Endotoxin Removal Service

Endotoxin removal is an important part of recombinant protein expression and purification as endotoxin-contaminated protein samples transfected into cells or injected into an animal host can initiate a strong immune response such as endotoxin shock, tissue injury, and even death. Endotoxin removal service of BiologicsCorp (Abbr. BIC) guarantees to effectively reduce the endotoxin level below 1 EU/µg throughout entire process and meet strict requirements for downstream animal and cell experiments.

Flow chart of endotoxin removal service

BIC offers one-stop endotoxin removal service including gene synthesis, protein expression and purification, and endotoxin detection and removal. This service can be addressed in the flow chart below:

Fermentation Service Process
Gene
synthesis
Protein
expression
Cell
disruption
Protein
purification
Endotoxin
removal
Endotoxin
detection

Deliverables & price

Endotoxin Level Options Timeline Price($) Deliverables
<1.0 EU/µg
GUARANTEED
Additional 1-2 weeks Start from 300$
  • Protein with requested endotoxin level
  • QC reports
<0.1 EU/µg Please inquire
<0.01 EU/µg

Notes: we can effectively reduce the endotoxin level below 1 EU/µg throughout the entire process. In case we fail, there is no charge to you

 

Endotoxin removal is one of the most difficult tasks in downstream processes during protein purification. The removal of endotoxins becomes more challenging when associated with labile biomolecules, such as proteins. The form below lists number of approaches typically utilized to reduce endotoxin contamination of protein preparations. The success of these techniques in separating endotoxin from proteins is strongly dependent on the properties of the target protein.

Methods Main features
Affinity chromatography
  • Affinity adsorbents: immobilized L-histidine, poly-L-lysine, poly(γ-methyl L-glutamate), and polymyxin B
  • The high selectivity eliminates the need for multiple purification steps and reduces production costs
  • Highly specific and efficient endotoxin removal combined with excellent target recovery
Ion exchange chromatography It has rapid separation, wide selection of AEC media, sodium hydroxide (NaOH) sanitisation and does not require any solvents
Hydrophobic chromatography Interacts with non-polar protein surfaces through van der Waals forces for endotoxin removal
Gel filtration chromatography and ultrafiltration
  • It uses composite polyacrylamide as the column which is highly porous
  • Ultrafiltration reveals relatively good endotoxin clearance from product solutions if the products are of low molecular weight
Triton X-114 phase separation
  • Involves separation of aqueous surfactant solution into micelle-rich and micelle-poor regions through excluded-volume interactions
  • External agent notably Triton X-114 is needed to maintain the inherent biological activity of protein while reducing endotoxin level by 100-fold
 

The commonly used FDA-approved techniques for endotoxin detection are the rabbit pyrogen test and Limulus Amoebocyte Lysate (LAL) assay.

Techniques Principles Features Detection range
Rabbit pyrogen test Measuring the rise in temperature of rabbits after intravenous injection of a test solution
  • High cost
  • Long turnaround time
>2.5EU/mL
Limulus Amoebocyte Lysate (LAL) assay LAL gel-clot assay Using the characteristics of limulus reagent can react with bacterial endotoxin, to detect the content of bacterial endotoxin
  • Semi-quantitative
  • Time consuming
>0.25EU/mL
Turbidimetric LAL assay
  • Quantitative
  • Accurate
  • Fast
0.01-100.0 EU/mL
Chromogenic LAL assay
  • Quantitative
  • Accurate
  • Fast
0.01-100.0 EU/mL
 

One-stop endotoxin removal service VS General endotoxin removal service

One-stop endotoxin removal service includes gene synthesis, protein expression and purification, and endotoxin detection and removal. Endotoxin control and removal starts from protein preparation experiment to ensure the low level of endotoxin. All the vessels, the instruments, water, reagents, liquid move straws used in the experiments need to do endotoxin removal. At the same time, the experiment is done in clean room.

General endotoxin removal service includes removing endotoxin with your target protein and quantitative and/or qualitative detection of endotoxin before and after endotoxin removal.

Generally, we guarantee to effectively reduce the endotoxin level to 1 EU/µg or less throughout controlling the endotoxin level from the protein preparation experiment.

Detection of endotoxin by LAL gel-clot assay:

  • Detection of endotoxin by LAL gel-clot assay:
  • Protein concentration: C=0.782mg/mL (determination by Bradford method)
  • Sensitivity of limulus reagent :λ=0.25EU/mL
  • The limit value of endotoxin of samples: L=1EU/μg
  • Maximum valid dilute double:MVD=C*L/λ

1. Results of endotoxin removal throughout the entire process controlling

Diluted multiples 50 400 1600 3000 3200
Results (solidification or not) Solidification No solidification No solidification No solidification No solidification

2. Results of endotoxin removal after protein preparation

Diluted multiples 50 400 1600 3000 3200
Results (solidification or not) Solidification Solidification Solidification No solidification No solidification

Analysis of experiment results: by controlling endotoxin throughout entire process, bacterial endotoxin level in the protein was much lower than that simply removing endotoxin after protein preparation.

Contact Us

  +1 (317) 703-0614
  +1 (855) 427-1516

* To get a price quotation, please fill the protein service pre-order form and email to Sales@BiologicsCorp.com.