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Purification of multispecific antibodies

A multi-specific antibody, specific technology, applied in the direction of anti-animal/human immunoglobulin, specific peptide, organic chemistry, etc., can solve problems such as inappropriate

Pending Publication Date: 2019-04-02
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Conventional purification techniques designed to remove process-related impurities (such as HCP, DNA, endotoxins, and other materials) that have very different characteristics and properties from antibodies may not be effective when used to remove impurities more similar to multispecific antibodies. appropriate

Method used

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  • Purification of multispecific antibodies
  • Purification of multispecific antibodies
  • Purification of multispecific antibodies

Examples

Experimental program
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preparation example Construction

[0463] Formulations and preparation methods of formulations

[0464] Also provided herein are formulations comprising multispecific antibodies purified by the methods described herein and formulations and methods of producing. For example, a purified polypeptide can be combined with a pharmaceutically acceptable carrier.

[0465] In some embodiments, polypeptide formulations can be prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the polypeptide with the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences 16th Ed., Osol, A. Ed. (1980)).

[0466] A "carrier" as used herein includes a pharmaceutically acceptable carrier, excipient or stabilizer which, in the dosages and concentrations employed, is nontoxic to cells or mammals to which it is exposed. A physiologically acceptable carrier is usually an aqueous pH buffered solution.

[0467] Acceptable carriers,...

Embodiment 1

[0497] Example 1: Assembly and purification of anti-X1 / anti-Y1 bispecific antibody

[0498] Bispecific antibodies against target proteins X1 and Y1 - anti-X1 / anti-Y1 bispecific or aX1 / Y1 bispecific were assembled as follows. Each half antibody (aX1 (knob) and aY1 (well)) was subjected to an affinity chromatography step independently using protein A resin (MabSelect SuRe, GE Healthcare). The Protein A step was done independently for each half antibody using similar process conditions but different loading density targets. The Protein A column was run at ambient temperature (15-30°C) and the load cooled to 12-18°C. Prepare the Protein A column by applying three column volumes of elution buffer followed by three column volumes of regeneration buffer. The column was then equilibrated, loaded, washed three times (equilibration buffer wash, potassium phosphate wash, equilibration buffer wash), eluted and regenerated for sufficient cycles to process the loaded material. If nec...

Embodiment 2

[0522] Example 2: F(ab') 2 Assembly and purification of bispecifics

[0523] Achieve 90% pure F(ab’) 2 Initial attempts at bispecificity resulted in low yields (less than 10% of starting material). There are several challenges to maintaining acceptable yields without loss of purity, including the instability of process intermediates and the presence of product-associated variants, such as homodimers, free light and heavy chains, and Unreacted Fab' leaving group. A new unit operation was developed to achieve the desired bispecific F(ab') 2 efficient assembly and purification. Such as figure 2 As shown in the schematic provided in , a bispecific F(ab')2 comprising two different Fab' molecules was assembled and purified.

[0524] First, the capture step is implemented as follows. Each Fab' was first captured from a separate E. coli extract supernatant. The supernatant containing one of the two Fab' half molecules was subjected to a capture step using CaptoL Pro...

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Abstract

The present invention provides methods of purifying multispecific antibodies. The methods comprise the sequential steps of performing capture chromatography, first mixed mode chromatography and secondmixed mode chromatography. In some aspects, the invention provides compositions of multispecific antibodies, which compositions have reduced levels of one or more product-specific impurities and / or process-specific impurities.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 62 / 351,908, filed June 17, 2016, the entire contents of which are incorporated herein by reference. [0003] Submit a sequence listing in an ASCII text file [0004] The contents of the following submitted ASCII text file are hereby incorporated by reference in their entirety: Computer Readable Form of the Sequence Listing (CRF) (File Name: 146392036340SEQLIST.TXT, Date of Record: June 9, 2017, Size: 32KB). field of invention [0005] Methods of purifying a multispecific antibody from a composition comprising the multispecific antibody and at least one impurity including at least one product-specific impurity are provided. In some embodiments, product-specific impurities are, for example, precursors, aggregates and / or variants of multispecific antibodies. Also provided are multispecific antibodies purified according to the methods, as well as com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/18C07K1/22C07K16/06C07K16/22
CPCC07K16/065C07K1/18C07K1/22C07K16/22C07K2317/31C07K2317/526C07K2317/54C07K2317/76C07K1/165C07K1/36A61P27/02A61P35/00C07K1/34C07K16/26C07K16/468C07K2317/56
Inventor G·S·吉塞E·罗森伯格B·萨利尔S·康拉德W·克恩莱因S·维尔曼A·比亚拉斯K·A·卡利亚斯-卡罗尔Y·伊格索
Owner F HOFFMANN LA ROCHE & CO AG
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