Methods for high-throughput screening of cell lines

A cell line, high-throughput technology, applied in the field of screening cell lines, can solve the problems of inability to produce post-translational modifications, waste of time and resources, and protein structure abnormalities

Inactive Publication Date: 2009-07-01
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other cell line clones may produce optimal protein expression levels but fail to produce fully functional protein mixtures due to structural malformations or inappropriate post-translational modifications of the proteins
These issues lead to wasted time and resources during the development of biologically relevant compounds

Method used

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  • Methods for high-throughput screening of cell lines
  • Methods for high-throughput screening of cell lines
  • Methods for high-throughput screening of cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] High-throughput screening of human Fc protein assays

[0113] 1. Preparation of anti-Aβ standard

[0114] Standard curve buffer was prepared in 16 assay plates (Corning / Costar, Corning, NY) by mixing 20 ul of medium R5CD1 and 24 ml of assay buffer (PBS w / 0.05% Tween 20 and 1% bovine serum albumin) (SCB). A 0.3 mg / ml intermediate standard was prepared using 6 ul 32.5 mg / ml reference standard and 644 ul SCB (prepared fresh each time). A lug / ml standard was placed in a well called A1 and an additional well called H1 of a 2ml deep well plate. In 16 assay plates, well A1 contained 6ul of 0.3 mg / ml intermediate standard and 1794ul of SCB. Repeat the process for hole H1. For each of the 16 assay plates, serial dilutions were prepared by taking 900ul of the solution in the wells and adding 900ul of SCB. Dilutions were dispensed at 50ul per standard well.

[0115] 2. Preparation of controls

[0116] Controls were prepared to create an intermediate control at a conce...

Embodiment 2

[0138] High-throughput purification and identification of suitable cell culture conditions

[0139] 1. Manual protein purification using centrifugation

[0140] The high-throughput titration screening method of Example 1 can be combined with the high-throughput purification method detailed below to facilitate potential improvements in cell culture.

[0141] Thin layer resin in 20% ethanol. Additional 20% ethanol solution was added to make up 50% of the final volume. The solution was mixed well and dispensed in 200uL aliquots per well of filter plates (Whatman 7700-2804, longdrip, 25um, 96 well x 800uL, Whatman LabSciences, Orange, NJ). Filter plates were stacked on top of empty microplates (Corning / Costar, Corning, NY) and centrifuged (Sorvall Legend RT) at 700 rpm (approximately equal to 104G) for 3 minutes to remove 20% ethanol. Then, 200 uL per well of RODI water was added and the plates were centrifuged for 3 minutes with an empty microtiter plate underneath. Repea...

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Abstract

Disclosed are methods for high-throughput screening of cell lines for use in protein expression in certain pharmaceutical, drug development, and biotechnological processes such that high productivity cell lines are identified for their ability to produce both desired levels of protein expression and appropriate quality of a protein-of- interest.

Description

[0001] This application claims priority to US Provisional Application 60 / 793,991, filed April 21, 2006, the specification of which is incorporated herein by reference in its entirety. technical field [0002] The present invention relates generally to the field of pharmaceuticals. More specifically, the present invention relates to methods of screening cell lines for their ability to produce proteins of sufficient quantity and quality on an industrial scale. Background technique [0003] High-throughput technology has become an important tool in pharmaceutical and biotechnology research. High-throughput analytical methods utilize automated procedures to rapidly analyze protein activity, levels of protein expression, gene expression, and the numerous chemical interactions that occur in biological systems. Data generated by these methods and techniques have been used in a wide range of fields such as cancer research, drug development, and crystallography (see, e.g., Abramovit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18
CPCG01N33/5005
Inventor J·H·周P·弗兰基S·科布N·巴伦
Owner WYETH LLC
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