Freeze-dried preparation containing monoclonal antibody

A freeze-dried monoclonal antibody preparation is prepared by adding to a solution of the monoclonal antibody gelatin or carboxylic acid or its salt, to prevent denaturation of the monoclonal antibodies during freeze-drying. Freeze-dried monoclonal antibody preparations having stablized antigen-binding activity result and may be used as an adminiculum for immunoauxotherapy for prophylaxis and treatment of bacterial infectious diseases and viral infectious diseases.

FIELD OF THE INVENTION 
The present invention relates to a freeze-dried preparation comprising a 
monoclonal antibody(or antibodies) as a main ingredient(s). 
PRIOR ART 
A monoclonal antibody is a homogeneous globulin protein having reactivity 
to only a specific epitope. Recent progress in technologies of cell 
fusion, cultivation and protein purification, etc. has made it possible to 
produce large amounts of monoclonal antibodies. As a result, monoclonal 
antibodies have come to be utilized in various fields, such as various 
analyses, diagnoses, treatments and prophylaxes. In particular, 
expectations of monoclonal antibodies as medicines for treatments and 
prophylaxes are increasing. Above all, their application to humans is 
expected to be developed further in future, and development of 
human-derived monoclonal antibodies which are favorable in point of 
antigenicity is being advanced. 
Hitherto, in this technical field, polyclonal antibodies such as 
immunoglobulin preparations have been used for the same purpose for 
medical diagnosis and treatment. While a monoclonal antibody is a 
homogeneous one having reactivity to only a specific epitope, a polyclonal 
antibody is a mixture of plural antibodies as is named so. Therefore, in a 
polyclonal antibody, plural molecules each having different properties act 
to mutually stabilize them so that such a polyclonal antibody is, as a 
whole, in a relatively stable state. However, in a purified monoclonal 
antibody, stabilization by the interaction between different molecules 
could not be expected so that such a monoclonal antibody is unstable to 
various physical and chemical actions irrespective of the immunoglobulin 
class of itself. 
Globulin proteins such as monoclonal antibodies and polyclonal antibodies 
are often heated for the purpose of inactivating viruses therein, 
especially when they are used for medical diagnosis and treatment. Such 
globulin proteins are unsuitable to storage in the solutions for a long 
period of time. Therefore, employment of freeze-drying has been common for 
a stable storage of such globulin protein molecules. In addition, the 
globulin proteins are often treated with acids or alkali substances, if 
desired. 
To such heat-treatment, freeze-drying and acid- or alkali-treatment, 
polyclonal antibodies are generally stable, but monoclonal antibodies 
would often be denatured and easily lose their activity by such treatment. 
In particular, IgM is less stable than any other monoclonal antibodies of 
other immunoglobulin classes (e.g., IgG, IgA and IgE). Regarding 
heat-treatment of antibodies, for example, JP-A 61-76423 (the term "JP-A" 
means an "unexamined published Japanese patent application") discloses the 
fact that monoclonal antibodies are unstable to heat-treatment and that, 
for the purpose of overcoming thermal instability, a hydrolysate of 
ovalbumin is added to a monoclonal antibody preparation. 
On the other hand, freeze-drying treatment involves a problem specific to 
monoclonal antibodies. Namely, in freeze-drying a monoclonal antibody, if 
a monoclonal antibody solution is freeze-dried without adding a stabilizer 
thereto, there occurs a problem of decrease of the antigen-binding 
activity of the monoclonal antibody due to denaturation of itself during 
freeze-drying. Therefore, it is necessary to prevent this problem. The 
problem is noticeable in freeze-drying a monoclonal antibody, while it is 
not so significant in freeze-drying a polyclonal antibody as a polyclonal 
antibody is stable because of the above-mentioned reasons. 
In preparing a freeze-dried product of a monoclonal antibody, addition of 
albumin of a heterologous protein to a solution of a monoclonal antibody 
before freeze-drying (for example, JP-A 60-146833, 61-78730 and 61-78731, 
and WO 90/11091) or addition of maltose of a saccharide thereto) (for 
example, WO 89/11297) is known. 
Immunoglobulin praparations of polyclonal antibodies are usually used at a 
relatively high concentration, and aggregates would often form in the 
solutions during storage or during the succeeding freeze-drying treatment. 
The aggregates are considered to cause a serious anaphylactoid side 
effect, when the globulin containing them is intravenously injected to 
human bodies. Therefore, for the purpose of preventing formation of such 
aggregates, addition of a heterologous protein to the stock solutions is 
known. For instance, addition of gelatin alone of a heterologous protein 
to an immunoglobulin solution (for example, JP-A 58-167518, vox.) Sang. 
(1983) 51, 81-86) or addition of both sucrose of a saccharide and gelatin 
thereto is known to be effective for preventing formation of aggregates in 
the stock solutions and also for maintaining antibacterial and antiviral 
activities (SU 700132). All the technologies as disclosed above are to 
prevent formation of aggregates in a high concentration solution of an 
immunoglobulin of a polyclonal antibody. None of them mention or discuss 
the matter of decrease of the antigen-binding activity of polyclonal 
antibodies due to the freeze-drying treatment. On the other hand, a 
monoclonal antibody is stored or freeze-dried in the form having a 
relatively low concentration. Even under such a low concentration, 
however, there is still a problem of denaturation of a monoclonal antibody 
during freeze-drying as well as a problem of decrease of its 
antigen-binding activity. The matter has not heretofore been identified as 
to whether or not addition of gelatin used to prevent formation of 
aggregates of immunoglobulins would be useful for solving this problem. 
On the other hand, it is widely known that carboxylic acids and their salts 
are used as a component of buffers for pH maintenance of various protein 
solutions. For instance, WO 89/11298 discloses the addition of maltose, 
sodium chloride or sodium phosphate, as a stabilizer, to a stock solution 
of a monoclonal antibody for the purpose of preventing formation of 
aggregates which precipitate in the solution. It also mentions use of 
sodium citrate, instead of sodium phosphate, as a component of the buffer. 
However, it merely indicates a technique of preventing formation of 
aggregates in a stock solution of a monoclonal antibody during storage, 
but it does not disclose a treatment for freeze-drying a monoclonal 
antibody, a treatment for preventing denaturation of a monoclonal antibody 
during freeze-drying it and also a treatment for preventing decrease of 
the antigen-binding activity thereof. WO 89/11297 discloses a technique of 
adding maltose, as a stabilizer, to a monoclonal IgG antibody solution to 
be freeze-dried and further adding, as a buffer component, 5 to 10 mM 
sodium acetate thereto so that the pH value of the solution is kept within 
an acidic range of being from 3 to 6. In this case, sodium acetate is 
obviously used as a component of a buffer solution. WO 89/11297 suggests 
nothing as to the fact that carboxylic acid or its salt would still act as 
a stabilizer for preventing denaturation of antibody during freeze-drying 
treatment thereof in the pH range where the carboxylic acid or its salt 
does not exert a buffer action. Regarding a pH range of an antibody 
solution, if an antibody solution having a low pH value is intravenously 
injected pain or injection often occurs. Where an antibody solution is 
used as an injection, it is desirable to have a pH value in an 
approximately neutral pH range. However, utilization of such an antibody 
solution in a neutral pH range is not suggested in WO 89/11297. 
For the purpose-of inactivating viruses which could contaminate 
immunoglobulins in preparing them from sera or plasma, immunoglobulins are 
often heated in the form of their solutions. For instance, JP-A 62-292731, 
61-194035, 61-191622 and 57-140724 disclose the addition of carboxylic 
acids to said globulin solutions for this purpose. JP-A 61-78730 and 
61-78731 disclose the addition of sodium acetate to immunoglobulin 
preparations and heating them in a dry state. However, all of them merely 
mention the addition of carboxylic acids for the purpose of stabilizing 
immunoglobulin preparations in the heat-treatment. It has not heretofor 
been known whether or not carboxylic acids and their salts would be useful 
for preventing denaturation of antibodies during freeze-drying and also 
for preventing a decrease of their antigen-binding activity owing to said 
denaturation. 
PROBIEM TO BE SOLVED BY THE INVENTION 
The object of the present invention is to provide stable freeze-dried 
preparations of monoclonal antibodies, which are free from denaturation of 
the monoclonal antibodies during freeze-drying and from a decrease of 
their antigen-binding activity owing to said denaturation. 
MEANS FOR SOLVING THE PROBLEM 
The present inventor shave found that gelatin, carboxylic acids or their 
salts are effective for stabilizing a monoclonal antibody in freeze-drying 
it. Namely, they have found that, by addition of gelatin to a solution 
containing a monoclonal antibody to be freeze-dried, denaturation of the 
monoclonal antibody during freeze-drying as well as decrease of its 
antigen-binding activity may be prevented and that, by the addition of 
carboxylic acid or its salt to a solution containing a monoclonal antibody 
to be freeze-dried, denaturation of the monoclonal antibody during 
freeze-drying as well as decrease of its antigen-binding activity owing to 
said denaturation may be prevented over a broad pH range and even at a pH 
value being outside the range where a buffer action is exhibited. On the 
basis of these observations, applicants have found that it is possible to 
prepare a stable and highly safe preparation of a monoclonal antibody. 
Specifically, the present invention provides a freeze-dried preparation 
containing a monoclonal antibody and geltain as well as a preparation 
prepared by freeze-drying a solution containing a monoclonal antibody and 
carboxylic acid or its salt and having pH between 6.1 and 8.1. 
The monoclonal antibody to be used in the present invention may be any 
monoclonal antibody that is generally obtained from human beings, mice, 
rats and others, and the origins and the producing means are not 
specifically defined. For instance, the monoclonal antibody for use in the 
present invention may be obtained from a culture supernatant as obtained 
by cultivating antibody-producing cells as prepared by known methods such 
as a cell fusion method or a transformation method, or by cultivating 
cells into which a cloned antibody gene has been incorporated, or from 
ascites, etc. of a mouse into which such antibody-producing cells have 
been transplanted. For purifying the monoclonal antibody obtained from 
such a cell culture supernatant or mouse ascites or the like, usable are 
various purification methods such as ammonium sulfate salting-out, ion 
exchange chromatography, gel filtration, affinity chromatography, 
ultra-centrifugation, adsorption chromatography and hydrophobic 
chromatography. The immunoglobulin classes of the monoclonal antibody to 
be used in the present invention are mostly IgG, IgM, IgA and IgE, but 
they are not specifically defined. A monoclonal antibody of any 
immunoglobulin class can be used in the present invention. Above all, IgM 
is less stable than those of other immunoglobulin classes. Therefore, the 
stabilizing method effective for the IgM class monoclonal antibody may 
easily be applied to monoclonal antibodies of other immunoglobulin 
classes. In fhe present invention, a single monoclonal antibody may be 
used or several monoclonal antibodies may be also used as a mixture of 
them with no problem. 
Gelatin may be grouped into two types (neutral type and acidic type) 
according to the methods of preparing it, of which the isoelectric points 
are different from each other. Both of them may be used in the present 
invention. In addition, chemically modified gelatins such as 
oxypolygelatin or modified liquid gelatins may be also used. 
As carboxylic acid, usable are, for example, citric acid, acetic acid, 
oxalic acid, succinic acid and fumaric acid. Citric acid is preferable. As 
salt of carboxylic acid, usable are, for example, sodium citrate, 
potassium citrate, sodium acetate, potassium acetate, sodium oxalate, 
potassium oxalate, sodium succinate, potassium succinate, sodium fumarate 
and potassium fumarate. Sodium citrate is preferable. 
For the purpose of stabilizing the monoclonal antibody or for the purpose 
of pH adjusting, isotonicating and buffering the monoclonal 
antibody-containing solution to be freeze-dried, inorganic salt, 
monosaccharide, disaccharide, sugar alcohol or amino acid may be further 
added, in addition to gelatin or carboxylic acid or its salt. 
As inorganic salt, usable are, for example, sodium chloride, potassium 
chloride and magnesium chloride. Sodium chloride is preferable. 
As monosaccharide, usable are, for example, glucose, mannose, galactose and 
fructose. Glucose or mannose is preferable. 
As disaccharide, usable are, for example, maltose, sucrose and lactose. 
Maltose or sucrose is preferable. 
As sugar alcohol, usable are, for example, sorbitol and mannitol. Mannitol 
is preferable. 
As amino acid, usable are, for example, glycine, alanine, valine, leucine, 
isoleucine, tyrosine, phenylalanine, serine, threonine, glutamine, 
glutamic acid, asparagine, aspartic acid, arginine, lysine, histidine, 
proline, tryptophan, methionine and cysteine. Glycine or arginine is 
preferable. 
For producing the freeze-dried preparation of the present invention, a 
monoclonal antibody solution containing gelatin or carboxylic acid or its 
salt may be freeze-dried. Preferably, the monoclonal antibody solution is 
added to a buffer containing gelatin or carboxylic acid or its salt having 
an adjusted pH value; or gelatin or carboxylic acid or its salt is added 
to a monoclonal antibody-containing solution. The concentration of the 
monoclonal antibody in the solution to be used in the present invention is 
from 0.01 mg/ml to 50 mg/ml, preferably from 0.1 mg/ml to 10 mg/ml. The 
amount of gelatin is from 1/100 to 100 parts by weight to one part by 
weight of the monoclonal antibody. Preferably, it is from 1/10 to 10 parts 
by weight to one part by weight of the same. The concentration of 
carboxylic acid or its salt to be added is from 2 mmoto 500 mM, preferably 
from 10 mM to 200 mM. 
The pH value of the solution for dissolving the monoclonal antibody to be 
freeze-dried is from 4.0 to 8.1 when gelatin is added thereto; or it is 
from 6.1 to 8.1, preferably from 6.5 to 7.8, when carboxylic acid is added 
or both gelatin and carboxylic acid are added. Adjustment of the pH value 
of the solution may be done using organic acids, inorganic acids, 
inorganic salts or other compounds which are generally used for pH 
adjustment, singly or in combination of two or more of them. As a compound 
usable for such pH adjustment, there are mentioned, for example, citric 
acid, sodium citrate, potassium citrate, phosphoric acid, sodium 
phosphate, potassium phosphate, hydrochloric acid, 
tris(hydroxymethyl)aminomethane, acetic acid, sodium acetate, potassium 
acetate, sodium hydroxide, boric acid, sodium borate, and potassium 
borate. The concentration of the buffer solution for dissolving the 
monoclonal antibody is from 5 mM to 500 mM, preferably from 10 mM to 500 
mM. As mentioned above, carboxylic acid or its salt may be also used for 
pH adjustment of a monoclonal antibody containing solution, and the 
above-mentioned amount of the acid or its salt indicates the total amount 
thereof in the solution including the amount for pH adjustment. 
The thus prepared monoclonal antibody solution may be stable when 
freeze-dried directly as it is. It is also possible to add thereto a 
surfactant such as TWEEN 20 or TWEEN 80, a human or bovine albumin, or a 
chelating agent such as EDTA, for the purpose of isotonicating the 
solution or of preventing adhesion of the monoclonal antibody to the 
container containing the solution. 
Freeze-drying of the monoclonal antibody solution may be carried out by any 
ordinary known method, and the drying temperature and the vacuum degree in 
the method may be selected suitably.

EXAMPLES 
Next, the present invention will be explained by way of the following 
examples, which are, however, not limitative. IgM is exemplified herein as 
a monoclonal antibody in the present invention. This is because, as 
mentioned above, IgM is less stable than antibodies of other 
immunoglobulin classes (e.g., IgG, IgA and IgE), and therefore the 
stabilizing effect to be verified in IgM may be easily applied to 
antibodies of other immunoglobulin classes. 
Example 1 
Cells of Epstein-Barr virus (EB virus) transformed cell line MP-5038 (FERM 
BP-1596) producing an IgM antibody reactive to Group E serotype 
Pseudomonas aeruginosa were cultured, and a human monoclonal antibody was 
purified from the culture supernatant by ammonium sulfate salting-out, gel 
filtration with SEPHACRYL S-300 (Pharmacia Co.) and column chromatography 
with a hydroxyapatite HPLC column (Mitsui Toatsu Chemicals, Inc.) and 
BLUE-SEPHAROSE (Pharmacia Co.). The monoclonal antibody as obtained by 
these methods had a purity of 99% or higher, as analyzed by 
SDS-polyacrylamide gel eletrophoresis and HPLC with a gel filtration 
column. The monoclonal antibody was dissolved in a phosphate-buffered 
physiological saline having an adjusted pH value of 7.4 (hereinafter 
referred to as PBS), to have a final concentration of 0.1 mg/ml. On the 
other hand, gelatin (high-grade gelatin; Nippi Co., type A (neutral 
gelatin) and type B (acidic gelatin)) was added thereto to have a final 
concentration of 0.001 to 1%. The resulting solution was then put in 2 
ml-volume polypropylene cryotubes (Corning Co.) under a sterilized 
condition, each in an amount of 0.5 ml, and frozen therein at -80.degree. 
C. These were freeze-dried in vacuo. After drying, the same amount, as 
that before freeze-drying, of a distilled water for injection was added to 
the freeze-dried product so that the product was dissolved. The 
antigen-binding activity of the monoclonal antibody in the resulting 
solution was measured. 
Measurement of the antigen-binding activity of the anti-Pseudomonas 
aeruginosa antibody was carried in the manner mentioned below. A 
lipopolysaccharide (LPS), as prepared from formalin-killed cells of Group 
E serotype Pseudomonas aeruginosa ATCC 27581 by Tanabe et al's method 
(Menekijikkensousahou C, (1978) 1793-1801), was dissolved in PBS to have a 
concentration of 1 mg/ml, and this was diluted by 500-fold with 0.1M 
phosphate buffer (pH 7.0). The thus diluted solution was then put in wells 
of a 96-well EIA plate (Immulon-600; Greiner Co.) in an amount of 50 
.mu.l/well. The plate was allowed to stand at 4.degree. C. overnight for 
coating, and it was then washed with PBS containing 0.05% TWEEN 20 
(hereinafter referred to as a "washing solution"). A PBS containing 0.5% 
bovine serum albumin (hereinafter referred to as a "blocking solution") 
was added to each well in an amount of 200 .mu.l/well, and the plate was 
then shaken at room temperature for one hour so that the non-specific 
protein-binding sites were saturated. After the blocking solution was 
removed, solutions of a sample to be tested, each having a multi-fold 
diluted concentration in order from a determined concentration, were put 
in the wells each in an amount of 100 .mu.l/well, and the plate was then 
shaken for 2 hours at room temperature. After washing with the washing 
solution four times, a peroxidase-labeled goat anti-human IgM antibody 
(Tago Co.) was diluted by 1000-fold with the blocking solution, and was 
put in each well in an amount of 100 .mu.l/well, and the plate was shaken 
at room temperature for 2 hours. After washing with the washing solution 
four times and then with 0.1M citric-acid buffer (pH 4.0) one time, a 
substrate solution containing 1 mg/ml of 
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 0.003% hydrogen 
peroxide in the same buffer was put into each well in an amount of 50 
.mu.l/well, and the plate was then shaken at room temperature. After 30 
minutes, 2% succinic acid was added to each well in an amount of 50 
.mu.l/well so that the enzymatic reaction therein was stopped. The 
absorbance at 414 nm was measured with a 96-well plate reader (Nippon 
InterMed Co.). Double logarithmic plotting was done between the reciprocal 
of the diluting magnification and the absorbance, and the diluting 
magnification to show the absorbance of being 0.1 was obtained and 
indicated as the antigen-binding activity of the sample tested. 
The results are shown in Table 1, as a relative activity on the basis of 
the antigen-binding activity of the sample before being frozen of 10.Where 
the monoclonal antibody was freeze-dried without addition of gelatin 
thereto, the antigen-binding activity noticeably decreased. As opposed to 
the case, when gelatin was added, the antigen-binding activity was well 
recovered even in the freeze-dried product, and the effect depended upon 
the concentration of the gelatin added. 
TABLE 1 
______________________________________ 
Gelatin Concentration 
Antigen-Binding Activity 
(%) Neutral Gelatin 
Acidic Gelatin 
______________________________________ 
0 2 2 
0.001 4 3 
0.003 6 6 
0.01 10 8 
0.03 10 10 
0.1 10 10 
1 10 10 
______________________________________ 
Example 2 
The same monoclonal antibody as that used in Example 1 was dissolved in 
various buffers each having a different pH value to each have a final 
concentration of 0.1 mg/ml. On the other hand, a neutral gelatin was added 
to each of them to have a final concentration of 0.01%. Each was put in 
polypropylene cryotubes each in an amount of 0.5 ml under a sterilized 
condition and frozen at -80.degree. C. These were then freeze-dried in 
vacuo. The same amount, as that before freeze-drying, of a distilled water 
for injection was added to the freeze-dried product so that the product 
was dissolved, and the antigen-binding activity of the monoclonal antibody 
in the resulting solution was measured. 
The results obtained are shown in Table 2, as a relative activity based on 
the activity of the sample before being frozen of 10. At every pH 
condition, the antigen-binding activity was well recovered. 
TABLE 2 
______________________________________ 
Gelatin Antigen-Binding 
Buffer (0.2M) 
pH Concentration (%) 
Activity 
______________________________________ 
Sodium Citrate 
4.0 0.01 10 
5.0 0.01 10 
6.0 0.01 10 
Sodium Phosphate 
6.2 0.01 10 
7.0 0.01 10 
8.1 0.01 10 
______________________________________ 
Example 3 
0.1 mg,ml, as a final concentration, of the same monoclonal antibody as 
that used in Example 1 was dissolved in 20 mM phosphate buffer (pH 7) not 
containing or containing 2 or 10 mM sodium citrate, whereupon the salt 
concentration of the resulting solution was adjusted to be 150 mM with 
sodium chloride. The monoclonal antibody solution was then put in 
polypropylene cryotubes under sterile conditions, each in an amount of 0.5 
ml, and frozen therein at -80.degree. C. These were freeze-dried in vacuo. 
The same amount, as that before freeze-drying, of a distilled water for 
injection was added to the freeze-dried product so that the product was 
dissolved. The antigen-binding activity of the monoclonal antibody in the 
resulting solution was measured. 
The results obtained are shown in Table 3, as a relative activity based on 
the activity of the sample before being frozen of 10. Where the monoclonal 
antibody was freeze-dried in the absence of sodium citrate, the 
antigen-binding activity of the freeze-dried monoclonal antibody 
noticeably decreased. As opposed to the case, when the monoclonal antibody 
was freeze-dried in the presence of sodium citrate, then the 
antigen-dinding activity of the freeze-dried monoclonal antibody was well 
recovered, depending upon the concentration of the sodium citrate added. 
TABLE 3 
______________________________________ 
Sodium Citrate Concentration (mM) 
0 2 10 
______________________________________ 
Antigen-Binding Activity 
2 5 9 
______________________________________ 
Example 4 
0.1 mg/ml, as a final concentration, of the same monoclonal antibody as 
that used in Example 1 was dissolved in 50 mM phosphate buffer (pH 6.1 to 
8.1) containing sodium citrate in a concentration of from 10 mM to 200 mM, 
whereupon sodium chloride was added thereto, if necessary, so that the 
salt concentration of the resulting solution became 160 mM. The resulting 
monoclonal antibody solution was then put in polypropylene cryotubes under 
a sterilized condition, each in an amount of 0.5 ml, and frozen therein at 
-80.degree. C. These were freeze-dried in vaccuo. The same amount, as that 
before freeze-drying, of a distilled water for injection was added to the 
freeze-dried product so that the product was dissolved. The 
antigen-binding activity of the monoclonal antibody in the resulting 
solution was measured. 
The results obtained are shown in Table 4, as a relative activity based on 
the activity of the sample before being frozen of 10. By adding sodium 
citrate at pH of from 6.1 to 8.1, the antigen-binding activity of all the 
freeze-dried products was well recovered. 
TABLE 4 
______________________________________ 
Antigen-Binding Activity 
pH of Sodium Citrate Concentration (mM) 
Solution 10 50 100 200 
______________________________________ 
6.1 10 10 10 10 
7.0 9 10 10 10 
8.1 9 10 10 -- 
______________________________________ 
Example 5 
0.1 mg/ml, as a final concentration, of the same monoclonal antibody as 
that used in Example 1 was dissolved in PBS. In addition, 0.003%, as a 
final concentration, of a neutral gelatin was added thereto, and further 
glucose, sucrose, mannitol, glycine or arginine was added thereto in an 
amount, as a final concentration, of from 0.001 to 0.1%. The resulting 
monoclonal antibody solution was then put in polypropylene cryotubes under 
a sterilized condition, each in an amount of 0.5 ml, and frozen therein at 
-80.degree. C. These were freeze-dried in vacuo. The same amount, as that 
before freeze-drying, of a distilled water for injection was added to the 
freeze-dried product so that the product was dissolved. The 
antigen-binding activity of the monoclonal antibody in the resulting 
solution was measured. 
The results obtained are shown in Table 5, as a relative activity based on 
the activity of the sample before being frozen of 10. The antibody 
activity was well recovered in all cases of the low molecular substances, 
depending upon the concentration of them. 
TABLE 5 
______________________________________ 
Concentra- 
Concentra- 
tion of Low 
Antigen-Binding Activity 
tion of Gela- 
Molecular man- argin- 
tin (%) Substance (%) 
glucose sucrose 
nitol 
glycine 
ine 
______________________________________ 
0.003 0.001 7 7 7 8 7 
0.003 0.003 8 8 7 8 8 
0.003 0.01 8 6 8 10 8 
0.003 0.03 8 8 8 10 8 
0.003 0.1 8 10 8 10 8 
0.003 0 6 6 6 6 6 
______________________________________ 
Example 6 
0.1 mg/ml, as a final concentration, of the same monoclonal antibody as 
that used in Example 1 was dissolved in PBS. In addition, 0.003%, as a 
final concentration, of a neutral gelatin was added thereto, and further 
0.5 or 1%, as a final concentration, of mannitol was added thereto. The 
resulting monoclonal antibody solution was then put in polypropylene 
cryotubes under a sterilized condition, each in an amount of 0.5 ml, and 
frozen therein at -80.degree. C. These were freeze-dried in vacuo. The 
same amount, as that before freeze-drying, of a distilled water for 
injection was added to the freeze-dried product so that the product was 
dissolved. The antigen-binding activity of the monoclonal antibody in the 
resulting solution was measured. 
The results obtained are shown in Table 6, as a relative activity based on 
the activity of the sample before being frozen of 10. The antigen-binding 
activity was well recovered in all the freeze-dried products, each 
containing a different concentration of mannitol. 
TABLE 6 
______________________________________ 
Concentration of Mannitol (%) 
0.5 1.0 
______________________________________ 
Antigen-Binding Activity 
10 10 
______________________________________ 
Example 7 
1 mg/ml, as a final concentration, of the same monoclonal antibody as that 
used in Example 1 was dissolved in 0.1M phosphate buffer (pH 7.0) 
containing a neutral gelatin (0.01%), sodium citrate (0.02M), mannitol 
(0.5%) and sodium chloride (0.05M). The resulting monoclonal antibody 
solution was then put in 10 ml-volume glass vials (Iwaki Glass Co.) under 
a sterilized condition, each in an amount of 1 ml, and frozen therein at 
-80.degree. C. These were freeze-dried in vacuo. The same amount, as that 
before freeze-drying, of a distilled water for injection was added to the 
freeze-dried product so that the product was dissolved. The 
antigen-binding activity of the monoclonal antibody in the resulting 
solution was measured. As a result, the freeze-dried monoclonal antibody 
products were found to have the same antigen-binding activity as that of 
the samples before being frozen. 
Example 8 
1 mg/ml, as a final concentration, of the same monoclonal antibody as that 
used in Example 1 was dissolved in 0.1M phosphate buffer (pH 7.0) 
containing sodium citrate (0.02M), sodium chloride (0.05M) and mannitol 
(0.5%). The resulting monoclonal antibody solution was then put in glass 
vials and frozen therein at -80.degree. C. These were freeze-dried in 
vacuo. The same amount, as that before freeze-drying, of a distilled water 
for injection was added to the freeze-dried product so that the product 
was dissolved. The antigen-binding activity of the monoclonal antibody in 
the resulting solution was measured in the same manner as in Example 1. As 
a result, the freeze-dried monoclonal antibody products were found to have 
the same antigen-binding activity as that of the samples before being 
frozen. 
Example 9 
Cells of human-human hybridoma MP5121 (FERM BP-2270) producing a human IgM 
reactive to Group A serotype Pseudomonas aeruginosa, which had been 
produced by cell fusion, were cultured, and the monoclonal antibody was 
purified from the culture supernatant in the same manner as in Example 1. 
The monoclonal antibody was dissolved in 0.1M phosphate buffer (pH 7.0) 
containing sodium citrate (0.02M), sodium chloride (0.05M) and mannitol 
(0.5%), to have a final concentration of 1 mg/ml. The resulting monoclonal 
antibody solution was then put in glass vials and frozen therein at 
-80.degree. C. These were freeze-dried in vacuo. The same amount, as that 
before freeze-drying, of distilled water for injection was added to the 
freeze-dried product so that the product was dissolved. The 
antigen-binding activity of the monoclonal antibody in the resulting 
solution was measured in the same manner as in Example 1, provided that 
the antigen LPS was extracted from Group A serotype Pseudomonas aerugionsa 
(ATCC 27577). As a result, the freeze-dried monoclonal antibody products 
were found to have the same antigen-binding activity as that of the 
samples before being frozen. 
Example 10 
Monoclonal antibodies were purified from culture super-natants of cells of 
human IgM-producing human-human hybridoma MP5097, MP5139, MP5114 and 
MP5156 (FERM BP-2268, 2272, 2269 2339, respectively), all of which had 
been produced by cell fusion. These monoclonal antibodies had reactivity 
with Pseudomonas aeruginosa and were reactive to Groups B, E, G and I 
serotypes Pseudomonas aeruginosa, respectively. Five kinds of monoclonal 
antibodies comprising 4 kinds of these monoclonal antibodies and the 
monoclonal antibody used in Example 9 were dissolved in 0.1M phosphate 
buffer (pH 7.0) containing sodium citrate (0.02M), sodium chloride (0.05M) 
and mannitol (0.5%), each in an amount, as a final concentration, of 5 
mg/ml. These monoclonal antibody solutions were put in glass vials and 
frozen therein at -80.degree. C. These were freeze-dried in vacuo. The 
same amount, as that before freeze-drying, of a distilled water for 
injection was added to each of the freeze-dried products so that each 
product was dissolved. The antigen-binding activity of each of the 
monoclonal antibodies in the resulting solutions was measured in the same 
manner as in Example 1, provided that as the antigen to each antibody, 
used were LPSs as extracted from ATCC 27577 (to Group A serotype), ATCC 
27578 (to Group B serotype), ATCC 27581 (to Group E serotype), ATCC 27584 
(to Group G serotype) and ATCC 27586 (to Group I serotype), respectively. 
As a result, the freeze-dried monoclonal antibody products were found to 
have the same antigen-binding activity to the five Pseudomonas aeruginosa 
LPSs of different serotypes, respectively, as that of the samples before 
being frozen. 
In accordance with the present invention characterized by addition of 
gelatin or carboxylic acid or its salt to a monoclonal antibody-containing 
solution to be freeze-dried, denaturation of a monoclonal antibody during 
freeze-drying can be prevented so that a monoclonal antibody-containing 
freeze-dried preparation having a stable antigen-binding activity can be 
provided. The present invention may be applied to a monoclonal antibody of 
any immunoglobulin class, including IgG, IgM, IgA and IgE. Especially, it 
can be applied to unstable IgM. The present invention may be well applied 
to any human-derived, mouse-derived and rat-derived monoclonal antibodies. 
Various numbers and kinds of monoclonal antibodies may be contained in the 
freeze-dried preparation of the present invention. 
The monoclonal antibody-containing freeze-dried preparation of the present 
invention may be used, like other immunoglobulin-preparations, as an 
adminiculum for immunoauxotherapy for prophylaxis and treatment of 
bacterial infectious diseases and viral infectious diseases. 
The following deposits were made under the terms of the Budapest Treaty on 
the International Recognition of the Deposit of Microorganisms for the 
Purposes of Patent Procedure at the Fermentation Research Institute, 
Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, 
Tsukuba-shi, Tbaraki-ken, 305, Japan: 
______________________________________ 
Depositor's Date of FERM Deposit 
Designation Deposit Number 
______________________________________ 
MP5038 Dec. 7, 1989 
BP-1596 
MP5097 Feb. 7, 1989 
BP-2268 
MP5139 Feb. 7, 1989 
BP-2272 
MP5114 Feb. 7, 1989 
BP-2269 
MP5156 Mar. 16, 1989 
BP-2339 
MP5121 Feb. 7, 1989 
BP-2270 
______________________________________