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Assay for determining potential to self-association of a protein using concentration-dependent self-interaction nanoparticle spectroscopy

A technology of nanoparticles and gold nanoparticles, which can be used in measurement devices, biological testing, and analysis by causing chemical reactions to occur in materials, which can solve problems such as instability

Active Publication Date: 2019-04-19
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some proteins are stable under certain conditions but not others

Method used

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  • Assay for determining potential to self-association of a protein using concentration-dependent self-interaction nanoparticle spectroscopy
  • Assay for determining potential to self-association of a protein using concentration-dependent self-interaction nanoparticle spectroscopy
  • Assay for determining potential to self-association of a protein using concentration-dependent self-interaction nanoparticle spectroscopy

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0193] Example 1: Materials

[0194] Monoclonal antibody (mAb) by Cells are generated and purified by protein A chromatography and the polishing step of anion exchange or hydrophobic interaction chromatography. The buffer components are obtained from Sigma-Aldrich, VWR or JT-Baker and are the highest grade available. Illustra NAP column (Cat. #17-0853-02) and XK26 / 100 Superdex200pg column (Cat. #90-1002-73) were purchased from GE Healthcare Life Sciences. Amicon centrifugal filtration device (Cat. #UFC905024) was purchased from EMD Millipore. Slide-A-LyzerTM G2 dialysis cassette (Cat. #87732) was purchased from ThermoFisher Scientific. 20nm gold nanoparticles (Cat.#HD.GC20) were purchased from BBI solutions. ThermoScientificTM NuncTM MicrowellTM 96-well microplate (Cat.#12-565-66) was used as a reaction vessel to obtain absorption spectra.

example 2

[0195] Example 2: High-concentration static light scattering method (HC-SLS).

[0196] In 10mM MES, pH 6.0, 50mM sodium chloride, through XK26 / 100Superdex 200pg column Each concentrated (100 g / L) monoclonal antibody (mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6) was purified on avant (GE Healthcare Life Sciences). The monomer mAb fraction was collected and concentrated using a series-connected Easy-LoadMAsterFlexL / S (Cole Parmer) pump and VivaFlow 2030,000 MWCO HY membrane. Divide 150 mL of each concentrated antibody into 3 parts, and pack them into 10,000 MWCO dialysis cassettes, and follow 2 L of 10 mM MES, pH 6.0, 250 mM sodium chloride; 10 mM MES, pH 6.0, 50 mM sodium chloride; and 10 mM MES , PH 6.0, 10mM sodium chloride solution exchange. A centrifugal filter unit with 50,000 MWCO was used to concentrate the solution to a final volume of approximately 15 mL in the corresponding buffer. The concentration was measured using SoloVPE (CTechnologies, Inc.). In 10mM MES, pH 6.0, an...

example 3

[0197] Example 3: Standard Self-Interacting Nanoparticle Spectroscopy (SINS).

[0198] Sub-aliquots of each antibody (mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6) under different salt conditions were added to separate 15 mL centrifuge tubes. 5mL of 1 optical density (1O.D) 20nm gold nanoparticle solution was added to the solution, so that the final protein concentration obtained was 50μg / mL. After waiting for 30 minutes, the absorption spectrum and λ max Recorded in SPECTRAmax 340PC (Molecular Devices).

[0199] Concentration-dependent self-interacting nanoparticle spectroscopy (CD-SINS). Prepare a 100 mM buffer solution at a suitable pH. Adjust the Illustra NAP column with 2.4 mL of 100 mM buffer solution (for example, MES or sodium phosphate, depending on the target pH). Use the adjusted desalting column to buffer exchange the concentrated (50-75 g / L) mAb stock solution. Use SoloVPE to measure the result Concentration. Then each antibody was diluted to 5.12mg / mL in 100mM buffer. ...

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Abstract

Methods for producing high concentration protein formulations having high stability are provided. Assays for selecting proteins and formulation conditions that have high self-repulsive attributes areused as an early step in the manufacturing process. Specifically, a protein concentration-dependent self-interaction nanoparticle spectroscopy method is employed as a protein colloidal interaction assay.

Description

Technical field [0001] The present invention generally relates to the selection of therapeutic proteins for development and commercialization; and more specifically, to the selection of proteins that have a high probability of easily reaching high protein concentrations while having a low probability of forming aggregates. The invention specifically relates to evaluating protein colloidal interactions and selecting proteins to formulate at high concentrations. Background technique [0002] Large biomolecules-"biotherapeutics"-are an important class of drugs. For example, monoclonal antibodies provide precise therapeutic specificity, long biologic half-life, and high safety characteristics. [0003] Given the size and complexity, biotherapeutics are difficult to produce and formulate. Most biotherapeutics are formulated into freeze-dried solid or liquid preparations, and the stability of these molecules is an important issue. Stability not only affects the shelf life of the final...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N33/68
CPCG01N21/25G01N21/31G01N2021/258G01N21/77G01N33/587G01N33/6845
Inventor M·马洛M·森尼特M·施耐德
Owner REGENERON PHARM INC
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