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Exciting advances in proteinomics, cellular and
molecular biology over the past two decades have
led to a plethora of well characterized complex
pharmaceutical compounds which are amenable to
scale up manufacturing using bacterial cell
culture systems as “biofactories.” The desired
end products from such cellular manufacturing
must be isolated and completely purified for
clinical usage. For those drug compounds not
requiring glycosylation it is most simple and
cost effective to utilize bacterium as the
source of the “biofactory” for cellular growth.
The desired pharmaceutical may then be either
released from the bacteria or isolated after
bacterial cell disruption.
A cardinal problem
encountered during bacterial cell culture is
coping with the presence of endotoxins. There is
no way to get around the fact that endotoxins
are inherent when dealing with bacterium, and it
is essential to assure removal of endotoxins as
part of the isolation and characterization of
any drug compound during industrial downstream
processing.
Endotoxins are
lipopolysaccharides (LPS) located on the outer
layer of the cell wall (cell membranes) of gram
negative bacteria and are most commonly isolated
from microorganisms of the Enterobactereaceae
family. These lipopolysaccharides are composed
of hydrophobic fatty acid and hydrophilic
carbohydrate domains. They make up the majority
of pyrogens essential for removal from
pharmaceutical products, biologicals for
injection and media used for tissue culture.
While endotoxins are associated with destruction
of the cell membrane during bacterial cell death
they are also continuously released during
bacterial cell growth and cell division.
The presence of small
amounts of endotoxin in recombinant protein
preparations when injected into patients may
cause systemic inflammatory reactions running
the spectrum from tissue injury, to endotoxin
shock and death. Highly toxic to mammalian
cells, endotoxin is one of the most potent
modulators of the immune system. In terms of
scope, a single bacterial organism of
Escherichia coli contains 2 million LPS
molecules per cell. As noted above, control of
LPS from Escherichia coli is important because
this bacteria is the biopharmaceutical workhouse
used by industry for the manufacture of many
recombinant DNA products such as proteins and
peptides not requiring complex glycosylation.
Pharmaceutical products
produced using E. Coli and other gram negative
bacterium as cellular factories are virtually
always contaminated with LPS and all measurable
endotoxin must be removed during the production
process. Low concentrations of the LPS molecule
bind to the CD 14 receptor of mammalian cells
which subsequently leads to the release of a
spectrum of pro-inflammatory mediators such as
tumor necrosis factor (TNF), and interleukins
IL-1 and IL-6. The maximum acceptable level of
endotoxin for intravenous applications is set at
5 endotoxin units (EU) per kg of body weight per
hour.
Endotoxins are very stable
molecules, resistant to extreme temperature and
pH changes and much more durable than proteins
or peptides. Recognition of this molecular
hardiness is essential when establishing a
purification process to assure endotoxin removal
while at the same time not altering the
physical, biological or chemical properties of
the desired pharmaceutical compound.
Besides LPS, gram negative
bacteria release peptides such as exotoxin A,
peptidoglycans, muramyl peptides and still
unidentified substances, all of which possess
biological properties which induce the secretion
of cytokines.
In aqueous solutions
endotoxins can self assemble into a variety of
shapes such as lamella, cubic and hexagonal
inverted arrangements with diameters up to 0.1
um and 1000 kDa. Within these assemblages
endotoxins achieve high stability. These
arrangements are extremely heat stable and are
not destroyed under regular sterilizing
conditions. The chemical stability of
lipopolysaccharides makes pyrogen removal so
difficult. Because endotoxins are so pH and heat
stable their removal is often the most difficult
portion of downstream processing in protein
purification.
High concentrations of acids
or bases are necessary to destroy LPS within a
reasonably short time. Naturally occurring LPS
has a Stokes radius which is smaller than the
purified endotoxin typically used to quality
filters, which further adds to the uncertainty
in developing effective LPS removal methods.
Endotoxins when forming into lamellar and
micellar forms may interact with proteins
through electrostatic interactions, making their
removal extremely difficult. Endotoxin can be
inactivated when exposed at temperatures of 250
C for more than 30 minutes or 180 C for more
than 3 hours. Acidic or alkali solutions of at
least 0.1 M strength may be used to destroy
endotoxin; however the removal of endotoxin from
basic proteins is more difficult than removal
from acidic proteins.
Levels of endotoxin are much
higher in recombinant proteins derived from
soluble or cytoplastic fractions than in
proteins derived from insoluble or inclusion
bodies. This finding is consistent with
guidelines followed during conventional
biopharmaceutical manufacturing processes, in
which lipopolysaccharides present in cell walls
are solubilized during the cell lysis procedure.
No one single purification
method has been demonstrated to completely
remove endotoxin. Commonly used purification
techniques include LPS affinity resins, two
phase extractions, ultrafiltration, hydrophobic
interaction chromatography, ion exchange
chromatography and membrane adsorbers. Each of
these methods when used to isolate and remove
endotoxin has met with varying success. Current
good manufacturing processes include batch
processing and column chromatography techniques
to isolate peptides and proteins as part of the
downstream purification process.
The superior method of
endotoxin purification may be using the so
called negative chromatographic method. This
allows binding of the endotoxin with the peptide
or protein passing the adsorber without
retention. Elution of endotoxin is not the
object, and therefore irreversible adsorption of
the endotoxin is desired. There is a need to
achieve a very low dissociation constant between
the endotoxin and the adsorbent. This is
critical as the amount of endotoxin to be
removed may be quite low in concentration
compared to the concentration of the desired
final product.
Ion exchange chromatography
is the most commonly used method for the removal
of pyrogens when the desired end product is a
recombinant protein. Limitations when using ion
exchange chromatography include handling and
usage problems such as packing, channeling, low
flow rates, long regeneration times,
compressibility and limited chemical stability.
When hydrophobic adsorbents
are used in protein solutions there is
hydrophobic binding between the adsorbent and
the lipophilic group of endotoxins. Important
mediators include the net charge and
hydrophobicity of the protein and the pH and
ionic strength of the solution.
Anionic exchange
chromatography is potentially useful for the
decontamination of positively charged proteins
such as urokinase but provides little benefit
when used with negatively charged proteins due
to significant loss of product through
adsorption. This chromatographic technique takes
advantage of the negative net charge of
endotoxins.
Ultrafiltration is useful
when used for small proteins such as myoglobin,
but proves ineffective when used with larger
proteins such as immunoglobulins (150,000 Da).
Of great concern is that proteins may be sheared
by physical forces during ultrafiltration, which
may impact both biological and immunologic
properties.
Affinity chromatography is
useful as part of a two step endotoxin
purification process. In one process the peptide
antibiotic Polymyxin B is placed in a
chromatography column and the solution
containing the desired cellular product (protein
or peptide) is run through the column, with the
endotoxin binding to the Polymyxin. This first
step is then followed by washing the column with
a nonionic detergent.
Such phase separation has
proven to be a useful approach towards endotoxin
removal. Surfactant agents such as Triton X-114
or zwitterionic surfactants containing both
negatively and positively charged moities have
been used to help dissociate endotoxin from
proteins in solution, with increased endotoxin
removal once freely suspended in solution in the
detergent phase and the upper aqueous phase
contained the desired protein Of note the
immunoactivity, physical integrity and
biological activity of the desired protein
appear to remain unchanged after this phase
separation.
Specially activated alumina
with surface modified chemical moieties has
proven to provide a superior tool for the
purification of endotoxins due to its amphoteric
property. No other commercially available agent
can provide such a rewarding pH response or
offer a better or cost effective method for the
removal of pyrogens from a protein or peptide
solution. It is clear that endotoxins develop
especially strong binding to adsorbants that
carry positively charged functional groups.
Electrostatic interactions play an important
role during endotoxin adsorption. Proteins are
also amphoteric. Since proteins are amphoteric
molecules, electrostatic interactions are not as
strong as for the mainly negatively charged
endotoxin. Owing to the globular structure of
proteins, charged and hydrophobic groups are
fixed and cannot be twisted towards functional
groups or surface structures of the adsorbants.
Additional benefits provided through the use of
activated alumina include low cost, limited
safety issues, extremely well defined chemical
characteristics and with minimal impact on the
bioactivity of protein when placed into a
standard manufacturing process.
Furthermore the protein
binding capacity of membrane adsorbers are much
lower than those of particulate sorbents. The
removal of pyrogens using specially designed
activated alumina can be performed using either
column chromatography or batch treatment. When
using column chromatography the final product is
achieved by filling a column with the alumina
modified to enhance pyrogen adsorbance,
prewashing the packed column with a suitable
buffer and then passing the pyrogen containing
solution through the column. In the method of
batch treatment the final product freed of
pathogens can be obtained by stirring the
pryogen adsorbent in a pyrogen containing
solution of the desired compound and then
removing the adsorbent.
DynaPharmaTM
Pyrogen for the removal of endotoxins and
pyrogens
The isolation of therapeutic
molecules from bacterial cell growth is hampered
by the need to remove endotoxins and pyrogens.
Many different commercial techniques are in
place in an attempt to remove these endotoxins,
which are lipopolysaccharide components of
bacterial cell walls.
DynaPharmaTM
Pyrogen, using either
column chromatography or batch processing
methods has proven to be a superior tool for the
isolation and removal of pyrogens. The surface
of activated alumina may be chemically modified
to help enhance pyrogen removal. Furthermore,
the amphoteric property of activated alumina
offers unique purification advantages allowing
Alumina P to be used either as a single step or
as part of a two step purification process for
the isolation and removal of endotoxin.
DAI offers a specially
designed superior Alumina which may prove to be
just what you need for the removal of endotoxins
and pyrogens from your bacterial cell culture
system.
Talk with a member of our
scientific team to discuss how we may be able to
best address your production needs.
Dyna-Pharma™
Pyrogen
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