JP2016028102A - Inhibitor for aggregation of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a safe inhibitor for the aggregation of protein that can inhibit the aggregation of protein and prepare a low viscous protein solution.SOLUTION: An inhibitor for the aggregation of protein includes a peptide surfactant.SELECTED DRAWING: Figure 1

Description

The present invention relates to a protein aggregation inhibitor containing a peptide surfactant.

Proteins are unstable in aqueous solutions and aggregate due to changes in natural conformation and protein association. In the current technology, aggregation is suppressed by adding a denaturing agent such as urea or a specific amino acid to an aqueous protein solution. Furthermore, as a means for improving protein solubility, a surfactant having a weak interaction with a protein such as Tween 80 or Triton X-100 is used. However, the existing technology has a problem in that the structure of the protein is denatured or the activity is inhibited.

There are many proteins that can serve as therapeutic agents for many diseases, but it is very difficult to formulate them while maintaining their pharmacological effects when developing protein drugs. In particular, antibody drugs that have been attracting attention in recent years are problematic due to the high viscosity of the preparation resulting from antibody aggregation, which hinders product development. In order to solve these problems, development of a protein aggregation inhibitor that is safe and has an extremely low influence on the structure and activity of the protein and consequently reduces the viscosity of the protein solution is awaited.

The peptide surfactant of the present invention is known to have an action of stabilizing membrane proteins such as G protein-coupled receptor bovine rhodopsin and a self-assembling property (Non-patent Documents 1 and 2). However, its use as a protein aggregation inhibitor has not been known so far.

X Zhao et al. , PNAS, Vol. 103, no. 47, 17707-17712 A Nagai et al. , J .; Nanosci. Nanotechnol. Vol. 7, no. 7, 1-7

The problem to be solved by the present invention is to provide a protein aggregation inhibitor that is safe, suppresses protein aggregation, and can prepare a protein solution with low viscosity.

As a result of intensive studies to solve the above problems, the present inventors have surprisingly found that a peptide surfactant is useful as a protein aggregation inhibitor and completed the present invention.

Therefore, the present invention
1. A protein aggregation inhibitor that contains a peptide surfactant and is safe and reduces the viscosity of the protein solution;
2. The protein aggregation inhibitor according to 1 above, wherein the peptide surfactant comprises a head composed of 4 to 10 hydrophilic amino acids and a tail composed of 1 or 2 hydrophobic amino acids,
3. The protein aggregation inhibitor according to 1 above, wherein the peptide surfactant is AAAAAAD or AAAAAAK,
4). The protein aggregation inhibitor according to the above 1, wherein the protein is a monoclonal antibody,
5. The protein aggregation inhibitor according to 2 above, wherein the protein is a monoclonal antibody,
6). 4. The protein aggregation inhibitor according to 3 above, wherein the protein is a monoclonal antibody.

In the present invention, a peptide surfactant includes 6 to 10 amino acid residues and has a length of about 2 to 3 nm, and is a conventional surfactant such as n-dodecyl-β. It exhibits the same characteristics as -D-maltoside (DM) and octyl-D-glucoside (OG).

The peptide surfactant of the present invention preferably comprises a head composed of 4 to 10 hydrophilic amino acids and a tail composed of 1 or 2 hydrophobic amino acids.

More specifically, GGGGDD (G4D2), GGGGGGDD (G6D2), GGGGGGGGDD (G8D2), GGGGGGGGGDD (G10D2), AAAAAAD (A6D), A6AA, A6A, A6A, A6A, A6A , VVVVVVDD (V6D2), VVVVVVK (V6K), VVVVVVKK (V6K2), LLLLDD (L2D2), LLLLLLD (L6D), LLLLLLDDD (L6D2), LLLLLLK (L6K), or L3LL (L6K) ), FFFFRR (F4R2), among which AAAAAAD (A6D) or AAAAAAAK (A6K) Preferably, AAAAAAK (A6K) is particularly preferred.

The method for producing the peptide surfactant is synthesized by a solid phase synthesis method. Since the peptide can be artificially synthesized, it does not contain biological substances and there is no risk of infection.

In the present invention, a protein aggregation inhibitor refers to a composition that can be used to suppress protein aggregation when a protein containing an antibody is formulated for medical application, and can reduce the viscosity of a protein solution. Say.

Comparison of viscosities of albumin solutions with different concentrations and 0.05% A6K solution

Aggregation inhibition of high-concentration albumin solution by low-concentration A6K solution High-concentration albumin solution is serially diluted with purified water or A6K solution, the viscosity of all solutions is measured, and the protein aggregation inhibition effect (viscosity reduction effect) of A6K is analyzed. As a result, it was confirmed that the aggregation of albumin was suppressed and the viscosity of the solution was reduced by the A6K solution.

<Viscosity measuring device>
Rotary dynamic viscoelasticity measuring device: HAAKE RheoStress 600
Sample stage: 20 mm diameter aluminum plate (attached to the device)
Measuring sensor: 20mm diameter cone-type aluminum cap (attached to the device)
<Material>
Albumin (derived from bovine serum; Sigma-Aldrich)
Concentration: 160 mg / mL
Peptide surfactant sequence: Ac-AAAAAAK-NH2, Celtek concentration: 10 mg / mL (stock solution)
·purified water

<Method>
-Preparation of albumin solution Gently add 320 mg of albumin powder to 1 mL of purified water and leave it to dissolve.
-Preparation of A6K solution Add 900 μL of purified water to 100 μL of stock solution to prepare 0.1% A6K solution.
Add 300 μL of purified water or 0.1% A6K solution to 300 μL of the albumin solution prepared on dilution of the albumin solution to prepare a solution with an albumin concentration of 160 mg / mL. The same operation is continuously performed to prepare 300 μL each of albumin concentrations of 80 mg / mL and 40 mg / mL.
-Setting the viscosity measuring device Set the sample stage temperature to 23 ° C, and fix the gap between the sample stage and the sensor to 0.5 mm. The CS-CR flow curve is selected as the measurement mode, and the shaft rotation speed is set to 0-100 r / s with a linear acceleration for 1 minute.
• Place 100 μL of each of the albumin solutions prepared for measuring the viscosity of the solution on the sample stage, and measure the viscosity. The viscosity is calculated from the measured shear stress and the rotation speed, and the obtained maximum viscosity is taken as the viscosity of each sample. The measurement was performed once for each sample.

<Result>
When each solution was visually confirmed, the albumin solution dissolved in purified water became cloudy over time, but the albumin solution to which the A6K solution was added was clear. The result of the viscosity measurement is shown in FIG. At three albumin concentrations, the concentration of the albumin solution was lower when the A6K solution was used than with purified water. Further, as the albumin concentration was lowered, the effect of reducing the viscosity of the albumin solution by the A6K solution was conspicuous.

Optimum A6K solution that reduces the viscosity of the albumin solution Three concentrations of A6K solution are added to the albumin solution having a constant concentration, and the effect of inhibiting the aggregation of the albumin solution is visually examined. The optimal A6K solution concentration is 0 .05% was confirmed.

<Material>
・ Albumin aqueous solution concentration: 200 mg / mL
Peptide surfactant sequence: Ac-AAAAAAK-NH2, Celtek concentration: 10 mg / mL (stock solution)
purified water

<Method>
-Preparation of A6K solution Purified water is added to the stock solution, and 500 μL of A6K solutions with the following three concentrations are prepared.
3 concentration: 10 mg / mL (1% solution)
5 mg / mL (0.5% solution)
1 mg / mL (0.1% solution)
-Examination of aggregation inhibitory effect of albumin solution To 100 μL of albumin solution, an equal amount of each of the four concentrations of the A6K solution prepared above is added, and the transparency of the solution is visually confirmed.

<Result>
The albumin solution added with 1% and 0.5% A6K solution became cloudy, but the albumin solution added with 0.1% A6K solution was clear.

<General consideration>
In combination with the results of Example 1 and Example 2, it was considered that the peptidic surfactant prepared at an appropriate concentration is useful for inhibiting protein aggregation.

Claims (4)

  An aggregation inhibitor that lowers the viscosity of a protein solution, comprising a peptide represented by the sequence AAAAAAAK.   The protein aggregation inhibitor according to claim 1, which is used in an aqueous solution having a concentration of 0.05 to 1%.   The protein aggregation inhibitor according to claim 2, which is used in an aqueous solution having a concentration of 0.05 to 0.1%.   A pharmaceutical comprising the protein aggregation inhibitor according to any one of claims 1 to 3.