Method for producing an IgM preparation for intravenous application

In the method for producing an immunoglobulin solution suitable for intravenous application with an IgM proportion of more than 5% by weight with respect to the immunoglobulin proportion, an IgM-containing immunoglobulin solution is treated with a protease. The intravenously well tolerated preparation obtained is characterized by not being chemically modified and by having low anticomplementary activity ACA.

This application claims priority to PCT/CH97/00388, filed Oct. 14, 1997. 
This invention relates to a method of producing an immunoglobulin solution 
suitable for intravenous application. Used as the starting material is a 
protein fraction obtained from human or animal blood which contains the 
immunoglobulins in concentrated form. 
As is well known, immunoglobulins play an important role in the immune 
system of man and mammal in fighting off infections. The immunoglobulins 
are divided up into different classes (e.g. IgG, IgA, IgM, IgD and IgE) 
with differing biochemical and physiological properties. Until 1980 only 
IgG was isolated and used as an IV-well-tolerated product for prophylaxis 
and therapy. In EP-A-0 013 901, EP-A-0 413 187 and EP-A-0 352 500 IgM 
preparations are described, which have been made intravenously well 
tolerated mainly through treatment with .beta.-propiolactone. EP-A-0 413 
188 describes a method in which the IV-tolerance is achieved through anion 
exchange chromatography with selective elution of the IV-tolerant 
fraction. 
Object of the present invention is the production of a highly purified, IgM 
concentrate suitable for intravenous administration for therapy and 
prophylaxis. The product should have a low anticomplementary activity 
(ACA), and demonstrate a low blood pressure drop in a rat model, but the 
IgM molecules should not be chemically modified, however. This object was 
obtained surprisingly through treatment of a IgM-containing immunoglobulin 
solution with a protease. 
The subject matter of the invention is therefore the method defined in 
claim 1. 
The protease treatment is preferably an incubation at raised temperature in 
the presence of pepsin, papain, plasmin or thermolysine. The proteases can 
also be chemically modified, immobilized on a substrate and/or produced 
through genetic engineering. The preparation according to the inventive 
method can be transferred into an IV-administrable solution. Such a 
solution displays a reduction of the ACA, of the blood pressure drop in a 
rat model and of the C1q binding activity. 
Suitable as starting materials for the method according to the present 
invention are immunoglobulin-containing solutions, such as, e.g. plasma, 
precipitate A or B from Kistler-Nitschmann-fractionation; Cohn fraction 
I/II/III; II/III; III; or other IgM-containing plasma fractions from human 
or animal plasma. For example, an immunoglobulin-containing fraction, such 
as precipitate B according to Kistler-Nitschmann, can be dissolved in a 
buffer, most of the impurities being removed through a precipitation with 
0.5 to 5% octanoic acid at pH 4 to 6, preferably pH 5. Afterwards the 
solution is incubated at low ionic strength for 1 to 48 hours, preferably 
9 hours, at a temperature of 20 to 50.degree. C., preferably 37.degree. C. 
with addition of at least 50 U/g of pepsin, preferably 600 U/G. 
For further purification, the solution can be subjected to an adsorption, 
for example with a gel containing a DEAE-group in batch or column method. 
If the IgM concentration in the end product is supposed to be increased 
further, the IgM solution is put on an ion exchanger (e.g. 
TMAE-Fraktogel.RTM.). Through a selective elution, e.g. by means of a salt 
gradient or pH gradient, the IgM fraction can be isolated. Through 
ultrafiltration and diafiltration, for example a gel filtration, the 
solution can be concentrated and the electrolyte content can be adjusted 
to a final, intravenously well tolerated formulation. The protein 
concentration can amount to 1 to 20%, preferably 3 to 6%. The product can 
contain in addition proteins, preferably albumin, as well as sugar, 
preferably glucose or sucrose, or amino acids. 
To assess the intravenous compatibility of immunoglobulin preparations, the 
anticomplementary activity (ACA) is usually used. To determine the ACA, a 
defined quantity of the product to be tested is incubated with a defined 
quantity of guinea pig complement and the remaining quantity of complement 
titrated. The ACA is indicated as consumption of CH50 per g of 
immunoglobulin. The indicated results of the ACA were determined to a 
large extent according to the method published by M. Mayer (Mayer, M. M. 
(1961), "Complement and Complement Fixation" in Experimental 
Immunochemistry, 2.sup.nd edition, pp. 133-240, C Thomas, Springfield, 
Ill.). Valid as a guide value for intravenously usable IgG products is an 
ACA of &lt;1000 CH50 per g of protein. 
To assess intravenous compatibility, the binding of the C1q complement 
components to the immunoglobulin can be further used. For determination, a 
defined quantity of test product is incubated with a defined quantity of 
purified, radioactively labelled C1q complement in buffer and in serum. 
The C1q binding activity of the test product is determined through 
precipitation in the presence of polyethylene glycol. The higher the 
radioactivity in the precipitate, the greater the C1q binding activity of 
the product. Finer predictions about the type of C1q binding and thereby 
the quality of the product can be achieved if the C1q is radioactively 
labelled with two different methods. On the one hand, under as mild as 
possible oxidative conditions with lactoperoxidase (LPO), and, on the 
other hand, under drastic oxidative conditions with chloramine T (CT). The 
tests were carried out to a large extent according to the method published 
by P. Spath (P. J. Spath, A. Corvetta, U. E. Nydegger, R. Buttler: "An 
Extended C1q-Binding Assay Using Lactoperoxidase- and 
Chloramin-T-iodinated C1q," Scand. J. Immunol. 18, 319-328, 1983). 
Expected of an intact, intravenously well tolerated preparation is that 
the C1q binding activity is as minimal as possible. A model for testing 
the IV compatibility of immunoglobulins is the rat model according to 
Bleeker et al. [W. K. Bleeker, J. Agterberg, G. Rigter, A. de Vries-van 
Rossen, J. C. Bakker: "An Animal Model for the Detection of Hypotensive 
Side Effects of Immunoglobulin Preparations,", Vox. Sang. 52:281-290 
(1987)]. Tolerance parameter in this model is blood pressure. 
Intravenously poorly tolerated products lead to a significant drop in 
blood pressure.

EXAMPLES 
Reference Example 1 
1 kg of precipitate B according to Kistler-Nitschmann was suspended in 4 kg 
of 0.1 mol/l acetate buffer, pH 5.1, and 2% octanoic acid was added at 
room temperature. 0.15 g of tricalcium phosphate was added per g of 
octanoic acid, and the precipitate filtered off. The filtrate was 
diafiltered against 20 mmol/l piperazine, 60 mmol/l NaCl, pH 5.8. The 
diafiltered solution was treated with 75 mg DEAE-Sephadex.RTM. per g of 
protein. Then the protein concentration was adjusted to 20 mg/ml, and the 
solution was treated with 1% Tweene.RTM. 80 and 0.3% TNBP 
(tri-n-butyl-phosphate) for 8 hours at 25.degree. C. The solution was then 
put on a TMAE-Fraktogel.RTM. column, and the IgM fraction was eluted with 
20 mmol/l piperazine, 160 mmol/l NaCl, pH 5.8. The end product was 
concentrated to 5% protein, and the pH value adjusted to 4.5. 
Reference Example 2 
1 kg of precipitate B according to Kistler-Nitschmann was suspended in 4 kg 
of 0.1 mol/l acetate buffer, pH 5.1, and 2% octanoic acid was added at 
room temperature. 0.15 g of tricalcium phosphate was added per g of 
octanoic acid, and the precipitate filtered off. The filtrate was 
diafiltered against 20 mmol/l NaCl diafiltered, and the solution brought 
to 20 mg/ml protein. The pH value was adjusted with 0.2 mol/l HCl to 4.0, 
and the solution incubated for 9 hours at 37.degree. C. After cooling down 
to 20.degree. C., the pH was adjusted to 5.8, and piperazine ad 20 mmol/l 
and NaCl ad 60 mmol/l added. The solution was subsequently treated with 1% 
Tween.RTM. 80 and 0.3% TNBP (tri-n-butyl-phosphate) for 8 hours at 
25.degree. C. The solution was then put on a TMAE-Fraktogel.RTM. column, 
and the IgM fraction was eluted with 20 mmol/l piperazine, 160 mmol/l 
NaCl, pH 5.8. The end product was concentrated to 5% protein, and the pH 
value adjusted to 4.5. 
Example 1 
1 kg of precipitate B according to Kistler-Nitschmann was suspended in 4 kg 
of 0.1 mmol/l acetate buffer, pH 5.1, and 2% octanoic acid was added at 
room temperature. 0.15 g of tricalcium phosphate was added per g of 
octanoic acid, and the precipitate filtered off. The filtrate was 
diafiltered against 20 mmol/l NaCl diafiltered, and the solution brought 
to 20 mg/ml protein. The pH value was adjusted with 0.2 mmol/l HCl to 4.0, 
and 600 U pepsin per g of protein were added. Then the solution was 
incubated for 9 hours at 37.degree. C. After cooling down to 20.degree. 
C., the pH was adjusted to 5.8, and piperazine ad 20 mmol/l and NaCl ad 60 
mmol/l added. The solution was subsequently treated with 1 % Tween.RTM. 80 
and 0.3% TNBP (tri-n-butyl-phosphate) for 8 hours at 25.degree. C. The 
solution was then put on a TMAE-Fraktogel.RTM. column, and the IgM 
fraction was eluted with 20 mmol/l piperazine, 160 mmol/l NaCl, pH 5.8. 
The end product was concentrated to 5% protein, and the pH value adjusted 
to 4.5. 
Example 2 
1 kg of precipitate B was prepared analogously to reference example 2, 
instead of 600 U of pepsin per g of protein of the solution, 1200 U of 
pepsin per g of protein being added, however, before the pH 4 incubation. 
I. Characterization of the Experimental Products 
The immunoglobulins IgG, IgA and IgM were nephelometrically determined with 
antisera. The total protein content was determined with the Kjeldahl 
method. 
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Protein IgG IgA IgM Isoagglutinins 
mg/g mg/g mg/g mg/g Anti-A Anti-B 
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Reference 
44.4 4.1 16.8 45.0 1:64 1:64 
example 1 
Reference 
48.1 8.6 19.3 43.5 1:128 1:64 
example 2 
Example 1 
51.3 7.0 22.0 50.5 1:128 1:64 
Example 2 
46.9 4.7 20.2 51.0 1:128 1:64 
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TABLE II 
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Tolerance Parameters 
Rat model 
Blood C1q-binding 
pressure 
Puffer Serum 
ACA drop LPO CT LPO CT 
Treatment CH50/g % % % % % 
______________________________________ 
Reference 
Without pH 4 
515 19 1.5 0.1 43 5.5 
example 1 
Reference 
pH 4 179 18 0 0.5 42 6.7 
example 2 
Example 1 
pH 4 with 125 7 0 0.3 34 3.9 
600 U pepsin 
Example 2 
pH 4 with 89 2 0 0.5 23 3.7 
1200 U pepsin 
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The addition of pepsin causes a reduction of the ACA, a lessening of the 
blood pressure drop in the rat model as well as a reduction in C1q binding 
activity.