Endotoxin Testing Analysis and Removal

About Bacterial Endotoxin Analysis

Endotoxins, also called lipopolysaccharides (LPS), are major contaminants found in commercially available proteins or biologically active substances. This bacterial endotoxin is a potent stimulator of the mammalian immune system in vivo, and it may decrease tissue culture cell viability and inhibit transfection efficiency in vitro. This often complicates the study of the biological effects of the main ingredient by causing false readings in cell-based assays, or it can cause side effects in host organisms such as endotoxin shock, tissue injury, and even death. Current FDA limits require eluates from medical devices to be less than 0.5 EU/mL, unless the device comes into contact with cerebrospinal fluid where the limit is then 0.06 EU/mL. Due to these reactions, it is essential to remove endotoxins from drugs, injectables, and other biological and pharmaceutical products.

Key Characteristics of Endotoxins

• Endotoxins (pyrogens) are found in the cell walls of Gram-negative bacteria
• Heat stable
• Mammals can be severely affected by small doses
• Also called Bacterial Endotoxin Test or BET

Usage of Endotoxin Testing

Endotoxin testing is necessary for the following:

• Injectables or parenteral products
• Radiopharmaceuticals and cytotoxic agents
• Water for injection (WFI)
• Validation of depyrogenation processes

Source of endotoxin contamination

• Water
• Raw compounds
• Instruments
• Method of storage between preparation and sterilization
• Inadequate depyrogenation cycle

Detection Methods

During the early days, one of the first tests used to determine if endotoxins were present was the Rabbit Pyrogen Test (RPT). A rabbit was inoculated with the test substance and then monitored it to see if a fever was induced. This test however, does not give a quantitative result, is time consuming, and is not suitable for products that may in themselves adversely affect the animal.

The most commonly used approach now is a Limulus Amoebocyte Lysate or LAL test.The LAL test focuses in particular on 2-keto-3-deoxyoctonoic acid, and it is this which is used as an indicator in the majority of endotoxin assays.

LAL is a reagent derived from the blood cells of the horseshoe crab. Unlike a mammal, the crab does not have a developed immune system. However, the LAL component in its blood will bind to and inactivate endotoxins. In the crab, the resulting clot alsoforms a protective barrier against bacterial infection. At its simplest, the LAL test consists of adding LAL reagent to the sample in a test tube if a gel or clothas formed. Then a positive result is recorded.

LAL Assay Options

• Gel-Clot: Qualitative or semi-quantitative method by comparing a sample against a dilution series of an endotoxin standards. Used to provide a pass/fail for a certain limitation and suitable with low sample numbers.
• Turbidimetric: Systems depend upon an increasing concentration of insoluble coagulin released as the test progresses. This changes the turbidity of the sample solution. The change in turbidity can be measured either as transmission or absorbance.
• Chromogenic Measurement: Chromogenic assays depend upon a chromogen which changes color if endotoxin is present in the sample. The higher the concentration of endotoxin, then the more chromogen is released.

Fast and Reliable Endotoxin Detection & Removal Services

Bio‑Synthesis offers rapid endotoxin testing solutions which ensures to our customers the safety and quality of their products.

Advantages of our endotoxin detection and removal services:

• Customized endotoxin detection and removal solutions for individual client needs
• Method development and/or transfer
• Quantitative endotoxin detection assay before and after endotoxin removal
• Reduce endotoxin levels to 0.5 EU/µg or less
• Greater than 95% protein recovery
• Time saving and cost effective solutions

Qualitative analysis of endotoxin: $175/test

Quantitative analysis of endotoxin: $225-$350/test (price vary based on sample type)

To help us better understand your project, please complete our Endotoxin Sample Testing Information Sheet. The more we understand your project needs, the more accurate feedback we will be able to provide you.

Endotoxin Removal Kits

Bacterial endotoxins (lipopolysaccharides, LPS) show strong biological effects in human, animal and cell cultures even at very low concentrations. Due to the high toxicity of bacterial endotoxins and DNA in vivo and in vitro, the removal of pyrogen from pharmaceutical products, recombinant proteins produced in E. Coli(or other gram-negative bacteria), and tissue culture media is of great importance for many applications.

Bio-Synthesis's Endotoxin Removal Kits are designed based on spin-column chromatography using Bio-Synthesis's innovative resin as the separation matrix. The sample can be easily purified from contaminating endotoxins within 20 minutes with a recovery rate of >90%. Depending on the sample types, endotoxin levels are reduced to less than 0.1 EU to 0.01 EU/µg of DNA or protein.

• Endotoxin-Free DNA, Protein Peptides: Reduce endotoxin levels from 0.1 to 0.01 EU/µg or less
• Fast and easy processing: Effectively remove endotoxins in 20 minutes
• High Recovery: Over 90% recovery
• Ready-to-use: No need for lengthy column, resin buffer preparation
• No endotoxin carry-over: Unlike other gel-based endotoxin removal procedures, which can lead to endotoxin carry-over. (Columns are single use only)
• Suitable for downstream applications: Purified protein is suitable for in vivo and in vitro introduction into cells and organisms, monoclonal and polyclonal antibody generation, mass spectrometry, gel electrophoresis, immunoassays, and Western blots and purified DNA is fully compatible with sequencing, cloning and transfections.

Ordering & Contact Information

• Please contact us for additional information or send an email to info@biosyn.com.
• You may also request an online quote.
• To contact us by phone, please call 1-972-420-8505 or Fax at 1-972-420-0442
• Orders may be placed using a purchase order (PO) or by credit card through our secure online ordering system.
• When using a credit card ( ) it will be billed under "Bio-Synthesis, Inc."