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EndoTrap® FAQs

What is EndoTrap®?
EndoTrap® is an affinity chromatography resin based on a bacteriophage-derived protein that binds endotoxins with high specificity. The ligand targets a conserved region of the lipopolysaccharide (LPS) inner core and binds endotoxins from Gram-negative bacteria. It is not an antibody, a synthetic peptide, or a Polymyxin B-based resin. The same matrix can be used in batch or column (flow-through) mode. For regulatory documentation, toxicological data, a Regulatory Support File, and a leakage ELISA for EndoTrap® HD are available.
How do batch mode and column mode work?
In batch mode, settled resin is added directly to the sample. Test contact times of about 3 to 20 minutes to find suitable conditions. In column mode, pack depyrogenated resin as a 50% slurry in sterile buffer, or use ready-to-use columns. Column mode is usually easier and more efficient than batch mode. For any format, optimize pH, ionic strength, temperature, and contact time for your application to maximize endotoxin removal and sample recovery.
How do ready-to-use flow-through columns work?
EndoTrap® is a flow-through system. On a 1 ml column you can process up to 50 ml of sample in total, loading up to 3 ml per pass (an optional funnel allows about 20 to 25 ml per load). One cleaning step means your complete working volume has passed through the column once; regenerate the column between steps. Pre-packed columns and bulk resin for self-packing are available from Lionex (see the product catalogue). Fully automated liquid chromatography systems may be used if run times stay within the limits for your product grade.
What flow rates should I use?
Gravity flow is typically about 0.5 ml/min. Do not exceed 1 ml/min on EndoTrap® red; higher rates can compress the gel bed. EndoTrap® HD tolerates higher flow rates. A peristaltic pump may be used at or below these limits; slower flow often improves endotoxin removal. Keep all wetted equipment pyrogen-free.
What is the binding capacity, and how many cleaning steps do I need?
EndoTrap® red: 2,000,000 EU/ml resin. EndoTrap® HD: > 5,000,000 EU/ml resin (1 EU = 100 pg LPS). For HD, aim not to exceed about 2.5 million EU per ml of resin per run. Repeat cleaning steps to reach lower endotoxin levels (red: at least three cycles; HD: up to ten). Each pass can reduce endotoxin by about two log steps (HD: about three log steps). For reliable planning, keep total LPS applied within about 30 to 50% of the stated capacity. With repeated use, typical targets include about 0.005 EU/ml for buffers, about 0.1 EU/ml for proteins (removal efficiency may fall at very low levels), and about 0.01 EU/ml for DNA. Performance depends on sample properties; if you do not reach your target after three cleaning steps (each followed by regeneration), contact Lionex technical support.
Can EndoTrap® be regenerated, stored, or sterilized?
Yes. Use the supplied regeneration buffer. EndoTrap® red can be reused at least three times; EndoTrap® HD more than ten times, without loss of endotoxin removal efficiency under recommended conditions. Store resin in regeneration buffer with 0.02% sodium azide to limit microbial growth. Do not autoclave the material. Washing with 30% ethanol is acceptable; avoid sodium hydroxide, which may denature the ligand.
When does my sample elute, and what if flow is slow?
The void volume of a 1 ml column is about 0.3 to 0.5 ml; sample usually elutes immediately afterward. If there is slight retention on the matrix, wash with equilibration buffer, collect fractions, and pool as needed. Gravity flow through a 1 ml column at about 0.5 ml/min takes roughly 20 minutes for 10 ml. If flow is much slower, check for air bubbles (for example after transport): centrifuge the closed column at about 1000 × g for 5 minutes, gently reseat the top frit with a pyrogen-free pipette, or remove bubbles from the bottom using a syringe and endotoxin-free tubing while the column contains buffer.
Which samples and conditions are compatible?
Substances that pass through the column can be processed, including proteins, peptides, antibodies, and plasmid DNA. EndoTrap® removes endotoxin from proteins with isoelectric points (pI) from about 5 to 9. EndoTrap® HD is also suitable for plasmid DNA, RNA, and plant extracts. A working concentration of about 1 to 10 mg/ml protein is recommended (higher concentrations have been tested). The resin is active at 4 °C and at room temperature. Up to 2 M urea at pH 7 is generally compatible. EndoTrap® HD can be used with up to 1 M NaCl; EndoTrap® red up to 250 mM NaCl (best below 100 mM).
What agents inhibit EndoTrap® or damage the ligand?
EndoTrap® HD is inhibited by EDTA and other calcium chelators (EGTA, HEDTA, NTA, citrate), ammonium sulphate, SDS, and other detergents. EndoTrap® red is inhibited by salt concentrations above 250 mM, GdnHCl, ammonium sulphate, SDS, and other detergents. Dithiothreitol (DTT) interferes with EndoTrap® red; EndoTrap® HD can be used with up to 10 mM DTT or pre-washed with DTT. Proteases may destroy the ligand: use conditions that reduce protease activity (for example 4 °C) or adjust the buffer (pepsin, for example, is inactive above pH 6).
Does EndoTrap® bind pyrogens and yeast extract?
Most pyrogenic activity relevant to bioprocessing comes from bacterial endotoxin (LPS). EndoTrap® binds conserved LPS structures from Gram-negative bacteria. Yeast cell walls do not contain endotoxin, but yeast extracts may be contaminated with LPS from bacteria in laboratory water systems; EndoTrap® can remove that endotoxin during downstream processing.

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