Use of hydrophobic-interaction-chromatography or hinge-region modifications for the production of homogeneous antibody-solutions

A hinge, antibody molecule technology, applied in the direction of peptides, immunoglobulins, chemical instruments and methods, etc., can solve the problems of negative impact on overall yield, increased effort required for downstream processing, etc.

Active Publication Date: 2006-10-04
BIOGEN MA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current production processes discard at least 50% of the total antibody produced, which has a negative impact on overall yield
Furthermore, the presence of both isoforms increases the effort required for downstream processing

Method used

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  • Use of hydrophobic-interaction-chromatography or hinge-region modifications for the production of homogeneous antibody-solutions
  • Use of hydrophobic-interaction-chromatography or hinge-region modifications for the production of homogeneous antibody-solutions
  • Use of hydrophobic-interaction-chromatography or hinge-region modifications for the production of homogeneous antibody-solutions

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0315] Preparations of dosage forms for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol / water solutions, emulsions or suspensions, including saline and buffered media. In the present invention, the pharmaceutically acceptable carrier includes but not limited to: 0.01-0.1M and preferably 0.05M phosphate buffer saline or 0.8% saline. Other common parenteral vehicles include phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluids, nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. There may also be preservatives and other additives such as antimicrobials, antioxidants, chelating agents, and ...

Embodiment 1

[0361] Example 1: Identification of A and B isoforms

[0362] The solution of antibody molecules contains two different isotypes. One of these, type A, comprises heavy chain molecules linked via at least one disulfide bond. Another type, B, comprises heavy chain molecules that are not linked via at least one disulfide bond. In intact gamma 1 MAbs, such as Rituxan(R), type B is absent or very infrequent. However, the frequency of type B is much higher compared to domain deleted (dd) constructs with similar hinges. These forms can be differentiated using denatured non-reduced SDS page. In domain-deleted antibody preparations, type A is a 120 kDa dimer and type B is a 60 kDa monomer ( figure 1 ). figure 2 A and 2B show densitometer plots of non-reducing SDS-PAGE gels of ddCC49(CH2) and ddCC49(Gly / Ser), respectively.

Embodiment 2

[0363] Example 2: Identification of hinge region heterology in CH2 domain-deleted Mab fragments

[0364]The hinge domain can be subdivided into three distinct regions: upper, middle, and lower hinge regions (Roux et al. J. Immunol. 1998 161:4083). The polypeptide sequences comprising these regions are shown in Table 1 for the IgG1 and IgG3 hinges. In addition to two conserved cysteine ​​residues, the IgG3 hinge region also contains a 15 amino acid motif repeated three times. Amino acid sequences from these regions were used to design synthetic IgG1 / IgG3 linker peptides. These consist of the IgG1 upper hinge residues corresponding to positions 226-238, the IgG1 middle hinge corresponding to positions 239-241, and a single IgG3 middle hinge repeat motif corresponding to positions 241EE-242, which binds position 243 or added proline at positions 243, 244, and 245 (Kabat numbering system), respectively, alanine, proline, followed by a flexible Gly / Ser spacer (Table 2). In addit...

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Abstract

The instant invention describes methods of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers. These forms can be separated from each other using hydrophobic interaction chromatography. In addition, the invention pertains to connecting peptides that result in the preferential biosynthesis of dimers that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage. The invention also pertains to compositions in which a majority of the dimers are linked via at least one interchain disulfide linkage or are not linked via at least one interchain disulfide linkage. The invention still further pertains to novel binding molecules, e.g., comprising connecting peptides of the invention.

Description

[0001] related application [0002] This application claims USSN 60 / 483877, entitled "Purification and Preferred Synthesis of Polypeptides," filed on June 27, 2003, and USSN 60 / 483877, entitled "Purification and Preferred Synthesis of Antigen-Binding Polypeptides," filed on October 3, 2003. 508,810 priority. The application also claims USSN 60 / 515,351, filed on October 28, 2003, entitled "Modified Antibody Molecules Comprising a Linker Peptide," and USSN 60 / 515,351, filed on October 30, 2003, entitled "Modified Antibody Molecules Comprising a Linker Peptide," Priority of USSN 60 / 516,030. This application is also related to USSNXX / XXXXXX filed 200406028 entitled "Modified Binding Molecules Comprising Linker Peptides". The entire contents of these applications are incorporated herein by reference. Background technique [0003] Antibodies are dimeric molecules; each monomer that makes up the dimer contains one light chain and one heavy chain. Solutions of antibody molecules e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00
Inventor 加里·R·布拉斯拉夫斯基斯科特·格拉泽杨祖宏珍妮弗·霍普保罗·钦
Owner BIOGEN MA INC
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