE) Reagent:

• Re-hydrate  following lot specific EU/ng ratio.
• Vortex vial for 5 minutes( As for manufactures recommendation)the first use and 1 minute for subsequent. usage.
• Vortex each dilution for 30 seconds and use dilutions as soon as possible.
• Storage of dilutions is only permitted based upon stability studies.
• Store Re-hydrate vial in refrigerator not the freezer.
• US Reference Standard Endotoxin (RSE) E.Coli -  O113 : H10 : K0

Combination of LAL and CSE:

• While LAL/ CSE Lots Remained the same Reaction times for each standard changed (verify the Co-A).
• CSE is freeze dried powder is available 10 EU/ng to 1000 EU/ng ampoules.
• CSE is intended for use to construct standard curves.
• This test use in-vitro laboratory purpose only, not for use in human and animals.
• The potency of CSE is referenced against the USP Referenced standard Endotoxin (RSE).

EU/ng Ratio:

• Referenced standard Endotoxin (RSE)------- convert------Controlled Standard Endo toxin (CSE).
• EU (Endotoxin Units)  = IU ( International Units).

EU/ng    X      ng/Vial   = EU/vial

EU/vial / mL vial  = EU/mL

Example : 1 

RSE = 10 EU/ng       Potency  = 10ng/Vial

=   10 EU/ng   X  10 ng/Vial   = 100 EU/Vial

=100 EU/Vial/ 5 mL of Vial  = 20 EU/mL

Example : 2

CSE = 0.5 ug/vial   (0.5 ug/vial x 1000 =500 ng/vial)      Potency : 14 EU/ng

 = 14 EU/ng x 500 ng/vial = 7000 EU/vial 

=    7000 EU/ml/ 5 ml LRW= 1400 EU/ml 

USP Reference to Glucans:

• Amoebocyte Lysate reacts to some beta -Glucans in addition to endotoxins. Amoebocyte Lysate preparation that do not react to glucans are available. They are prepared by removing the G-Factor reacting to glucans from Amoebocyte Lysate or by inhibiting the G-Factor reacting system of Amoebocyte Lysate and may used for endotoxin testing in the presence of glucans.
• Only (1-3) β-D Glucans have high reactivity with LAL.
• Because most  β-D Glucans are not soluble in water, some are soluble in alkaline solution (NaOH), DMSO.
• Most glucans interference can be eliminated using ES- Buffer to re-hydrate the LAL reagent.
• Use ES-Buffer containing β-D Glucans blocker.

Dilutions:

• Dilutions can be calculated as follows.
  
  
  Dilutions =   What we have  / What we want
  
  
  Example :  Need 2 EU/mL from 20 EU/mL tube make an 10 fold dilution.
  
  
  = 20 EU/mL / 2 EU/mL  
  
  
  = 10 Dilution
  
  
  1mL of sample  add 9 mL of diluent (LRW) this dilution is called 10 fold dilutions.
  
  

  

  
  

  Dilutions Verification:

   = Total Volume of the Solution ( Sample + Diluent) / Volume of the sample
  
  
  = 1 ml + 9mL  / 1     = 10
  
  
• Related: Gel- Clot Test

Gel- Clot Test

• 1977 FDA grants approval of first commercial LAL (Limulus  Amybiocyte Lysate) reagent.
• Gel-clot technique based on the gel formation.
  
  
  
• Kinetic turbimetric based on the turbidity.
• Kinetic chromogenic based on the colour formation.
• "Frederick Bag" Gel-clot method is developed.
• Gel-clot method is pass or fail test.
• LAL (Limulus  Amybiocyte Lysate) + Endotoxin  = Clotting
• LAL test is in-vitro test for semi quantitative analysis of endotoxin ( it means given the results less than or more than of specified quantity).

Apparatus:

• Depyrogenated dilution tubes(16 X 100 mm)

·         Depyroginated Assay tubes(10 X 75 mm), (polystyrene test tubes)

·         Calibrated Micropipettes.

·         Dipyrogenated Tips( Endotoxin free pipette tips)

·         pH Indicator

·         calibrated stop watch

Equipment:

Calibrated Heating Block.

Materials:

·         CSE (Controlled Standard Endotoxin)

·         LAL ( Limulus  Amybiocyte Lysate)

·         LRW ( LAL Reagent Water)

·         Test sample

Sample Preparation :

• Dispense 100 µL of sample and 100 µL of LAL  in to 10 x 75 mm assay tubes ( Endotoxin free dilution tubes- Borosilicate glass or equivalent  polystyrene plastic).

•  Mix the sample solution.

pH Checking:

·         The LAL reaction requires a neutral pH and is time and concentration dependent.

·         Sample and Lysate mixture take with the help of the depyrogenated tip  (Measure pH with one to one ratio of sample dilution and LAL to obtain an accurate pH reading).

·         Put the one drop on pH indicator

·         Read the pH

·         Lysate having some pH adjustment buffers .

·         Not maintain in the pH , please add the sterile 0.1N Tri-NaoH, 0.1 NTri-Hcl.(Tri sodiumhydroxide, tri hydoxy chloride)

·         The combination of LAL and prepared product will have a neutral pH 6-8.    

   

Observation:

·         Temperature 37°C ±1°C for 60 ± 2 min avoiding vibrations.

·         Check the result 180° smoothly & gently

·         Check the PPC results inverse position.

Select Lambda ( λ ) 

• Labelled LAL sensitivity.
• Choose one of the following LAL reagents (0.25 EU/mL,  0.125 EU/mL, 0.0625 EU/mL, 0.03125 EU/mL and 0.015625 EU/mL)
• Work in 2 fold dilutions for gel-clot tests.
• Run standard series from 2λ, λ, λ/2 and λ/4.
• 2λ - must always clot.
• λ/4 - must never clot.
• Negative controls must not clot.

PPC preparation 2 methods:

1. Hot Spike method :

In reaction Test tube 0.01 mL of 20λ + 0.1 mL (100 µL)of test Solution  + 0.1 mL(100 µL) of LAL.

 

2. 50/50 method or Half strength method  or Double Strength method:

• Use equal volumes of 4λ and sample.
• Sample should be 2x desired dilution/concentration.
• Accounts for 1: 2 dilution.
• It means 50 µL of 4λ + 50 µL of test solution + 100 µL LAL.

Example : if want 500 dilution,

you can do 250 dilution, take 250 dilution of 50 µL sample and 50 µL LRW.

this dilution equal to 500 Dilution.

Routine Test Control:

• Each test has four controls.

1. Negative Water Control (NWC)
2. Positive Water Control (PWC)
3. Negative Product Control (NPC)
4. Positive Product Control (PPC)

Results observation:

End point sought by 180° inversion

positive =  firm gel

Negative = anything other than firm gel.

Related : Pyrogen test ( Rabbit Test or Sham Test)

What is the Lipopolysaccharide (LPS) ?

• Lipopolysaccharide (LPS) also known as Lipoglycon and Endotoxin. 
• It consist of Lipid and Polyacrylamide composite, Lipid-A and core polyacrylamide of O-Antigen or O-side  chain.
• As  in peptidoglycan biosynthesis LPS molecules are assembled at the plasma or inner membrane.
  
  
  
  
  
• Outer core and inner core joined by covalent bond they are found in outer membrane of Gram negative bacteria.
• Lipid A contains endotoxin and sugars such as heptose.
• Lipopolysaccharide (LPS) is also Exogenous Pyrogen ( external fever inducing substances).
• Lipid A more than 99.9% of pyrogen activity.
• Lipid A composed of B-1, 6-glycosamine disaccharide units with aminohydrogens and fatty acids.
• Single Bacterial cell has been estimated to contain about 3.5 million Lipopolysaccharide (LPS) molecules.
• Lipopolysaccharide (LPS) is always toxic and pyrogenic and also causes endotoxic shock.
• Lipopolysaccharide (LPS) layer also called the outer membrane is the outer most layer present in the cell wall of gram negative bacteria. It is a characteristics feature of gram negative bacteria.
• Exception : some Gram positive bacteria, Ex : Listeria monocyotogenes has been found to contain an authentic lipopolysaccharide.Bacillus thuringenesis, Bacillus cereus and Bacillus anthrax are Gram positive bacteria, These bacteria are capable of produce crystal proteins ( proteinaceous inclusions) called delta endotoxins, that have insecticide action. 
• Lipopolysaccharide (LPS) is extremely heat stable and pH stable, these are pass the 0.2 um membrane filter.

Related : peptidoglycan

Why are using Seed lot Techniques for microbial culture?

• Seed lot Technique is recommended for storage of the microbial culture for long time.
• As per USP general chapter 51 ( Antimicrobial effectiveness Testing) and 1117 (Microbiological best laboratory practices) seed lot techniques is better for maintain the reference cultures (master seed) in the laboratory.
• Seed lot technique used to avoid the microbial contamination  and cross contamination.
• To reduce the possibility of phenotypic variation, genetic drifts, mutations and contamination as much as possibles the number of passages  should be minimized ( five passages).
• Seed lot technique to prevent  the unwanted drift of properties which might ensure from repeated subcultures or multiple generation.
• Seed lot technique reference strain is sub cultured to several replicates at one time, all of which are with one passage( seed stock is sub cultured).
  
• Stock cultures can be sub cultured for reference cultures and working cultures, Reference cultures monthly basis  and working cultures weekly basis.
• Frozen cultures should be stored at -30 degree C or below, until use. If stored at -70 degree C or below in lyophilized form. Slants may be stored at 2 to 8 degree C for up to a week.
  

Related : What is the meaning of passage? how many passages are acceptable in the laboratory?

What is the meaning of passage and how many passages are acceptable in the laboratory?

What is the Meaning of passage?

• Whenever viable organisms growth on fresh medium is called passage (transfer of organisms grow on fresh medium), either on solid agar or in broth.
• Sub-culturing is considered to be a transfer/ passage.
• Freeze-dried cultures by thawing or rehydrating is not a passage. 
• It means solid media to liquid media or liquid media to semi solid media transfer not a passage, every transfer of organisms grow on fresh medium is called passage. It means new generation.
• Every organism multiplication (generation) some time required, at least that time your providing for organism multiplication (generation) is called passage.
• The USP recommends Seed lot system is better for microbial culture maintenance.
• Microbial cultures are commercially available. 

Refer : Microbial culture collection

• Cultures are used in performance testing of products, as possitive and negative controls, as indicator organisms and identification standards.
• Culture are called different names control strains, standard cultures, reference strains, test strains and quality control strains. These terms are generally be used interchangeablly, though the performance seems to be reference strains or reference culture.

How many Passages are acceptable in the laboratory?

• As per USP general chapter 51 (Antimicrobial Effectiveness Testing) says, The viable microorganisms used in the test must not more than five passage removed from the original strain.
• Because increasing passages (more than five passages) are cause to phenotypic variations, genetic drift, mutation and risk of contamination.
• Frozen cultures should be stored at -30 degree C  or below, until use. If stored at -70 degree C or below in lyophilized form. Slants may be stored at 2 to 8 degree C for up  to a week.

Related : Seed lot technique

Restricted Access Barrier System (RABs)

• Restricted Access Barrier System ( RABs) for critical area protection .
• This RABs used for higher grade of protection of the working area already fitted with a simple Laminar Air Flow surrounded by two portable straps.
• The RABs allowed a positive pressure level of 15 Pa to be reached, dramatically increasing aseptic condition of the critical area contained.
  
• RABs provides a physical barrier between workers and production areas.
• RABs are becoming increasingly popular within aseptic processing, the reason being that they offer effective product protection by providing a high level of separation between operations and the critical aseptic core.
• RABs will provide ISO 5 class Laminar Air Flow with an ISO 7 background.
  

1. A barrier to prevent human intervention directly in to the critical zone.
2. Airflow for an ISO 5 at least in the critical zone.
3. Glove ports and transfer ports used for interventions.
4. High level disinfection.
5. Highly automated process and well-defined procedures for rare open door interventions.

Related: Isolator Technology

Isolator Decontamination cycle verification

• Decontamination is function, verify the decontamination process correct or not.
• Different decontamination methods can be used to eliminate bio burden from isolator system and supplies.
• Decontamination purpose chemicals used to treat isolators are Peracetic acid, chlorine dioxide, ozone and hydrogen peroxide (H2O2).
• Decontamination process  inside the temperature of isolator also important, particularly for hydrogen peroxide vapor decontamination.
• Verify the concentration and distribution of the decontamination chemicals. when applied in gaseous or vapor form, the distribution may be evaluated using chemical indicators, spectroscopic methods or electronic sensors.
• Gas and vapor decontamination methods may require fans in the isolator to distribute the chemical evenly.
• If the isolator utilizes a recirculating unidirectional air flow system, distribution fans may not be required, but this should be evaluated on a case-by-case basis.
• Distribution checks are done with the isolator fully loaded with equipment and supplies and setup of these units is defined and documented.
• After decontamination, decontamination agents need to be removed from the isolator. Aeration, open loop and external exhaust system is major role decontamination agents removing process.
• Verification process check the chemical indicators and biological indicators.
• If required after exposure of Biological indicators verify the fraction negative or total kill analysis method. This process at least 3 log reduction kills is conformed in three consecutive validation studies.
• The operator establishes a frequency for re-decontamination of the isolator. The frequency may be as short as a few days on as long several weeks, depending on the maintenance effort. 
• Decontamination agent does not adversely effect on product containers (prove as per validation).
• The ability of the isolator system to maintain an aseptic environment throughout the defined operational period must be validated.
• Microbiology monitoring programme ususally a routine sampling verify the decontamination process.

Related : Isolator Technology

What is importance Isolator integrity test or Isolator Chamber leak test?

• Is also called glove integrity testing system.
• Gloves or half-suits is the most critical parts of an isolator.
• Is used to test glove integrity in conformity with the pressure decay method ISO 14644-7, Annex E.5(test in positive pressure).
• The glove testing unit only needs compressed air supply used to blow the gloves.
• This leak test of an isolator chamber using a dedicated glove flange cover equipped with a differential pressure transmitter and temperature sensor.
• In isolator technology the loss of glove integrity or accidental introduction of material that has not been decontaminated are among the most probable causes of detectable microbial contamination.
• Very small leaks in glove are difficult to detect until the glove is
  stretched during use.
• they are several commercially available glove leak test  detectors.
  
• Continuous nonviable particle monitoring within the isolator enclosure is ideal, because it can quickly detect filter failure.
• Viable monitoring results take a more time.
• A pair of sterile gloves is frequently worn under the isolator gloves.
•  Isolator glove can provide a additional level of safety against glove leaks or can act as hygienic measure.

Related : Isolator Technology

What is the uses of isolator technology ?

• Isolator  technology used since the mid-1980s.
• In recent years, isolator technology has found a broad acceptance in healthcare manufacturing.
• Isolators are very useful in sterile manufacturing where microbial contamination and cross contamination is a major problem.
• Contaminated drugs have caused grave (Ex. permanent injury, death ) consequence for the consumer.
• Isolator system protect during handling direct contact between the analyst and test articles.
• Isolator are constructed of flexible plastics (Such as polyvinyl chloride) rigid plastics, glass or stainless steel.
• Aseptic manipulations within the isolator are made with half-suits or gloves and sleeves.
• Pharmaceutical industry compounding isolator are used for maintaining sterility of the product.
• Isolators are routinely found with the pharmaceutical industry and are widely used in aseptic compounding applications.
• Air velocity and changes are far less important in isolator or closed RABs than in clean rooms because personnel are more carefully separated from the product, product containers and closures.
• Total particulate counting in an isolator is likely to provide an immediate of changes in contamination levels.
• Isoloaters provide a working space that is detached from the surrounding environment.
•  Positive pressure isolator is most common, negative pressure isolator used in  handle of toxic product.
  
• Enclosed isolator are always positively pressurized units with High Efficiency Particulate Air (HEPA) filters that do supply ISO 5 airflow in a unidirectional or turbulent manner to the interior.
• Air is generally re-circulated and cleaning may be automated or manual.
• Isolator are designed to provide continuous and complete isolation of the inside of the isolator from the external  room environment, only installed gloves or robotic arms are used to manipulate the product.
• This ensures that the environment is maintained as contamination-free to safeguard patients who will later be administrated the drug. 
• Isolator system microbial monitoring is much less important in establishing their efficiency in elimination bio burden. 
• As per USP general chapter 1116  RABs, clean rooms aseptic air sampling frequency each operational shift, but isolator air sampling frequency once per day.
• Bio-decomposition occurs via an automated cycle that generally utilizes Vaporized Hydrogen Peroxide (H2O2).
• The interior of the isolator is treated with sporicidial chemicals that result in the elimination of all viable bio burden on exposed surfaces.
• Isolator work operation not required to wear sterilized clean room gown.
• A pair of sterile gloves is frequently worn under the isolator gloves.
• A second glove worn under isolater, can provide an additional level of safety against glove leaks or can act as a hygienic measure.
• Isolator technology should be significantly, lower sill and can be expected to be <0.1% on the basis of published monitoring results.
• One isolator to another isolator material transfer purpose Rapid transfer port (RTP's) are available.
• RTP gaskets disinfectant frequently and changed at the recommended frequency and periodically checked for damage.

Related : Isolator Decontamination cycle verification

Sterility Test Isolator:

• Sterility test isolator, which are generally a range of two glove and four glove isolator.
• They are specifically built for sterility testing and are designed for sterility testing in pharmaceutical products.
• Sterility isolator mainly used for avoid the cross contamination of product testing.
• Sterility isolator give the results  most accurate.

Two types of Isolaters available.

1. Closed isolators: Which are systems with no direct opening to the external environment, are normally used for maintained the sterility of the product.
2. Opened isolators: Which allow the egress of material through a defined openings that precludes the entry of contamination by means of air over pressure may be used.

• Closed isolators and opened isolators both are maintained positive pressure relative to the surrounding environment, and over pressure of 20 Pa or more are typical.
• The user should never exceed the maximum pressure recommended by the isolator manufacturer. 

Related : Isolator Integrity test or Isolator chamber leak test

Different stages used in Isolator technology:

1. Sterility testing isolator
2. Isolator for API dispensing and Aseptic operation
3. Isolator for Dispensing & Weighing 
4. Six stage research and development isolator
5. Isolator for Dispensing & milling
6. Isolator for Compact mixer -Granulation dryer 
7. Isolator for Deduster and metal check
8. Bottle filling and vial filling machine isolator
9. Isolator for cytotoxic manipulation.