Hyglos endotoxin detection presentations and visit our trade show booths during 2014

This year Hyglos celebrates 5 successful years as a company dedicated to the Research & Development for improved endotoxin detection!

Attend to Hyglos scientific presentations and visit our trade show booths during 2014:

 

PDA Europe Pre-Conference Workshop on Bacterial Endotoxin Testing with a focus on Biopharmaceuticals - moderated by Dr. Wolfgang Mutter, Hyglos
February 17, Berlin (Germany)

PDA Europe Conference on Pharmaceutical Microbiology 2014
February 18-19, Berlin (Germany)

Aktuelle Trends bei Endotoxin- und Pyrogentests
March 26-27, Mannheim (Germany)

analytica
April 1-4, Munich (Germany)

PDA Annual Meeting
April 7-9, San Antonio (USA)

Bio Korea 2014
May 28-30, Kintex (Korea)

SIMPOSIO AFI 2014
June 11-13, Rimini (Italy)

BIO International Convention
June 23-26, San Diego (USA)

The Bioprocessing Summit 2014
August 18-22, Boston (USA)

Scanlab 2014
September 9-11, Copenhagen (Denmark)

aseptikon
September 30-October 1, Frankfurt (Germany)

BioJapan 2014
October 15-17, Yokohama (Japan)

PDA 9th Annual Global Conference on Pharmaceutical Microbiology
October 20-22, Bethesda (USA)

PharmaLab 2014
November 14-15, Düsseldorf (Germany)

 

Innovative Endotoxin Removal in Biomanufacturing Processes

EndoTrap HD® manufactured by Hyglos GmbH (former Profos AG) is a new revolutionary tool especially designed to meet the needs of pharma and biotech industry for the efficient removal of endotoxins in biomanufacturing processes. EndoTrap® HD fulfils all requirements for industrial applications, and it can easily be integrated in/at early or late-stage purification processes of biological substances such as proteins, drugs, antibodies, or vaccines.

Background
Endotoxins (lipopolysaccharides, LPS) are major contaminants found in commercially available proteins or biologically active substances. LPS are highly potent immune stimulators able to cause side effects in host organisms such as endotoxin shock, tissue injury, and even death. Due to these reactions, it is essential to remove LPS from injectable drugs and other biological as well as pharmaceutical products,
Purification very often represents the most time consuming and cost intensive step of the whole production process. Therefore optimization of the downstream process is essential in order to achieve time and cost efficiency. Generally, high LPS concentrations can be reduced to about 100 EU/mL without special treatment, but many applications require an even lower-limit value as described within the pharmacopoeia — e.g., 5 endotoxin units (1 EU ≈ 100 pg) per kilogram of body weight per hour for intravenous applications.

New Solution for Endotoxin Removal
Reducing the LPS level to <1 ng/mg (10 EU/mg) is a very difficult task, particularly without a significant loss of target substance. Frequently, LPS removal (e.g., from protein solutions) is insufficient with non–LPS-specific standard methods such as ultrafiltration (disadvantage: not suitable for large proteins), ion-exchange chromatography (disadvantages: possibly high loss of protein due to negatively charged proteins; reduced LPS removal efficiency due to positively charged proteins, two-phase extraction (disadvantage: traces of detergents interfere with high-resolution mass spectrometry, interact with eukaryotic cell membranes and have toxic effects). Only methods with the highest LPS removal capacity combined with excellent recovery rates of the target substances are acceptable. EndoTrap® HD meets all these specific requirements, and is highly suitable as a final polishing step to remove all remaining LPS contaminations.

EndoTrap® HD Features

• Highly specific and efficient LPS capture from complex mixtures
• Average sample/protein recovery >95%
• Compatible with commonly used buffer additives and high-salt conditions
• Excellent chromatographic characteristics (reusable 10 times)
• Low ligand leakage (comparable to leakage of protein A from purification columns)
• Ligand leakage monitoring with Hyglos Leakage ELISA
• Available Regulatory Support File (RSF); neither cytotoxic nor immunomodulatory effects of the EndoTrap® HD components were observed.

About Hyglos GmbH
Hyglos’ core competency is to exploit the principles of bacteriophage biology and expert use of proprietary phage–protein technology for pharmaceutical, diagnostic, and research applications.

Mycoplasma Detection Simplified by Endotoxin Testing Solutions

Mycoplasma detection is often difficult due to its lack of visable appearance, therefore it can be afflicting your valuable cell and affecting your results without your knowledge. Cells contaminated by mycoplasma species can have changes in proliferation, metabolism, gene synthesis and processing, and even adhersion properties. The solution for quick and easy mycoplasma detection is Endotoxin Testing Solution's PCR Mycoplasma Detection Kit. The PCR Mycoplasma Detection Kit allows for fast and reliable identification of mycoplasma contamination in cell cultures.  Mycoplasma DNA in the cell culture supernatant is amplified via PCR and visualized using gel electrophoresis. In addition to the short detection process (less than 2 hours), the easy handling and high sensitivity makes this PCR Mycoplasma Detection Kit a convenient tool for routine examination of cell cultures and media

 

Features

-direct addition of cell culture supernatant to PCR reaction -- no DNA isolation/purification steps required.

-ready-to-use primer mix  — reduces variability.

-able to detect numerous mycoplasma species – high sensitivity.

-included positive control to verify negative results.

-rapid results in 2 hours.

Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus-Amebocyte lysate test: an in vitro study

Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus-Amebocyte lysate test: an in vitro study

Simone Schaumberger, Andrea Ladinig, Nicole Reisinger, Mathias Ritzmann and Gerd Schatzmayr

Endotoxins are part of the cell wall of Gram-negative bacteria. They are potent immune stimulators and can lead to death if present in high concentrations. Feed additives, which bind endotoxins in the gastrointestinal tract of animals, could help to prevent their negative impact. The objective of our study was to determine the potential of a bentonite (Bentonite 1), a sodium bentonite (Bentonite 2), a chemically treated smectite (Organoclay 1) and a modified attapulgite (Organoclay 2) to bind endotoxins in vitro. Polymyxin B served as positive control. The kinetic chromogenic Limulus Amebocyte lysate test was adapted to measure endotoxins. Firstly, a single sorption experiment (10 endotoxin units/mL (EU/mL)) was performed. Polymyxin B and organoclays showed 100% binding. Secondly, the adsorption efficiency of sorbents in aqueous solutions with increasing endotoxin concentrations (2,450 - 51,700 EU/mL) was investigated. Organoclay 1 (0.1%) showed a good binding efficiency in aqueous solution (average 81%), whereas Bentonite 1 (0.1%) obtained a lower binding efficiency (21-54%). The following absorbent capacities were calculated in highest endotoxin concentration: 5.59 mg/g (Organoclay 1) > 3.97 mg/g (Polymyxin B) > 2.58.mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Thirdly, a sorption experiment in artificial intestinal fluid was conducted. Especially for organoclays, which are known to be unspecific adsorbents, the endotoxin binding capacity was significantly reduced. In contrast, Betonite 1 showed comparable results in artificial intestinal fluid and aqueous solution. Based on the results of this in vitro study, the effect of promising clay minerals will be investigated in in-vivo trials.

AMB Express 2014, 4:1  doi:10.1186/2191-0855-4-1
Published: 2 January 2014

http://www.amb-express.com/content/4/1/1/abstract

Recombinant Factor C Technology in Endotoxin Detection - EndoLISA® & End...

ICH Guideline Q4B Annex 14: Bacterial Endotoxins Test

ICH guideline Q4B Annex 14 to note for evaluation and recommendation of pharmacopoeial texts for use in the ICH regions on bacterial endotoxins tests – general chapter has been adopted by the European Medicines Agency.

The document allows for Ph.Eur. 2.6.14. Bacterial Endotoxins, JP 4.01 Bacterial Endotoxins Test, and USP General Chapter <85> Bacterial Endotoxins Test, can be used as interchangeable in the ICH regions. The proviso is that the gel-clot test remains the reference test.

Furthermore, the USP, JP, and Ph.Eur. reference standards are considered interchangeable as they have been suitably calibrated against the WHO (World Health Organization) International Standard for Endotoxin.

 

Document can be viewed here ICH Guideline Q4B Annex 14: Bacterial Endotoxins Test

FDA Adopts New Standard to Allow for Interchangeable Endotoxin Testing Between ICH Regions

FDA Adopts New Standard to Allow for Interchangeable Endotoxin Testing Between ICH Regions

Pharmaceutical Microbiology: Endotoxin detection: techniques and developments

Pharmaceutical Microbiology: Endotoxin detection: techniques and developments: American Pharmaceutical Review has issued a special supplement dedicated to endotoxin testing. The supplement features interesting ...

Recombinant Factor C Endotoxin Detection Presentations from Hyglos and Lonza

 

 

 

 

 

Comparison of the pyrogen tests in rabbits and with limulus lysate

In the past years an assortment of samples of plasma proteins, enzymes, vaccines and blood substitutes were tested comparatively in rabbits (pyrogen test, European Pharmacopoeia) and with the LAL test (Pyrogent, Byk-Mallinckrodt, Inc.). Specificity and sensitivity were tested with endotoxins and lipid A of gram-negative bacteria. The limulus amebocyte lysate (LAL) test gave similar results or was tenfold more sensitive than the assay in rabbits. More than 300 samples of drugs were examined by both tests. All preparations positive in the rabbit test were positive in the LAL test too. In the testing of plasma proteins the LAL test was more sensitive. The examination of 45 samples of vaccines for pyrogens gave the same result in both assays. Streptokinase does not inhibit the LAL test unspecifically. The LAL test is not an alternative but an additional method in the detection of lipopolysaccharides in drugs

Comparison of the pyrogen tests in rabbits and with limulus lysate

Ronneberger HJ.

Dev Biol Stand. 1977;34:27-36

 

History of the LAL Test : Validation and Regulatory Acceptance

 

Abstract

Bacterial endotoxins gram-negative bacteria are the most relevant substances inducing hyperthermia in humans (pyrogens). Since endotoxins may contaminate pharmaceutical preparations during production process, purity is assured by monitoring the increase in body temperature of rabbits exposed to such preparations. This bioassay can be replaced by the LAL-test in which the clotting reaction of blood cells of the horseshoe crab limulus polyphemus is measured after contact with bacterial endotoxins. Although this reaction is significantly more sensitive to endotoxins than hyperthermia in rabbits, the LAL-test had to undergo 25-30 years of validation to achieve regulatory acceptance. Although endotoxin induced blood clotting in Limulus is quite similar to the same reaction in humans, acceptance of the assay as an alternative to testing in rabbits was delayed, since chemicals present in pharmaceutical preparations may interfere with the LAL clotting reaction. In addition, fever can be induced also by substances other than endotoxins. Therefore it has to be proven for each new preparation that the LAL-test can replace the rabbit pyrogen test in a case by case validation according to guidelines, as e.g. the German guideline, which was released in 1993. During the past decade some laboratories of the pharmaceutical industry in Germany were able to replace 95% of the rabbit pyrogen tests by the LAL-test without increasing the risk for humans.

History of the LAL Test: Validation and Regulatory Acceptance

Liebsch M.

ALTEX. 1995;12(2):76-80.

PMID:

11178419

[PubMed - as supplied by publisher]

Limulus Amebocyte Lysate from Wikipedia

Limulus Amebocyte Lysate Wikipedia Link

Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantification of bacterial endotoxins.


Fred Bang reported in 1956 that gram-negative bacteria, even if killed, will cause the blood of the horseshoe crab to turn into a semi-solid mass. It was later recognized that the animal's blood cells, mobile cells called amoebocytes, contain granules with a clotting factor known as coagulogen; this is released outside the cell when bacterial endotoxin is encountered. The resulting coagulation is thought to contain bacterial infections in the animal's semi-closed circulatory system.
In 1970 the U.S. Food and Drug Administration (FDA) approved LAL for testing drugs, products and devices that come in contact with the blood. Prior to that date, much slower and more expensive tests on rabbits had been used for this purpose.
Blood is removed from the horseshoe crab's pericardium; the crabs are returned to the water. LAL manufacturers have measured mortality rates of 3% in bled crabs, however recent studies indicate that this number may be closer to 15%^[1] or even 30%.^[2] The blood cells are separated from the serum using centrifugation and are then placed in distilled water, which causes them to swell and burst ("lyse"). This releases the chemicals from the inside of the cell (the "lysate"), which is then purified and freeze-dried. To test a sample for endotoxins, it is mixed with lysate and water; endotoxins are present if coagulation occurs.^[3]

There are three basic LAL test methodologies: gel-clot, turbidimetric, and chromogenic. The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. In the United States, the FDA has published a guideline for validation of the LAL test as an endotoxin test for such products [1].
The LAL cascade is also triggered by (1,3)-β-D-glucan. Both bacterial endotoxins and (1,3)-β-D-glucan are considered "Pathogen-Associated Molecular Patterns", or PAMPS, substances which elicit inflammatory responses in mammals ^[4]

One of the most time consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition and enhancement.^[5] Agents such as EDTA and heparin are known to affect the assay if they are present in sufficient concentrations. All assays, independent of methodology are standardized using endotoxin in water. Therefore, unless the sample is water, some components of the solution may interfere with the LAL test such that the recovery of endotoxin is affected. If the product being tested causes the endotoxin recovery to be less than expected, the product is inhibitory to the LAL test. Products which cause higher than expected values are enhancing. Overcoming the inhibition and enhancement properties of a product is required by the FDA as part of the validation of the LAL test for use in the final release testing of injectables and medical devices. Proper endotoxin recovery must be proven before LAL can be used to release product.

1. http://www.pbs.org/wnet/nature/episodes/crash-a-tale-of-two-species/the-benefits-of-blue-blood/595/
2. http://www.mass.gov/dfwele/dmf/publications/mortality_in_female_horseshoe_crabs_abstract.pdf
3. The History of Limulus and Endotoxin, Marine Biological Laboratory. Retrieved 24 September 2008.
4. [Sandle, T. (2013). Pharmaceutical Product Impurities: Considering Beta Glucans, American Pharmaceutical Review, 16 (5) Supplement S1: 16-19]
5. Interference with the LAL Test and How to Address It, LAL Update, October 2005