From: Profos AG [noreply@profos.de]
Sent: Thursday, October 09, 2008 1:20 PM
To: mariol@mail.ls.huji.ac.il
Subject: Endotoxin removal methods - advantages & disadvantages
Profos Newsletter

Profos changed the way to remove endotoxins from aqueous solutions

EndoTrap has become the chromatography system of choice for removing endotoxin from biological solutions during the last years. Since 2003 the Profos AG successfully provides products to remove endotoxins from aqueous solutions for the bioresearch and bioprocess markets. EndoTrap eliminates common limitations connected with present methods e.g. ultrafiltraion, ion exchange chromatography, or two phase extraction.

Advantages and disadvantages of current methods for endotoxin removal:
Many different processes have been developed for the removal of LPS from proteins based on the unique molecular properties of the LPS molecules. The success of the techniques in separating LPS from proteins depends strongly on the properties of the target molecule (e.g. net charge, hydrophobicity) and the solution conditions (e.g. pH, ionic strength). Only affinity chromatography is LPS specific.

Current methods for endotoxin removal Advantages / Disadvantages
ultrafiltration advantages:
- easy handling
- suitable for small proteins
disadvantages:
- not LPS specific
- not suitable for large proteins
two phase
extraction
advantage:
- cheap procedure
disadvantages:
- not LPS specific
- traces of solvents have to be removed from the target sample as they
  > interfere with high resolution mass spectrometry
  > interact with eukaryotic cell membranes
  > and have toxic effects
heat sterilization
advantages:
- easy handling
disadvantages:
- not LPS specific
- not applicable to bioreagents like recombinant proteins that are destroyed at high temperatures as well
ion exchange
chromatography
advantages:
- easy handling
- reusable
- no limitation considering the molecular weight
disadvantages:
- not LPS specific
- negatively charged proteins may co-adsorb onto the matrix and cause a significant loss of target protein
- positively charged proteins could form complexes with LPS, causing the proteins to drag LPS along the column and minimizing the LPS removal efficiency
affinity
chromatography
advantages:
- easy handling
- specific LPS capture
- reusable
- no limitation considering the molecular weight
disadvantage:
- not suitable for viscous solutions

Affinity chromatography is based on the specific adsorption of a molecule to a ligand. Almost all biological molecules can be purified on the basis of specific interaction between their chemical or biological structure and a suitable affinity ligand.
EndoTrap affinity chromatography is one principal method for cleaning biological solutions from endotoxin contaminations. The EndoTrap ligand is a protein by nature, which binds to the bead-matrix by stable covalent bonds. However, leakage of minute of amounts of ligand is a matter of fact for all affinity materials and testing on these contaminants is often required for regulatory purpose.
Profos provides leakage data for EndoTrap blue and EndoTrap red and a Leakage ELISA for EndoTrap HD.

Advantages and disadvantages of current affinity chromatography methods for endotoxin removal:

Affinity
chromatography
Advantages / Disadvantages
Ligand:
Polymyxin B
*
advantages:
- specific LPS capture (Lipid-A)
- easy handling
- many customer publications available
disadvantages:
- Polymyxin B is an antibiotic substance
(you have to consider that you will contaminate your target molecule with an
antibiotic substance!)

- most products based on Polymyxin B have low LPS binding capacity
- pH limitation
- temperature limitation
- the removal procedure needs often incubation times
- regeneration often with DOC which is toxic for cells
- no leakage data available
- not suitable for viscous solutions
Ligand:
"solid phase
reagent"
*
advantages:
- specific LPS capture
- nearly no temperature limitation
disadvantages:
- low LPS binding capacity
- pH limitation
- for the removal procedure incubation step is recommended
- regeneration with 1M NaOH, therefore intensive washing steps necessary
- additional equipment necessary: peristaltic pump
- sample must be filtered before being applied
- no leakage data available
- not suitable for viscous solutions
Ligand:
polylysin *
advantages:
- high LPS binding capacity
- nearly no temperature limitation
disadvantages:
- pH limitation
- complex protocol for the removal procedure
- regeneration limitation (only 5-times)
- no leakage data available
- not suitable for viscous solutions
- no customer publications available
Ligand:
peptides
*
advantages:
- high LPS binding capacity (Lipid-A)
- nearly no temperature limitation
- nearly no pH limitation
- reusable
disadvantages:
- only for small sample volume (1 ml sample / 1 ml resin)
- regeneration with DOC which is toxic for cells
- no leakage data available
- not suitable for viscous solutions
- no customer publications available
Ligand:
EndoTrap blue or
EndoTrap red


(protein-based
LPS-binding ligand)
advantages:
- high LPS binding capacity (inner core region of LPS)
- up to 50 ml sample / 1 ml resin
- nearly no temperature limitation
- nearly no pH limitation
- best sample recovery (compared to competitive products)
- best LPS removal (compared to competitive products)
- reusable
- low ligand leakage
- leakage data available
- many customer publications available (link)
disadvantages:
- not suitable for viscous solutions
Ligand:
EndoTrap HD

launched June 2008

advantages:
- can be used in early or late biomanufacturing process step
- high LPS binding capacity (inner core region of LPS)
- nearly no temperature limitation
- nearly no pH limitation
- best sample recovery
- best LPS removal
- reusable
- robust and chemically defined matrix
- excellent chromatographic characteristics
- low ligand leakage
- Leakage ELISA available
- Regulatory Support File available
disadvantages:
- not suitable for viscous solutions
* Product information according website or protocol. We can not give any guarantee for this information.

EndoTrap represents a benchmark in the field of endotoxin removal technology based on affinity chromatography (e.g. binding capacity) in the meantime. Of course there exist various products for the removal of endotoxin but we are not aware of any other product that combines all important advantages like EndoTrap. Through its efficiency and its favourable and unique price performance ratio EndoTrap is the first choice for the removal of endotoxins during research and biomanufacturing process.

EndoTrap Customer Feedback
Customer Feedback
(EndoTrap blue,
EndoTrap red)
- "Probably the best system of recent years."
- "... We are not aware of any other method or commercially available product, which allows the removal of endotoxins from native protein samples as the last step of the protein purification protocol with the same efficiency and recovery rates as we have experienced with the EndoTrap-family products. It is also worth mentioning the adequate, competent, and immediate support from the Profos AG team. The way of interaction is very straight forward, uncomplicated, and solution oriented."
- "... We compared the EndoTrap red columns with several other LPS removal procedures and found this to be the best system in our hands."
Our EndoTrap
customers
among others
are from ... *
Biotechnology Companies (like AstraZeneca, Cytos Biotechnology, ESBATech, Integrated DNA Technologies and Micromet), Big Pharma (like Baxter and Eli Lilly), Research Institutes (like DKFZ, INSERM, NCI-Frederick, RKI and The Scripps Research Instituteand over 400 universities (like Charité, Johns Hopkins University and TU Munich) world-wide.
* EndoTrap customers who agreed to mention them as reference.

Within only four years (customer publications considered: November 2004 - October 2008) more than 90 papers were published by the R&D market (universities, research institutes, biotech companies).
The newest product within the EndoTrap-family was launched in June 2008 (EndoTrap HD for biomanufacturing processes) and underlines that Profos has taken the next step to become a global market leader in the field of endotoxin removal systems and high-quality endotoxin-free products.

Overview about the differences between the EndoTrap products:

  EndoTrap blue EndoTrap red EndoTrap HD
LPS binding capacity 2.000.000 EU/ml 2.000.000 EU/ml 5.000.000 EU/ml
Support matrix highly cross-linked 4% agarose highly cross-linked 4% agarose hydrophilic,cross-linked methacrylic polymer
pH stability 4-9
6-9 4-9
Ionic strength
up to 600 mM NaCl up to 250 mM NaCl up to 1.000 mM NaCl
Tested kind of substances which can
be applied
proteins, peptides, enzymes, antibodies, plasmid DNA, phage particles, buffers proteins, peptides, enzymes, antibodies, phage particles, buffers proteins, peptides, enzymes, antibodies, plasmid DNA, phage particles, buffers
Package size ready-to-use columns (gravity flow) or slurry
ready-to-use columns (gravity flow) or slurry only available as slurry
Intended use Laboratory scale, Research use only
Laboratory scale, Research use only For biomanufacturing & large scale process, Biomanufacturing & Research use.
Note need 100 µM Ca2+ for efficient binding of LPS   need 100 µM Ca2+ for efficient binding of LPS
      Leakage ELISA & Regulatory Support File available


Profos is located in the BioPark I and II in Regensburg, one of the most important biotechnology centers in Germany. With more than 1700 sqm of office, lab and production space the facility houses state of the art equipment and instrumentation for biochemistry and molecular biology research.
Profos AG is certified according to ISO 9001:2000 and 13485, the internationally recognized quality management system standard developed by the International Organization for Standardization (ISO), for its Regensburg facilities of Research & Development, Production and Marketing & Sales.


Profos AG
Josef-Engert-Str. 11
93053 Regensburg
Germany

email: mailto:stephanie.steck@profos.de
homepage: http://www.profos.de/

This newsletter is a service of Profos AG.

Sitz der Gesellschaft: Regensburg
Registergericht: Regensburg HRB 8175
Aufsichtsratvorsitzender: Dr. Hellmut Beckstein
Vorstand: Dr. Wolfgang Mutter (Vorsitzender); Dr. Bernd Buchberger; Dr. Stefan Miller; Oliver Glück

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