Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1994-06-13
2001-03-27
Marx, Irene (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C424S094640, C424S094100
Reexamination Certificate
active
06207151
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to protein-containing aqueous solutions, methods for increasing the protein concentration of aqueous solutions and a protein preparation and to techniques applicable in the preparation of pharmaceuticals for clinical use using physiologically active proteins.
2. Description of the Prior Art
In using a protein as a homogeneous component, it is extremely important to dissolve the protein in a solvent. For example, where a certain amount of a protein is fractionated from a composition which contains that protein or where analysis of the protein is made, the composition containing the protein must be homogeneous. Furthermore, when a protein is dissolved in water for administration as a pharmaceutical such as an injectable preparation, the protein has to be completely dissolved.
In general, the solubility of proteins in aqueous solvents is strongly affected by hydrophilic or hydrophobic residues present on the surface of the protein and by charges on the protein. When the protein is only slightly dissolved because of the presence of hydrophobic residues on the surface of the protein, it is possible to increase the solubility by adding a surfactant.
On the other hand, when the pH of an aquaeous solvent is near the isoelectric point of the protein to be dissolved, which readily causes isoelectric precipitation, solubility of the protein can be increased by Increasing the salt concentration and the ionic strength of the aqueous solvent. In this case, a surfactant does not contribute to the increase of the protein solubility. Furthermore, when the pH of an aqueous solvent is near the isoelectric point of a protein and the salt concentration is low, the protein is soluble only at a relatively low concentration. Therefore, in order to dissolve the protein in a relatively high concentration, either a method in which a pH separate from the isoelectric point is used or a method in which the salt concentration is increased is generally used.
However, in some cases, It Is necessary to dissolve a protein at a sufficiently high concentration without increasing the salt concentration at the pH near the isoelectric point. An example is when a physiologically active protein having an isoelectric point near neutral pH is to be administered, in a form of a solution at a pH near neutral, to a patient who should maintain his or her salt Intake as low as possible. In this case, the only possible technique has been either to use the protein in a lower concentration or to inevitably administer salt, which is a serious practical problem to be solved in the formulation of protein active pharmaceuticals.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a highly concentrated aqueous protein solution. Another object of the present invention is to provide a method in which a protein can be dissolved at a high concentration in an aqueous solution. Another object of the present invention Is to provide a protein preparation having excellent solubility.
In achieving the above objectives, the present inventors found that a protein present in a solution binds to an ion exchanger if the pH of the solution is appropriate, that the binding of the protein to the ion exchanger was prone to take place especially at a low salt concentration of the solution, and that the solubility of a complex of the protein and ion exchanger is much better than the solubility of the protein itself in the solution especially at a pH near the isoelectric point. This phenomenon resembles one in which a surfactant dissolves a hydrophobic protein by forming a complex of the protein and the surfactant.
Based on this finding, the present inventors additionally determined in the course of further investigations that anionic polymers or salts of anionic polymers exert a favorable effect mentioned above, and thus completed the present invention.
The present invention provides an aqueous solution containing a protein in which an anionic polymer or a salt thereof coexists with the protein; a method for increasing the protein concentration of an aqueous solution containing the protein by arranging an anionic polymer or a salt thereof to coexist with the protein; and a pharmaceutical composition containing an anionic polymer or a salt thereof and a protein.
A pharmaceutical preparation containing a saccharide, a type of anionic polymer, such as polysulfate or a sulfonated sugar and t-PA (tissue-type plasminogen activator) has been disclosed in Japanese Patent Laid-open No. 236730/1986. However, the disclosed technique is to improve t-PA activity and is fundamentally different, in terms of technological idea, from that of the present invention which more broadly is to improve the solubility of proteins in general.
The present invention enables a protein to dissolve in an aqueous solution, especially in an aqueous solution which has a low salt concentration and a pH near the isoelectric point of the protein, to a degree the conventional techniques have never been able to achieve.
Conventionally, in order to dissolve a protein at a pH near the protein's isoelectric point, it is essential to significantly (1) decrease the protein concentration or (2) increase the salt concentration. The resulting protein solution is extremely inconvenient when a physiologically active protein has to be administered in the form of a solution at a pH near the isoelectric point of the protein to a patient whose salt intake is restricted. In order to dissolve the protein without increasing the salt concentration, it is essential to select a pH separate from the pH near the isoelectric point of the protein even if the selected pH is undesirable for the use in pharmaceuticals such as injections or other parenteral dosage form.
By contrast, according to the present invention, proteins can be dissolved without increasing the salt concentration at a pH near the isoelectric point of the protein so that a protein-containing aqueous solution with a low salt concentration at a pH near the isoelectric point of the protein can be provided. The present Invention provides an advantageous technique as compared to conventional ones.
The present invention is particularly suitable for preparing low salt pharmaceutical preparations for injection of a physiologically active protein having an isoelectric point near neutral.
DETAILED DESCRIPTION AND THE PREFERRED EMBODIMENTS
Examples of proteins to be used in the present invention include physicochemically simple proteins. conjugated proteins and induced proteins as well as proteins having relatively large amounts of hydrophobic groups. In particular, this invention is suitably applied to proteins such as globulin or t-PA which tend to drastically decrease their solubility at a pH ranging around the isoelectric points. Examples include proteins having an isoelectric point apart from the extremely acid region, preferably pH 4 or higher, and desirably a pH 5 or higher. These proteins may be obtained by extraction and purification from naturally occurring biological bodies or parts thereof, by chemical synthesis or by using genetic recombinant DNA techniques from cell culture. The protein obtained as mentioned above can be used after modification. Examples of these proteins include gamma-globulins such as immunoglobulins A, G and E, lactoglobulin, urokinase, pro-urokinase and tissue-type plasminogen activator (t-PA). A more preferable example is t-PA having a solubility of 2.0 mg/ml or less and preferably 1.0 mg/ml or less, at a pH of about 7.3 in 1/15 M phosphate buffer. In particular, the present invention is specifically adapted for proteins having physiological activities.
The proteins can be used alone or as a mixture of two or more proteins or different types of proteins.
Examples of anion residues of anionic polymers used in the present invention include carboxyl, carboxymethyl, sulfuric and phosphoric groups. Examples of polymer backbones of the anionic polymers include sugars such as sugar alcohol, cellulose, amylose, amino acid
Ishiwari Hisahiro
Kawashima Nobuhiro
Sakai Kiyoshi
Shimazaki Yukio
Suzuki Miki
Marx Irene
Mitsui Chemicals Inc.
Nixon & Vanderhye
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