Process for the purification of antibodies using affinity resins comprising specific ligands

An antibody purification and affinity technology, applied in the field of antibody purification using affinity resin containing specific ligands, can solve the problems of long process time, increased labor cost, pollution, etc.

Inactive Publication Date: 2011-10-12
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Conventional chromatography involving multiple steps suffers from one or more of the following drawbacks: low overall yield, high buffer consumption, long process time and investment in process equipment, increased labor costs
However, affinity resins with protein ligands are very expensive to produce and less chemically and conformationally stable compared to small synthetic ligands, and with purified antibodies either from protein fragments derived from protein ligands or from proteins with protein ligands Risk of contamination by other biological materials in the production of affinity resins

Method used

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  • Process for the purification of antibodies using affinity resins comprising specific ligands
  • Process for the purification of antibodies using affinity resins comprising specific ligands
  • Process for the purification of antibodies using affinity resins comprising specific ligands

Examples

Experimental program
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preparation example Construction

[0166] Preparation of affinity resin

[0167] Affinity resins can in principle be prepared in two quite different ways: (i) by synthesizing the ligand in free form and then immobilizing the ligand directly or via a linker to the solid phase material (see above), or (ii) by functionalizing the solid phase material followed by sequential synthesis of ligands. For the first approach, immobilization techniques are readily available in the art, eg as described in Hermanson et al. (see above). For the second way, techniques are also available, such as solid-phase peptide synthesis known in the art and its derivatives (Fields, G.B., etc. (1992) Principles and practice of solid-phase peptide synthesis (principle and practice of solid-phase peptide synthesis) Practice). In Synthetic Peptides: A User's Guide (Grant, G.A. Ed.), pp. 77-183, W.H. Freeman; Fields, G.B. Ed. (1997) Solid-phase peptide synthesis (solid-phase peptide synthesis). Methods in Enzymology 289 and Dorwald, F.Z. Org...

Embodiment 1

[0264] Example 1. Using PCA to select the structural unit (BUILDING BLOCK) of the combinatorial library

[0265]Combinatorial libraries were designed by using two parallel methods. A virtual combinatorial library containing 229,957 members was first generated. For each virtual library member, a set of 118 physicochemical descriptors was calculated according to the method of Cruciani et al. [Ref. Cruciani, C. et al., Molecular fields in quantitative structure-permeation relationships: the VolSurf approach. Journal of Molecular structure- Theochem, 2000.503(1-2): pp. 17-30; Cruciani, G., M.Pastor, and W. Guba, VolSurf: a new tool for the pharmacokinetic optimization of lead compounds. European Journal of Pharmaceutical Sciences, 2000.11: Pages S29-S39]. The resulting models were analyzed using multivariate statistics. To simplify the model, these 118 descriptors are designed as two principal components.

[0266] Then optimize the chemical diversity of the combinatorial libra...

Embodiment 2

[0268] Example 2: Synthesis of combinatorial libraries

[0269] The general structure of a combinatorial library of affinity ligands designed to bind to Fc fragments is shown in Figure 1. The scaffold is selected from aliphatic di-amino-carboxylic acids, and building blocks 1 and 2 are independently selected from natural amino acids, unnatural amino acids, and carboxylic acids.

[0270]

[0271] Figure 1. Schematic representation of the general structure of a trimeric ligand (A) and a tetrameric ligand (B). picture.

[0272] Combinatorial libraries were synthesized by employing one-bead-one-compound split-and-mix solid-phase synthesis. 770 different ligands were synthesized on approximately 20,000 beads of optically encoded amino-functional polyethylene glycol-acrylamide (PEGA) beads. To maintain access to all compounds throughout the synthesis, encoded bead technology was used to read the optically encoded beads used for the synthesis [WO 2005 / 061094, WO 2005 / 062018. T...

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Abstract

The present invention relates to a novel process for the purification of antibodies, e.g. monoclonal antibodies. The process utilizes an affinity resin comprising a solid phase material having immobilized thereto one or more low-molecular weight synthetic ligands. The affinity resins enable the separation of antibodies from even closely related proteins.

Description

field of invention [0001] The present invention relates to novel purification methods for antibodies, such as monoclonal antibodies. The method utilizes an affinity resin comprising a solid phase material on which one or more low molecular weight synthetic ligands are immobilized. The affinity resin is capable of separating antibodies from very closely related proteins. Background of the invention [0002] In recent years, monoclonal antibodies (mAbs) have become the main focus of many large biotech companies. mAbs are tested as biologic drugs for the treatment of various diseases, especially immune and autoimmune diseases and cancer. As the number of mAb drugs on the market is expected to increase dramatically in the future, the need to reduce production costs also increases. In current production, the purification step for mAb production accounts for approximately 70% of the production cost. [0003] Antibodies can currently be purified from fermentation supernatants b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22
CPCC07K1/22C07K16/065
Inventor P·M·圣希莱雷A·L·尼尔森
Owner NOVO NORDISK AS
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