Purification of monoclonal antibodies

A method of chromatographically separating monoclonal antibody type IgG from mouse ascites fluid utilizing a particular chromatographic packing of silica gel bearing bound polyethylenimine functions.

The invention relates to a method of purifying the monoclonal antibody 
content of a mouse ascites fluid sample. More particularly, the present 
invention is concerned with a method of separating and purifying 
monoclonal antibody type IgG from mouse ascites fluid utilizing liquid 
column chromatography on a particular stationary porous phase of silica 
gel bearing bound polyethylenimine (PEI) functions and gradient elution of 
the monoclonal antibody from the polyethylenimine bound column with 
aqueous buffer of from about pH 6.0 to about pH 8.3. 
The particular stationary porous phase of silica gel bearing bound 
polyethylenimine functions, that is, the chromatographic packing utilized 
in this invention, are of two types. 
The preferred type is described in U.S. patent application Ser. No. 
555,368, filed by Hugh Ramsden on even date herewith and entitled 
"Polyethylenimine Bound Chromatographic Packing", the content of which is 
incorporated herein by reference. Relevant text of this application is 
reproduced below. 
The second type is the adsorbed cross-linked PEI-silica gel stationary 
phase described by G. Vanecek & F. E. Regnier, Anal. Biochem. 121, 156-159 
(1982) and A. J. Alpert & F. E. Regnier, J. Chromatogr. 185, 375-392 
(1978), which type is commercially available from SynChrom, Inc. of 
Linden, Ind., under the brand name "SynChropak". Alpert and Regnier have 
shown that polyethyleneimine (PEI) may be adsorbed to silica gel surfaces 
and crosslinked to form a stable polyamine layer. The structure of PEI 
provides sufficient primary and secondary amino groups that adjacent 
adsorbed PEI molecules on the surface of silica gel may be crosslinked by 
multifunctional oxiranes into a polymeric layer. Through the use of a 
hydrophilic crosslinker such as diglycidylethylene glycol, a hydrophilic 
coating may be produced. 
DETAILED DESCRIPTION OF (RAMSDEN) INVENTION 
The non-crosslinked covalently bound PEI silica gel products of the present 
invention are conveniently prepared in accordance with the following 
steps: 
A. reacting particulate silica gel having an average particle diameter of 
from about 3 to about 70 microns and an average pore size of from about 50 
to about 1000 Angstrom units in an inert organic solvent slurry with a 
lower alkanolic solution of polyethyleniminopropyl trimethoxy silane 
having an average molecular weight of from about 400 to about 1800, said 
reaction being conducted at ambient to refluxing temperature for about 2 
to about 50 hours; 
B. recovering the resultant solid fraction from the reaction mixture; and 
C. heating said solid fraction at a temperature and for a time sufficient 
to dry and completely bond the silane to the silica gel. 
As used herein, the term "covalently bound" or "covalently bonded" means 
that the PEI moieties are covalently attached to the silica gel by way of 
chemical interaction resulting in a propyl-silyl (Pr-Si) linkage; and the 
term "non-crosslinked" means that the imino and amino groups on adjacent 
covalently bound PEI moieties are not crosslinked, or reacted with a 
crosslinking agent, to form a polymeric layer. 
Without being bound thereby, it is believed that the reaction proceeds to 
completion in two steps as follows: 
Step 1: Silica hydroxyls and the methoxy groups on the silane react to form 
Si-O-Si bonds and free methanol, with some residual methoxy groups 
remaining unreacted: 
##STR1## 
Step 2: Completion of the reaction with the residual methoxy groups is 
effected during heat curing by (a) and (b): 
##STR2## 
Silica gel, consisting of amorphous silica, is commercially available in 
irregular and spherical (preferred) particulate forms and in several 
commercial grades with mesh sizes ranging from 3 through 325 (ASTM). 
Rather than relying upon a numerical indication of mesh size, however, 
more accurate indicia for purposes of this invention are the average 
diameter and average pore size of the silica gel particles, respectively, 
from about 3 to about 70 microns and from about 50 to about 1000, 
preferably 250-500, Angstrom units. For end product use in packing HPLC 
chromatographic columns, a silica gel starting material of from about 3 to 
about 10 microns is preferred, and, for packing low pressure 
chromatographic columns, from about 40 to about 70 microns is preferred. 
Among the inert organic solvents suitable for preparing the silica gel 
slurry are aliphatic hydrocarbons such as, for example, hexane, heptane 
and the like; aromatic hydrocarbons such as, for example, benzene, 
toluene, xylene and the like; lower alkanols such as, for example, 
ethanol, isopropanol, butanol and the like; chlorinated methanes such as, 
for example, methylene chloride, chloroform, carbon tetrachloride and the 
like (Caution: such chloro solvents may react at higher temperatures-); 
and such other inert solvents as tetrahydrofuran, glyme, diglyme and the 
like. In general a 1:5 ratio of silica gel in grams to solvent in 
milliliters affords a suitable slurry. Due to the fine, insoluble nature 
of the particulate silica gel a slurry rather than a true solution is 
obtained. 
Polyethyleniminopropyl trimethoxy silane, also known as 
(N-trimethoxysilylpropyl)-polyethylenimine, is the reaction product of 
polyethylenimine and aminopropyltrimethoxy silane and can be represented 
by the following formula: 
##STR3## 
wherein, for purposes of this invention, n is an integer from about 4 to 
about 37, or, if expressed in terms of average molecular weight, from 
about 400 to about 1800. 
The silane (I) is used in the reaction with the silica gel in the form of a 
lower C.sub.1 -C.sub.6 alkanolic solution using sufficient alkanol to 
solubilize the silane. A fifty percent w/w isopropanolic solution is 
preferred. In general, about 25-100 grams of the silane, or, 
alternatively, about 50-200 ml of a fifty percent w/w alkanolic solution 
of the silane, is used to react with each 100 grams silica gel. The 
reaction may be conducted at ambient temperature although elevated 
temperatures up to the refluxing temperature of the reaction solvent 
system may be utilized to enhance the rate of reaction. The reaction 
proceeds readily to substantial completion (Step 1) within 2-50 hours. 
Stirring during admixture of the reactants is advantageously employed 
although the reaction thereafter may continue without further stirring. 
Anhydrous conditions are not critical, it having been found that the 
presence of a small amount of water, for example, about 0.1-1.0 ml per 50 
ml of the slurry solvent, does not adversely affect the reaction. 
The resultant solid fraction is recovered from the reaction mixture by 
conventional physical means, for example, filtration, centrifugation, etc. 
In general, a filtering means sufficient to retain a particle size of 5 
microns is suitable whereas centrifuging is suitable for a particle size 
of 3 microns. 
The recovered solid fraction is then heat cured at a temperature and for a 
time sufficient to dry and completely bond the silane to the silica gel 
covalently. In general, from about 1-4 hours at about 
40.degree.-120.degree. C. has been found sufficient. The thus-obtained 
covalently bound, non-crosslinked final product preferably contains from 
about 0.5 to about 3.8 percent nitrogen. 
For purposes of this invention, the chromatographic packing, hereinafter 
sometimes referred to as "PEI-silica gel" for purposes of convenience, is 
utilized wherein the starting silica gel is limited to one having an 
average particle diameter of from about 3 to about 40 microns. The average 
pore size may be from about 50 to about 1000 Angstrom units, although an 
average pore size greater than 250 Angstroms is preferred. 
Accordingly, the instant invention provides a method of obtaining 
essentially homogeneous monoclonal antibody type IgG from a sample of 
mouse ascites fluid containing said monoclonal antibody by employing 
liquid chromatographic means wherein the chromatographic packing comprises 
particulate silica gel having an average particle diameter of from about 3 
to about 40 microns and an average pore size of from about 50 to about 
1000 Angstrom units to which polyethylenimine functions are bound, either 
in adsorbed crosslinked form according to Regnier et al., ibid, or in 
covalently bound non-crosslinked form according to Ramsden, ibid. 
Regarding the latter, the PEI-silica gel chromatographic packing is the 
reaction product of the aforementioned particulate silica gel with 
polyethyleniminopropyl trimethoxy silane having an average molecular 
weight of from about 400 to about 1800. 
It has now been found that such chromatographic packing is particularly 
suitable for use in liquid chromatography, particularly high performance 
liquid chromatography (HPLC), for binding type IgG monoclonal antibodies 
and other proteins in mouse ascites fluid containing same, thereby 
allowing preferential separation of the bound proteins by gradient elution 
using appropriate aqueous buffers. There are thus obtained a number of 
protein components which are originally present in the ascites fluid 
including the essentially homogeneous monoclonal antibody of interest. As 
used herein "essentially homogeneous" means that &gt;90% of the protein 
present in the quantitatively recovered IgG antibody eluant fraction is 
the particular IgG monoclonal antibody. The percent purity may be verified 
by known procedures in the art, such as, for example, by sodium dodecyl 
sulfate polyacrylamide gel electrophoresis, see U. K. Laemmli, Nature, 
227, 680 (1970). 
The instant invention is suitable for use with mouse ascites fluid 
containing monoclonal antibody of all sub-classes of the IgG type, for 
example, IgG.sub.1, IgG.sub.2a and IgG.sub.1+3 and the like. The 
methodology of preparing such mouse ascites fluid containing monoclonal 
antibody is common to the art, for example, see "Monoclonal Antibodies, 
Hybridomas; A New Dimension in Biological Analyses", by R. H. Kennet et 
al., published by Plenum Press, N.Y. 1980; and "Methods in Enzymology" by 
G. Galfred and C. Milstein, edited by J. J. Langone and H. Van Vunakis, 
Vol. 73, p. 31-46, publ. by Academic Press, 1981. 
The isolation of monoclonal antibody in highly purified form is obviously 
desirable. For example, for in vivo therapeutic purposes, monoclonal 
antibody as pure and as concentrated as possible is required to minimize 
adverse side effects and to maximize the intended therapeutic purpose. 
Similarly, for in vitro diagnostic purposes, such purified and 
concentrated monoclonal antibody is desirable to maximize the sensitivity 
and specificity of the particular diagnostic test. 
Before the mouse ascites fluid can be used for this invention, it is 
pre-treated to remove interfering particulate matter and is equilibrated 
to the appropriate ionic strength and pH necessary to achieve binding of 
the monoclonal antibody to the PEI-silica gel support. The particulate 
matter can be removed by conventional clarifying means, for example, by 
filtration or by centrifugation at a force sufficient to pelletize the 
particulate material. Equilibration of the particulate-free mouse ascites 
fluid can be achieved by any means common to the state-of-the-art, for 
example, by chromatographic desalting with an appropriate buffer on 
molecular sieves of appropriate type and pore size such as those 
commercially available under the brand name "Sephadex", by dialysis 
against an appropriate buffer, and the like, to equilibrate the mouse 
ascites fluid to a pH greater than the pI (that pH at which the monoclonal 
antibody carries no net ionic charge in its environment) of the particular 
IgG monoclonal antibody and to an ionic strength equal to or less than the 
ionic strength of the lower ionic strength buffer used for gradient 
elution in the subsequent chromatographic treatment of the mouse ascites 
fluid. 
Chromatographic columns suitable for use in liquid chromatography, 
preferably HPLC, are packed with the previously described porous 
PEI-silica gel solid support. Suitable steel or glass chromatographic 
columns include those having dimensions of about 5-100 cm in length and 
internal diameters of about 1-100 mm. Selection of the proper 
chromatographic parameters such as, for example, column packing technique, 
column size, column temperature, pressure and the like, are readily 
determined by one of ordinary skill in the art. 
The packed column is equilibrated in a chromatograph by passing an 
appropriate buffer solution through the column. After this 
buffer-equilibration step, the column is used to make the chromatographic 
separation of the proteinaceous components of the mouse ascites fluid 
which, as noted, previously has been freed of particulate matter and has 
been equilibrated to the appropriate ionic strength and pH. A sample of 
such pre-treated mouse ascites fluid is then applied to the 
buffer-equilibrated column to bind its component proteins to the 
PEI-silica gel packing. 
The bound proteins can then be selectively eluted by conventional gradient 
elution techniques, taking into consideration the interdependent 
chromatographic parameters of time, flow-rate and gradient shape to 
generate gradients of increasing ionic strength or decreasing pH. Anionic 
buffers, for example, potassium phosphate, tris-acetate, ammonium acetate 
and the like, of from about pH 6.0 to about 8.3, can be used to generate 
such gradients to elute the bound proteins from the polyethyleneimine 
function. For example, the gradient can be advantageously formed from 
about one-half hour to about four hours with a flow rate of from about 0.1 
mL/min to about 2 L/min. 
The resolved proteins can be identified by any means common to the 
state-of-the-art, for example, by monitoring the ultraviolet absorbance at 
280 nm. The eluent fractions containing the separated proteins can be 
collected by use of a fraction collector. The eluent fraction containing 
the homogeneous monoclonal antibody can be identified by means 
well-established in the art such as, for example, by a radioimmunoassay 
developed for the particular antibody, by other antibody-antigen 
reactions, or by polyacrylamide gel electrophoresis. 
The process of this invention has been found to be independent of the total 
volume of the mouse ascites fluid containing the monoclonal antibody and 
there is no limiting factor except for the amount of PEI-silica gel used 
as the chromatographic packing, that is, the process is operable so long 
as the capacity of the solid chromatographic support is not surpassed. 
In accordance with the method of the present invention, therefore, a sample 
of mouse ascites fluid containing monoclonal antibody type IgG is 
chromatographically separated to provide said antibody in essentially 
homogeneous form. As more fully described heretofore, the preferred method 
comprises purifying a sample of particulate-free mouse ascites fluid 
containing such monoclonal antibody by: 
a. equilibrating said sample of particulate-free mouse ascites fluid to an 
ionic strength equal to or less than the ionic strength of the lower ionic 
strength buffer, used for gradient elution in the subsequent 
chromatographic separation and recovery step and to a pH greater than the 
pI of the particular IgG monoclonal antibody; and 
b. separating and recovering said monoclonal antibody type IgG from said 
sample by employing liquid chromatographic means wherein the 
chromatographic packing consists essentially of the reaction product of 
particulate silica gel having an average particle diameter of from about 3 
to about 40 microns and an average pore size of from about 50 to about 
1000 Angstrom units with polyethyleniminopropyl trimethoxy silane having 
an average molecular weight of from about 400 to about 1800.

The invention will be more easily understood with the aid of the examples 
which follow below which are given solely as an illustration of the 
present invention and are not limitative of the same. 
EXAMPLE 1 
A slurry of 20 grams silica gel with average particle diameter of 5.25 
microns and average pore size of 330 Angstroms, commercially available 
from The Sep A Ra Tions Group, Hesperia, CA, as a spherical silica under 
the trademark "Vydac A", Catalog No. 101T9B5, in 100 ml toluene and 2 ml 
water is prepared and stirred for 10 minutes at room temperature. To this 
is added with stirring 39.4 grams of a 50% w/w isopropanolic solution of 
polyethyleniminopropyl trimethoxy silane having an average molecular 
weight of 500 and the mixture is stirred for an additional 5 minutes. The 
mixture is then allowed to stand overnight at room temperature. The 
mixture is next filtered using a 1.0 micron filter funnel. The filtrate is 
washed with 50 ml toluene twice and 50 ml methanol twice, then air dried 
on the funnel and finally oven dried at 80.degree.-85.degree. C. for about 
3 hr. 30 min. to yield the covalently bound, non-crosslinked PEI-silica 
gel product; about 2.85% N. 
EXAMPLE 2 
A 2 mL sample of mouse ascites fluid containing a monoclonal antibody 
specific for bacterial lipopolysaccharide cell wall and belonging to the 
subclass IgG.sub.1, as determined by conventional subclass specific 
antisera, was centrifuged at 15,600.times.gravity for 5 minutes. The 
supernatant fluid was then equilibrated to 10 mM potassium phosphate 
buffer, pH 6.73, by dialysis overnight (about 17 hours) against 2 1-liter 
changes of the 10 mM potassium phosphate buffer, pH 6.73. A stainless 
steel chromatographic column, 25 cm.times.0.46 mm, was packed with 3.8 g 
of the PEI-silica gel product obtained from Example 1 and equilibrated 
with 0.01M potassium phosphate buffer, pH 6.73, by pumping this buffer 
through the column at a flow rate of 1 mL/min for 20 minutes. A 0.25 mL 
equilibrated sample of mouse ascites fluid was applied to the 
polyethyleneimine-bound silica gel column and protein separation was 
achieved by gradient elution using a 60 minute linear gradient from 0.01M 
potassium phosphate, pH 6.73, to 0.25M potassium phosphate, pH 6.8, at a 
flow rate of 1 mL/min. Protein elution was detected using UV absorbance at 
280 nm with full scale absorbance set at 0.64 absorbance units. A series 
of 280 nm absorbing peaks were detected ranging in retention time from 
about 3 minutes to about 25 minutes. The protein peak at about 16 minutes 
was identified as the antilipopolysaccharide monoclonal antibody by 
co-chromatographing with a homogeneous sample of the same monoclonal 
antibody using the identical gradient profile as outlined above. 
The protein peak eluting at about 16 minutes was evaluated to be greater 
than 90% pure by two criteria. First, the collected protein peak gave a 
symmetrical peak after chromatogaphy on the polyethyleneimine-bound silica 
gel column, according to F. B. Regnier, Science 222, 245 (1983). Second, 
sodium dodecyl sulfate polyacrylamide gel electrophoresis, performed 
essentially as described by U. K. Laemmli, Nature, 227, 680 (1970), gave 
two major coomassie blue bands corresponding to molecular weights of 
50,000 daltons (heavy chain of IgG) and 25,000 daltons (light chain of 
IgG) and a third faint coomassie blue band representing less than 10% of 
the total stained protein. 
EXAMPLE 3 
A 2 mL sample of mouse ascites fluid containing a monoclonal antibody 
specific for myosin and belonging to the subclass IgG.sub.2a as determined 
by conventional subclass specific antisera, was centrifuged at 
15,600.times.gravity for 5 minutes. The supernatant fluid was then 
equilibrated to 10 mM potassium phosphate buffer, pH 6.73, by dialysis 
overnight against 2 1-liter changes of the 10 mM potassium phosphate 
buffer, pH 6.73. A stainless steel chromatogaphic column, 25 cm.times.0.46 
mm, was packed with 3.8 g of the PEI-silica gel product obtained from 
Example 1 and equilibrated with 0.01M potassium phosphate buffer, pH 6.73, 
by pumping this buffer through the column at a flow rate of 1 mL/min for 
20 minutes. A 0.25 mL equilibrated sample of mouse ascites fluid was 
applied to the polyethyleneimine-bound silica gel column and protein 
separation was achieved by gradient elution using a 60 minute linear 
gradient from 0.01M potassium phosphate, pH 6.73, to 0.25M potassium 
phosphate, pH 6.8, at a flow rate of 1 mL/min. Protein elution was 
detected using UV absorbance at 280 nm with full scale absorbance set at 
0.64 absorbance units. A series of 280 nm absorbing peaks were detected 
ranging in retention times from about 3 minutes to about 25 minutes. The 
protein peak at about 16 minutes was identified as the antimyosin 
monoclonal antibody by co-chromatographing with a homogeneous sample of 
the same monoclonal antibody using the identical gradient profile as 
outlined above. The protein peak eluting at about 16 minutes was evaluated 
to be greater than 90% pure by the two criteria set forth in Example 2. 
EXAMPLE 4 
A 2 mL sample of mouse ascites fluid containing an IgG monoclonal antibody 
of unknown specificity was centrifuged at 15,600.times.gravity for 5 
minutes. The supernatant fluid was then equilibrated to 10 mM potassium 
phosphate, pH 6.73, buffer, by dialysis overnight against 2 1-liter 
changes of the 10 mM potassium phosphate buffer, pH 6.73. A stainless 
steel chromatographic column 25 cm.times.0.46 mm was packed with 3.8 g of 
the polyethyleneimine silica gel product of Example 1 and equilibrated 
with 0.01M potassium phosphate buffer, pH 6.73, by pumping this buffer 
through the column at a flow rate of 1 mL/min for 20 minutes. A 0.25 mL 
equilibrated sample of mouse ascites fluid was applied to the 
polyethyleneimine-silica gel column and separation was achieved by 
gradient elution by using a 60 minute linear gradient from 0.01 potassium 
phosphate, pH 6.73, to 0.25M potassium phosphate, pH 6.8. Protein elution 
was detected using UV absorbances at 280 nm with full scale absorbance set 
at 0.64 absorbance units. A series of 280 nm absorbing peaks were detected 
ranging in retention times from about 3 minutes to about 25 minutes. The 
protein peak at about 16 minutes was identified as the monoclonal antibody 
by co-chromatographing with a homogeneous sample of the same monoclonal 
antibody using the identical gradient profile as outlined above. 
The protein peak eluting at about 16 minutes was evaluated to be greater 
than 95% pure by polyacrylamide gel electrophoresis performed as described 
by O. Gabriel, "Methods in Enzymology", edited by W. B. Jakaby, Vol. 22, 
p. 565-578, publ. by Academic Press, 1971, which gave a single coomassie 
blue band. The mobility of this band corresponds to that observed for 
standard monoclonal antibodies. 
EXAMPLE 5 
A stainless steel chromatographic column, 25 cm.times.4.6 mm, was packed 
with 3.8 g of polyethyleneimine bound silica gel of Example 1 and 
equilibrated with 0.01M potassium phosphate, pH 6.73, by pumping this 
buffer through the column at a flow rate of 1 mL/min for 20 minutes. A 6 
uL equilibrated sample of the same mouse ascites fluid utilized in Example 
2 was applied to the PEI-silica gel column and separation was achieved by 
gradient elution using a 120 minute linear gradient from 0.01M potassium 
phosphate, pH 6.73, to 0.25M potassium phosphate, pH 6.8, at a flow rate 
of 1 mL/min. Protein elution was detected using UV absorbances at 280 nm 
with full scale absorbance set at 0.01 absorbance units. A series of 280 
nm absorbing peaks were detected ranging in retention times from about 3 
minutes to about 50 minutes. The protein peak at about 28 minutes was 
identified as the antilipopolysaccharide monoclonal antibody by 
co-chromatographing with a homogeneous sample of the same monoclonal 
antibody using the identical gradient profile as outlined above. 
EXAMPLE 6 
A stainless steel chromatographic column, 25 cm.times.4.6 mm, was packed 
with 3.8 g of polyethyleneimine bound silica gel of Example 1 and 
equilibrated with 0.01M potassium phosphate, pH 8.3, by pumping this 
buffer through the column at a flow rate of 1 mL/min. for 30 minutes. A 6 
uL equilibrated sample of the same mouse ascites fluid utilized in Example 
2 was applied to the PEI-silica gel column and separation was achieved by 
gradient elution using a 60 minute linear gradient from 0.01M potassium 
phosphate, pH 8.3, to 0.25M potassium phosphate, pH 8.3, at a flow rate of 
1 mL/min. Protein elution was detected using UV absorbance at 280 nm with 
full scale absorbance set at 0.01 absorbance units. A series of 280 nm 
absorbing peaks were detected ranging in retention times from about 3 
minutes to about 50 minutes. The protein peak at about 12 minutes was 
identified as the antilipopolysaccharide monoclonal antibody by 
co-chromatographing with a homogeneous sample of the same monoclonal 
antibody using the identical gradient profile as outlined above. 
EXAMPLE 7 
A stainless steel chromatographic column, 25 cm .times.4.6 mm, was packed 
with 3.8 g of polyethyleneimine bound silica gel of Example 1 and 
equilibrated with 0.01M potassium phosphate, pH 6.0, by pumping this 
buffer through the column at a flow rate of 1 mL/min. for 20 minutes. A 6 
uL equilibrated sample of the same mouse ascites fluid utilized in Example 
2 was applied to the PEI-silica gel column and separation was achieved by 
gradient elution using a 60 minute linear gradient from 0.01M potassium 
phosphate, pH 6.0, to 0.25M potassium phosphate, pH 6.0, at a flow rate of 
1 mL/min. Protein elution was detected using UV absorbance at 280 nm with 
full scale absorbance set at 0.01 absorbance units. A series of 280 nm 
absorbing peaks were detected ranging in retention times from about 3 
minutes to about 14 minutes. The protein peak at about 12 minutes was 
identified as the antilipopolysaccharide monoclonal antibody by 
co-chromatographing with a homogeneous sample of the same monoclonal 
antibody using the identical gradient profile as outlined above. 
EXAMPLE 8 
A stainless steel chromatographic column, 25 cm.times.4.6 mm, was packed 
with 3.8 g of polyethyleneimine bound silica gel of Example 1 and 
equilibrated with 0.01M ammonium acetate, pH 6.79, by pumping this buffer 
through the column at a flow rate of 1 mL/min. for 30 minutes. A 6 uL 
equilibrated sample of the same mouse ascites fluid utilized in Example 2 
was applied to the PEI-silica gel column and separation was achieved by 
gradient elution using a 30 minute linear gradient from 0.01M ammonium 
acetate, pH 6.79, to 1.0M ammonium acetate, pH 6.79, at a flow rate of 1 
mL/min. Protein elution was detected using UV absorbance at 280 nm with 
full scale absorbance set at 0.01 absorbance units. A series of 280 nm 
absorbing peaks were detected ranging in retention times from about 3 
minutes to about 25 minutes. The protein peak at about 12 minutes was 
identified as the antilipopolysaccharide monoclonal antibody by 
co-chromatographing with a homogeneous sample of the same monoclonal 
antibody using the identical gradient profile as outlined above. 
EXAMPLE 9 
A SynChrom, Inc., SnyChropak AX300 chromatographic column (25 cm.times.4.1 
mm) was equilibrated with 0.01M potassium phosphate, pH 6.73, by pumping 
this buffer through the column at a flow rate of 1 mL/min for 20 minutes. 
A 6 uL equilibrated sample of the same mouse ascites fluid of Example 2 
was applied to the column and separation was achieved by gradient elution 
using a 60 minute linear gradient from 0.01M potassium phosphate, pH 6.73, 
to 0.25M potassium phosphate, pH 6.8, at a flow rate of 1 mL/min. Protein 
elution was detected using UV absorbance at 280 nm with full scale 
absorbance set at 0.01 absorbance units. A series of 280 nm absorbing 
peaks were detected ranging in retention times from about 3 minutes to 
about 30 minutes. The protein peak at about 20 minutes was identified as 
the antilipopolysaccharide monoclonal antibody by co-chromatographing with 
a homogeneous sample of the same monoclonal antibody using the identical 
gradient profile as outlined above.