Copolymer 1 related polypeptides for use as molecular weight markers and for therapeutic use

• US 20050038233A1
• Filed: 03/02/2004
• Published: 02/17/2005
• Est. Priority Date: 09/25/1998
• Status: Active Grant

• Current Assignee: Alexander Gad, Dora Lis
• Original Assignee: Alexander Gad, Dora Lis
• Inventors: Lis, Dora, Gad, Alexander
• Granted Patent: US 7,074,580 B2
• Time in Patent Office: Days
• US Class Current: 530/388.100
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/02 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/2026 IL-4
• CPC Class Codes: A61K 38/2066 IL-10
• CPC Class Codes: A61K 38/215 IFN-beta
• CPC Class Codes: A61L 27/22 Polypeptides or derivatives thereof , e.g. degradation products
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: G01N 2800/285 Demyelinating diseases; Multipel sclerosis
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/505 involving T-cells
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6812 : Assays for specific amino acids
• CPC Class Codes: G01N 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6848 : Methods of protein analysis involving mass spectrometry
• CPC Class Codes: Y10T 436/10 : Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/105831 : Protein or peptide standard or control [e.g., hemoglobin, etc.]

Abstract

The present invention provides molecular weight markers for accurate determination of the molecular weight of glatiramer acetate and other copolymers. The present invention further provides a plurality of molecular weight markers for determining the molecular weight of glatiramer acetate and other copolymers which display linear relationships between molar ellipticity and molecular weight, and between retention time and the log of the molecular weight. The molecular weight markers also optimally demonstrate biological activity similar to glatiramer acetate orcorresponding copolymers and can be used for treating or preventing various immune diseases.

Claims

1-122. -122. (Canceled)

123. In a process for obtaining a pharmaceutical product containing an aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine, wherein the mixture has a desired average molecular weight and wherein during the process a batch of an aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine, is tested using a gel permeation chromatography column to determine whether the mixture has the desired average molecular weight for inclusion in the pharmaceutical product, the improvement comprising

calibrating the molecular weight obtained using the gel permeation chromatography column by subjecting a plurality of molecular weight markers, each of which is a polypeptide consisting essentially of alanine, glutamic acid, tyrosine and lysine and having a predetermined amino acid sequence, to chromatography on the column to establish a relationship between retention time on the column and molecular weight.

124. The process of claim 123, wherein the mixture of polypeptide that is tested is glatiramer acetate.

125. The process of claim 124, wherein the glatiramer acetate has an average molecular weight from 4000 to 13,000 Daltons.

126. The process of claim 125, wherein in the glatiramer acetate the molar fraction of analine is 0.427, of glutamic acid is 0.141, of lysine is 0.337 and of tyrosine is 0.093.

127. The process of claim 123, wherein the gel permeation chromatography column comprises a cross-linked agarose-based medium, with an exclusion limit of 2×

10⁶ Daltons, an optimal separation range of 1000 to 3×

10⁵ Daltons, and a bead diameter of 20-40 μ

m.

128. The process of claim 127, wherein the gel permeation chromatography column is Superose 12.

129. The process of claim 123, wherein in the molecular weight markers the molar fraction of analine is 0.38 to 0.5, of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to 0.10 and of lysine is 0.3 to 0.4.

130. The process of claim 129, wherein in the molecular weight markers the molar fraction of analine is 0.422 to 0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to 0.093 and of lysine is 0.333 to 0.349.

131. The process of claim 123, wherein one of the molecular weight markers is selected from the group consisting of

132. The process of claim 123, wherein the plurality of molecular weight markers is

133. The process of claim 124, further comprising a step of lyophilizing of the glatiramer acetate.

134. A process for obtaining a pharmaceutical composition containing an aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine, wherein the mixture has a desired average molecular weight, which comprises obtaining a batch of an aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine;

determining the average molecular weight of the mixture of polypeptides in the batch using a molecular weight-calibrated gel permeation chromatography column; and

including in the pharmaceutical product the mixture if the mixture is determined to have the desired average molecular weight,

wherein the calibration of the molecular weight obtained using the gel permeation chromatography column comprises subjecting a plurality of molecular weight markers to chromatography on the column to establish a relationship between the retention time on the column and molecular weight, wherein each of the markers is a polypeptide consisting essentially of alanine, glutamic acid, tyrosine and lysine and has a predetermined amino sequence.

135. The process of claim 134, wherein the batch of the aqueous mixture of polypeptide is glatiramer acetate.

136. The process of claim 135, wherein the glatiramer acetate has an average molecular weight from 4000 to 13,000 Daltons.

137. The process of claim 136, wherein in the glatiramer acetate the molar fraction of analine is 0.427, of glutamic acid is 0.141, of lysine is 0.337 and of tyrosine is 0.093.

138. The process of claim 134, wherein the gel permeation chromatography column comprises a cross-linked agarose-based medium, with an exclusion limit of 2×

10⁶ Daltons, an optimal separation range of 1000 to 3×

10⁵ Daltons, and a bead diameter of 20-40 μ

m.

139. The process of claim 138, wherein the gel permeation chromatography column is Superose 12.

140. The process of claim 134, wherein in the molecular weight markers the molar fraction of analine is 0.38 to 0.5, of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to 0.10 and of lysine is 0.3 to 0.4.

141. The process of claim 140, wherein in the molecular weight markers the molar fraction of analine is 0.422 to 0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to 0.093 and of lysine is 0.333 to 0.349.

142. The process of claim 134, wherein one of the molecular weight markers is selected from the group consisting of

143. The process of claim 134, wherein the plurality of molecular weight markers is

144. The process of claim 135, further comprising a step of lyophilizing of the glatiramer acetate having the desired average molecular weight distribution.

145. A process for determining the average molecular weight of an aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine, which comprises subjecting the mixture to chromatography on a molecular weight-calibrated gel permeation chromatography column so as to determine the average molecular weight of the mixture, wherein the calibration of the molecular weight obtained using the gel permeation chromatography column comprises subjecting a plurality of molecular weight markers to chromatography on the column to establish a relationship between retention time on the column and molecular weight, wherein each of the markers is a polypeptide consisting essentially of alanine, glutamic acid, tyrosine and lysine and has a predetermined amino acid sequence.

146. The process of claim 145, wherein the aqueous mixture of polypeptide is glatiramer acetate.

147. The process of claim 146, wherein the glatiramer acetate has an average molecular weight from 4000 to 13,000 Daltons.

148. The process of claim 147, wherein in the glatiramer acetate the molar fraction of analine is 0.427, of glutamic acid is 0.141, of lysine is 0.337 and of tyrosine is 0.093.

149. The process of claim 145, wherein the gel permeation chromatography column comprises a cross-linked agarose-based medium, with an exclusion limit of 2×

10⁶ Daltons, an optimal separation range of 1000 to 3×

10⁵ Daltons, and a bead diameter of 20-40 μ

m.

150. The process of claim 149, wherein the gel permeation chromatography column is Superose 12.

151. The process of claim 145, wherein in the molecular weight markers the molar fraction of analine is 0.38 to 0.5, of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to 0.10 and of lysine is 0.3 to 0.4.

152. The process of claim 151, wherein in the molecular weight markers the molar fraction of analine is 0.422 to 0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to 0.093 and of lysine is 0.333 to 0.349.

153. The process of claim 145, wherein one of the molecular weight markers is selected from the group consisting of

154. The process of claim 145, wherein the plurality of molecular weight markers is

155. A process for determining whether an aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine, has a desired average molecular weight, which comprises subjecting the mixture to a calibrated gel permeation chromatography column to determine the average molecular weight of the mixture and comparing the average molecular weight so determined to the desired average molecular weight, wherein the calibration of the molecular weight obtained using the gel permeation chromatography column comprises subjecting a plurality of molecular weight markers to chromatography on the column to establish a relationship between retention time on the column and molecular weight, wherein, each of the markers is a polypeptide consisting essentially of alanine, glutamic acid, tyrosine and lysine and has a predetermined amino acid sequence.

156. The process of claim 155, wherein the mixture of polypeptide that is tested is glatiramer acetate.

157. The process of claim 156, wherein the glatiramer acetate has an average molecular weight from 4000 to 13,000 Daltons.

158. The process of claim 157, wherein in the glatiramer acetate the molar fraction of analine is 0.427, of glutamic acid is 0.141, of lysine is 0.337 and of tyrosine is 0.093.

159. The process of claim 155, wherein the gel permeation chromatography column comprises a cross-linked agarose-based medium, with an exclusion limit of 2×

10⁶ Daltons, an optimal separation range of 1000 to 3×

10⁵ Daltons, and a bead diameter of 20-40 μ

m.

160. The process of claim 159, wherein the gel permeation chromatography column is Superose 12.

161. The process of claim 155, wherein in the molecular weight markers the molar fraction of analine is 0.38 to 0.5, of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to 0.10 and of lysine is 0.3 to 0.4.

162. The process of claim 161, wherein in the molecular weight markers the molar fraction of analine is 0.422 to 0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to 0.093 and of lysine is 0.333 to 0.349.

163. The process of claim 155, wherein one of the molecular weight markers is selected from the group consisting of

164. The process of claim 155, wherein the plurality of molecular weight markers is