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An optimized method for protein capture by multi-column continuous flow chromatography

An optimization method, flow chromatography technology, which is applied in the field of protein chromatography separation, can solve problems such as high personnel requirements, long time-consuming, and high difficulty, and achieve the effect of accelerating optimization speed, improving efficiency, and reducing optimization difficulty

Active Publication Date: 2021-09-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, optimization with the help of a mechanism model requires solving partial differential equations, which takes a long time and requires high personnel, making it difficult in practical applications

Method used

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  • An optimized method for protein capture by multi-column continuous flow chromatography
  • An optimized method for protein capture by multi-column continuous flow chromatography
  • An optimized method for protein capture by multi-column continuous flow chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Optimum operation bar number solution

[0050] (1) Experimentally obtained penetration curve

[0051] Use the Praesto Jetted A50 medium from Purolite Company, fill a 5ml chromatographic column, use immunoglobulin with a concentration of 5g / L to load the sample, and the retention time of the sample is 2min, and carry out the penetration experiment. After the concentration of the penetration protein reaches 4.5g / L Stop loading the sample, the breakthrough curve is as attached image 3 shown.

[0052] (2) Establish a linear relationship connecting sample loading time and sample loading retention time

[0053] Integrating the breakthrough curve at the set breakthrough percentage of 0.5 (that is, 50% breakthrough), the single-column loading is 79.8 g / L. The elution regeneration time t can be obtained by conventional batch chromatography experiment optimization RR The time is 55min, the connection flushing time is t CW It is 8min. Substituting the above single...

Embodiment 2

[0066] Example 2 Solution to Optimal Loading Retention Time

[0067] (1) Experimentally obtained penetration curve

[0068]Use the Praesto Jetted A50 medium from Purolite Company, fill a 5ml chromatographic column, use immunoglobulin with a concentration of 4g / L to load the sample, and the retention time of the sample is 4min, and carry out the penetration experiment. When the concentration of the penetration protein reaches 3.6g / L Stop loading the sample, the breakthrough curve is as attached Figure 5 shown.

[0069] (2) Establish a linear relationship connecting sample loading time and sample loading retention time

[0070] Integrating the breakthrough curve at the set breakthrough percentage of 0.7 (ie, 70% breakthrough), the single column load is 90.1 g / L. The elution regeneration time t can be obtained by conventional batch chromatography experiment optimization RR 55min, connection flushing time t CW It is 8min. Substituting the above single-column loading into th...

Embodiment 3

[0087] Embodiment 3 maximum productivity solution

[0088] Substitute the optimal loading retention time obtained in Example 2 into the following formula:

[0089]

[0090] The maximum productivity obtained is 28.6g / L / h.

[0091] Under the conditions of loading protein concentration of 4g / L, optimal loading retention time of 2.8min, and set breakthrough percentage of 0.7, the three-column continuous flow chromatography protein capture experiment of Praesto Jetted A50 medium was carried out, and it was found that the loading time t C and elution regeneration time t RR Basically equal, the productivity of continuous flow chromatography at this moment is 27.6g / L / h, near the maximum productivity 28.6g / L / h of prediction, higher than the productivity under other conditions, confirms the effectiveness of the inventive method.

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Abstract

The invention discloses an optimization method for capturing proteins by multi-column continuous flow chromatography, which comprises the following steps: step 1, under the conditions of a set loading protein concentration and arbitrary loading retention time, a protein penetration experiment is carried out, Obtain the protein breakthrough curve; step 2, under the penetration percentage of the target setting, integrate the breakthrough curve to obtain the single column load, and establish a linear relationship between the loading time and the loading retention time; step 3, use the step 2, to solve the optimal number of operating columns for continuous flow chromatography to capture proteins; step 4, use the linear relationship obtained in step 2 and the optimal number of operating columns to obtain in step 3 to solve the optimal number of columns for continuous flow chromatography to capture proteins Optimal sample loading retention time; step 5, use the optimal sample loading retention time obtained in step 4 to solve the maximum productivity of the protein captured by continuous flow chromatography.

Description

technical field [0001] The invention relates to protein chromatography separation technology in the fields of biochemical engineering and bioengineering, in particular to an optimization method for capturing proteins by multi-column continuous flow chromatography. Background technique [0002] Monoclonal antibody (referred to as monoclonal antibody) is the most important biotechnology drug, which has the characteristics of good targeting, high curative effect, and small toxic and side effects. In recent years, with the growth of upstream cell expression and the expansion of production scale, the productivity and economy of the downstream separation and purification process have attracted more and more attention. Traditional monoclonal antibody separation mainly uses three-step single-column batch chromatography based on protein A affinity capture, which has limited process efficiency and is difficult to match the rapidly growing upstream process. On the other hand, Protein ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/08
CPCB01D15/08B01D15/1871B01D15/1828C07K1/16C07K1/22C07K1/00C07K1/20
Inventor 林东强史策姚善泾
Owner ZHEJIANG UNIV
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