ENDOTOXIN REMOVAL

DEFINITION
Endotoxin are single most important class of pyrogen
ie. A substance that causes a fever when injected into
humans
and animals.

Endotoxins are complex aggregates of LPS

(Lipopolysacchrides) and may contain
protein material.

PROPERTIES:
They show pyrogenicity (so they need to be controlled and

minimized in any processes involving a parental drug.

In aqueous solutions endotoxin can exist in various state of
aggregation
Whereas individual molecules have M.Ws~ 10-20 KDa
Aggregation can have M.Ws as high as 1MDa
Divalent cations, as (Mg, Ca) are found to stabilize the
aggregation structure LPS
Whereas detergents structure into subunits
These endotoxin can easily pass through a 0.2um sterilizing-grade
membrane filter(ie. Where size exclusion is the sole retention
mechanism ).
Traditionally endotoxins have been removed by Reverse Osmosis
or UF membrane of 10,000 NMWL cut off.

Causes of Endotoxin Contamination:

They are continuously shed from the outer membrane
of viable gram negative bacterial
Released when bacterial cell dies
Gram negative bacteria are present in Envirnoment and
a source of concern in WFI, buffers, and other fluids
that goes into parental drugs making
It can be removed by using 0.2 um sterilizing grade
filter but Endotoxins can still be a concern in WFI
distribution system as LPS itself is difficult to remove
or inactivate because it is extremely Heat and pH
stable.

Amount of Endotoxin Required to Generate a

Pyrogenic Reaction

Amount is on the order 0.1 ng per Kg of body weight.

[ 0.1ng ~ 1 EU (Endotoxin unit)]

E.g.. Given typical gram negative bacterium contains

10-15 g of LPS (0.1 ng would require 105 bacterial cells
precursor.

Endotoxin limits for Various Products:

WFI

< 0.25 EU/ ml

LVPs

< o.5 EU/ml

Hlb Vaccine

< 50 EU/ml (< 25 EU/dose)

Factor IX

< 0.1 EU/ml ( <5 EU/dose)

Globulin

<0.91 EU/ml (< 5 EU/dose

Solution for Toxic

Control

Size Exclusion:
Family of UF products ie. PelliconTM,
ProstaicTM, Spirals
Used for solution where a 10,000 NMWL
cut off membrane

Charge Interaction:
Endotoxins are negatively charged at pH above
2
A positive membrane surface can remove
endotoxin.
e.g. Charged Durapore membrane surface has a net
positively charge on its surface
Note :
In the presence of competing negative ions, such as in
buffer filtration, a charged membrane cannot be used
because its losses its adsorption properties.

Hydrophobic Interaction:
Using the hydrophobic interaction of the
lipid A component of the endotoxin and
hydrophobic membrane filter, device
containing hydrophobic Durpore can be
used for removing endotoxin from buffer
ions solution.