Methods of evaluating protein formulation stability and surfactant-stabilized insulin formulations derived therefrom

• US 6,737,401 B2
• Filed: 10/18/2001
• Issued: 05/18/2004
• Est. Priority Date: 06/28/2001
• Status: Expired due to Term

• Current Assignee: Medtronic Minimed Incorporated (Medtronic Plc)
• Original Assignee: Metronic Minimed Incorporated
• Inventors: Gulati, Poonam S., Van Antwerp, William P., Gross, Todd M., Kim, Seonyoung
• Primary Examiner(s): Tate, Christopher
• Assistant Examiner(s): TELLER, ROY R
• Application Number:
  US09/981,968
• Publication Number:
  US 20030054979A1
• Time in Patent Office: 943 Days
• Field of Search: 514/2, 514/3, 514/4, 530/300, 530/303, 530/350
• US Class Current: 514/6.5
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 47/18 Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61M 5/14 Infusion devices, e.g. infusing by gravity; Blood infusion; Accessories therefor
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

Embodiments of the invention are directed to a method of estimating the physical stability of a protein formulation. A particular embodiment of the invention places the protein formulation under an agitational stress that causes the protein to aggregate at an accelerated rate. In one embodiment, the change in protein aggregation is monitored spectroscopically using Thioflavin-T. Embodiments of the invention then utilize a survival curve analysis to ascertain the relative physical stability of the different protein formulations under study. This method was used to develop novel surfactant-stabilized insulin formulations in a rapid, cost efficient manner, thus illustrating the utility of the inventive method to the discovery and development of pharmaceutical protein formulations.

Claims

1. A method of evaluating the physical stability of a protein formulation, wherein the protein is susceptible to changes in its native conformation that yield non-native conformers of the protein that are preferentially bound by a spectroscopic agent, the method comprising:

(a) preparing a statistically relevant number of identical samples of a protein formulation to yield a first sample type, wherein the protein within the protein formulation is susceptible to changes in its native conformation yielding non-native conformers of the protein;

(b) preparing a statistically relevant number of identical samples of at least one other protein formulation that differs from the first sample type to yield a second, or more, sample types, wherein the protein within at least one other protein formulation is susceptible to a changes in its native conformation yielding non-native conformers of the protein;

wherein the samples prepared in steps (a) and (b) further comprise a spectroscopic agent that exhibits a change in spectra when bound to a non-native conformer of the protein;

(c) applying a controlled stress on all sample types, wherein the controlled stress applied is an interfacial tension or a physical, photochemical or thermal stress which causes the protein to exhibit a change in its native conformation;

(d) monitoring the observable signal produced by the spectroscopic agent in the samples types to yield time-dependent data that are related to a degree of protein conformational change for each sample type;

(e) applying a survival analysis to the data obtained for each sample type; and

(f) comparing the survival analysis for each sample type to determine the relative physical stability of the protein formulations under evaluation.

2. The method of claim 1, wherein the controlled stress is agitation comprising shaking at 480-1200 rpm for 3-9 days.

3. The method of claim 1, wherein the protein is insulin and the non-native conformer of the protein is a fibril form of insulin.

4. The method of claim 1, wherein the protein formulation comprises an insulin analogue.

5. The method of claim 1, wherein the spectroscopic agent that exhibits a change in spectra when bound to a non-native conformer of the protein is Thioflavin-T.

6. The method of claim 1, wherein the sample volume of the protein formulations of steps (a) and (b) are from about 1 μ

l to about 1000 μ

l.

7. The method of claim 6, wherein the sample volume of the protein of steps (a) and (b) are from about 50 μ

l to about 500 μ

l.