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Process for obtaining antibodies

A technology for recombinant antibodies and expression vectors, applied in separation methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as difficulty in obtaining high recovery percentages, and achieve the effect of saving production time and costs

Active Publication Date: 2011-07-20
UCB SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Low yields per fermentation or culture are often a specific issue noted during the primary extraction phase; antibody expression is high intracellularly, but high recovery percentages during the primary extraction phase are clearly difficult to achieve

Method used

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  • Process for obtaining antibodies
  • Process for obtaining antibodies
  • Process for obtaining antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the effect of non-lysing treatment (pretreatment)

[0045] (a) Antibody A (a Fab') was expressed in Escherichia coli W3110 cells using vector pTTOD inserted with DNA encoding antibody A. Fermentation (in DD53) at 25°C until OD 600 For 111.6 and ready to collect. Collect an aliquot of the culture by centrifugation in 50 ml at room temperature and resuspend the cell pellet in 5 ml culture supernatant + 29 ml H 20 and 5 ml of 1M Tris, pH 7.4, containing 100 mM EDTA. The resuspended cell pellet was pretreated and stirred at 60rpm as figure 1 the time shown in . After pretreatment, the resuspended cell pellet was subjected to heat treatment at 50°C while stirring at 170 rpm for 14 hours. After heat treatment, the resuspended cell pellet was clarified by centrifugation in a Beckman J.6 centrifuge at 4200 rpm for 30 min at 4°C. Fab' in the supernatant containing functional antibody A was determined using protein GHPLC analysis in 20 mM phosphate buffer. An...

Embodiment 2

[0049] Example 2: Effect of non-cracking treatment (pressure)

[0050] (a) Antibody A was expressed in E. coli as described in Example 1. As described in Example 1, in the culture supernatant, H 20 Resuspended cell pellets were prepared in Tris / EDTA and thereafter pressure treated at different pressures using a French cell press. Heat treatment was performed and the yield of functional antibody was calculated as described in Example 1.

[0051] An increase in functional antibody yield was observed at all pressures except 500 psi compared to the case where no pressure was used ( figure 2 ). Increased yields of functional Antibody A were obtained by using this non-lysing treatment, particularly at 1000 psi and 2000 psi. Some cell lysis was seen at 4000 psi, but this was not significant (ie less than 20%).

[0052] (b) Antibody B (a Fab') was expressed in Escherichia coli W3110 cells using vector pTTOD inserted with DNA encoding antibody B. Fermentation was carried out as ...

Embodiment 3

[0060] Example 3: Determination of Cell Lysis Using FACS Analysis

[0061] Antibody A (a Fab') was expressed in Escherichia coli W3110 cells using vector pTTOD into which DNA encoding antibody A was inserted. The cell slurry was prepared to original volume in tris / EDTA extraction buffer as described in Example 1 and passed through a MG homogenizer (single pass) at different pressures (see Figure 5 ).

[0062] The feed stream from the homogenizer was collected and diluted to OD in phosphate buffered saline 600is 0.2. To each sample (500 μl) was added the fluorescent dye propidium iodide (200 μg / ml) (2 μl). Propidium iodide binds DNA but cannot cross the intact plasma membrane. Each reaction was evaluated using a Becton Dickinson FACSCalibur to determine lysed and non-lysed cells. Harvested cultures and Opsi controls are included and total intact cells before stress treatment are indicated. Figure 5 The data shown in indicate that pressure treatment up to and including 4...

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Abstract

The present invention relates to the manufacture of recombinant antibodies of therapeutic quality. In particular, the invention relates to methods for increasing the yield of functional antibody from large scale fermentations whereby a cultured host cell sample is subjected to a non-lysing treatment.

Description

technical field [0001] The present invention relates to methods for increasing the production or isolation yield of functional recombinant antibodies, and in particular therapeutic antibodies. These methods are particularly suitable for large-scale industrial production of therapeutic antibodies. Background technique [0002] Recombinant DNA technology has been rapidly developed and is particularly suited for the production of antibodies, especially therapeutic antibodies. The systems for expressing recombinant genes are well known to those skilled in the art. They include expression in mammalian cells, insect cells, fungal cells, bacterial cells and transgenic animals and plants. The choice of expression system depends on the characteristics of the encoded protein, such as post-translational modifications. Other considerations include time and especially the cost involved in producing the desired quantity of material of desired quality. These latter considerations are o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00C12N1/00B01D15/00C12P21/00
CPCC07K2317/55C07K16/00
Inventor M·塞迪夫M·斯皮塔利
Owner UCB SA
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