Sequential protein isolation and purirication schemes by affinity chromatography

A protein separation, protein technology, applied in the field of protein separation, can solve the problems of lack of specificity and selectivity

Active Publication Date: 2007-09-12
PROMETIC BIOSCIENCES LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But these methods lack the specificity and selectivity needed to isolate proteins used to manufacture multiple biopharmaceuticals from the same raw material, such as human plasma

Method used

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  • Sequential protein isolation and purirication schemes by affinity chromatography
  • Sequential protein isolation and purirication schemes by affinity chromatography
  • Sequential protein isolation and purirication schemes by affinity chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: Definition of Straight-line Cascade Sequence Schemes 1-7

[0086] The linear cascade is composed of five affinity columns: albumin (HSA), fibrinogen (Fg), IgG, plasminogen (Pg) and PON1 / ApoA1. First, the columns were run sequentially in four different sequences to determine the sequence most suitable for linear cascading. To simplify ongoing sample analysis, only the undiluted flow-through was collected as the load for the next column in the sequence. Specifically, this helps keep the concentration of background proteins constant throughout the experiment. It also simplifies the comparison of output to input at each column. Samples of the load and each flow-through were analyzed to monitor the recovery of target and non-target proteins in the flow-through. Each column was tested using these values ​​to determine its ability to capture the target protein with minimal downstream target protein hold-up. Use the sequence that best fits this criterion as th...

Embodiment 2

[0132] Example 2: Affinity capture of plasma proteins

[0133] The following column order was used in this experiment:

[0134] vWF / FVIII→plasminogen→fibrinogen→IgG→UF / DF→HSA→UF / DF.

[0135] Plasma preparations: Four liters of frozen pooled plasma were obtained from -20°C storage.

[0136] Plasma pools were thawed at 37.0°C ± 2°C in a water bath. Once the plasma is thawed, quickly remove it from the water bath. Plasma was adjusted to 20 mM Tris, 50 mM NaCl using a 50x dilution of 1 M Tris, pH 7.5 and a 40x dilution of 2M NaCl. Plasma was mixed well and filtered using SartoPure 300 PP2 (8 micron) sterile filters.

[0137] a. Von Willebrand Factor / Factor VIII (vWF / FVIII) Affinity Capture

[0138] A 7 cm x 10.6 cm packed bed column was prepared containing 410 mL of the affinity sorbent developed for the capture of vWF / FVIII affinity capture. The column is usually stored in a storage solution containing 0.1N NaOH when not in use for long periods of time. Was...

Embodiment 3

[0164] Example 3: Evaluation and Selection of Plasma Buffer Systems Used in the Linear Cascade Method

[0165] This experiment was performed to determine the most suitable buffer system for a linear cascade continuous plasma protein purification protocol. It was observed that the pH climbed up to two units above the plasma pH (pH ~ 7.5) during IgG column loading. Several pH changes were observed during IgG loading, which appeared to depend on the degree of protein depletion of the load. This means that certain plasma proteins have a buffering capacity and removal of these proteins from the loading solution may increase the probability of pH shifts. Buffer systems for plasma must maintain protein concentration and activity in plasma. The buffer system must maintain the pH within an acceptable range of approximately 7.5 ± 0.4 throughout the process.

[0166] To ensure that no pH changes occur during subsequent manipulations, plasma loads for subsequent manipulations must be b...

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Abstract

The invention discloses methods for sequential protein isolation and purification from a biological sample by affinity chromatography. Affinity chromatography is conducted using ligands or ligand support complexes that selectively and specifically bind to proteins in the biological sample. The ligands or ligand support complexes were contacted sequentially in a predetermined order with the biological sample to allow each ligand or ligand-support complex to sequentially bind a protein from the biological sample.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to Provisional Patent Application Serial No. 60 / 602,868, filed August 20, 2004, the entire contents of which are hereby incorporated by reference. technical field [0003] The present invention relates to methods for isolating proteins from biological samples. In particular, the present invention relates to methods for the continuous recovery of highly pure proteins from biological samples. Background technique [0004] Protein processing and development require efficient processing with a minimum number of steps and maximum output to achieve the desired purity. Protein isolation and purification methods present unique challenges due to the diversity of proteins, the varying nature of possible contaminants and / or impurities associated with each protein preparation, and the large quantities of protein typically required for the manufacture of biopharmaceuticals. Traditional purificatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23J1/06A61K35/14C07K1/22A61K35/16C07K17/00
CPCC07K14/775C07K14/745C07K1/22
Inventor 史蒂文·詹姆斯·伯顿巴尔德夫·贝恩斯约翰·柯林蒂莫西·基思·海斯德文胡·陈克里斯托弗·布莱恩特大卫·约翰·哈蒙德
Owner PROMETIC BIOSCIENCES LTD
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