Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method of purifying therapeutic proteins

a technology of therapeutic proteins and purification methods, applied in the field of purification methods of therapeutic proteins, can solve the problems of insufficient blood clotting, risk of haemorrhage, and destabilisation of therapeutic proteins in solution

Inactive Publication Date: 2014-06-05
CSL BEHRING GMBH
View PDF5 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for reducing the levels of plasminogen, tissue plasminogen activator, and other proteases in solutions containing fibrinogen and / or Factor VIII and von Willebrand factor (VWF). This may help to improve the stability and shelf life of these solutions.

Problems solved by technology

In some instances, the presence of impurities, such as proteases, will destabilise the therapeutic protein in solution, particularly during storage.
A deficiency in the level of any one or more of the proteins involved in coagulation, including fibrinogen, Factor VIII, and / or von Willebrand factor (VWF) whether congenital or acquired, can lead to insufficient clotting of blood and the risk of haemorrhage.
As a consequence, such preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.
However, existing precipitation and chromatographic techniques amenable to commercial scale manufacturing processes typically produce fibrinogen preparations that comprise contaminating proteins such as zymogens or proteases (e.g., prothrombin, tissue plasminogen activator (tPA) and plasminogen), which can destabilize fibrinogen in solution.
As a consequence, fibrinogen preparations are relatively unstable in aqueous solution, with long-term storage limited to lyophilized or frozen preparations.
However, the use of specific affinity resins is not amenable to large scale commercial processes.
Reasons for this include the affinity resins themselves not being sufficiently robust to be used repeatedly and generally add significantly to the both processing time and costs.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method of purifying therapeutic proteins
  • Method of purifying therapeutic proteins
  • Method of purifying therapeutic proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Purification of Fibrinogen Through HEA, PPA and MEP Hydrophobic Charge Induction Chromatographic (HCIC) Resin

[0167]Human pooled plasma cryoprecipitate was used as the starting material (i.e., fibrinogen-containing feedstock). Briefly, the pooled plasma cryoprecipitate was solubilised in an extraction buffer containing 20 mM Tri-sodium citrate, 200 mM epsilon-amino caproic acid (ε-ACA), 60 IU / mL heparin and 500 mM NaCl (pH 7.2±2) at 31±2° C. for 30 minutes (1 g cryoprecipitate per 4 g of buffer). Aluminium hydroxide 2% (w / w) was then added to the solubilised cryoprecipitate at a concentration of 25% (w / w). After which the aluminium hydroxide gel was removed by either centrifugation or depth filtration and the fibrinogen-containing supernatant was recovered for further chromatographic purification through a HCIC chromatographic resin.

[0168]The fibrinogen-containing supernatant was applied to chromatography columns that were packed with 1.8 mL of either HEA, PPA or MEP Hypercel resin. ...

example 2

Level of Impurities in a Fibrinogen Solution Reduced Through HEA Hypercel

[0170]Approximately 48.5 mL of fibrinogen containing solubilised cryoprecipitate prepared according to Example 1 was applied onto a 5 mL HEA Hypercel column which was pre-equilibrated in 25 mM Tris at either pH 6.5, 7.0, 7.5, 8.0 or 8.5. HCIC purification was performed in negative mode with respect to fibrinogen, in which fibrinogen was allowed to drop-through in the unbound flow-through fraction, whilst t-PA, plasminogen and Factor II remained bound to the resin. FIG. 4a shows step recovery of fibrinogen, plasminogen, t-PA and Factor II during post-chromatographic purification using HEA Hypercel. The results show that pH had little or no effect on the binding of plasminogen and Factor II to the HCIC resin, whereas the binding of t-PA to the resin appeared to be most effective at the lower pH range of 6.5-7.0. Recovery of fibrinogen was greater than 90% in the drop-through fraction under different pH conditions...

example 3

Level of Impurities in a Fibrinogen Solution Purified Through HEA Hypercel

[0172]Approximately 500 mL of the fibrinogen-containing supernatant obtained post alhydrogel adsorption step generated in accordance with Example 1 was loaded onto an XK 16 / 30 column packed with 36 mL of HEA Hypercel resin, pre-equilibrated in 25 mM Tris pH 7.0. The drop-through fraction was collected for fibrinogen, plasminogen, t-PA, and Factor II testing. A summary of the results is provided in Table 1 below.

TABLE 1FibrinogenVolumeProteinby ClaussPlasminogent-PAFactor II(mL)(mg / mL)(mg / mL)(ng / mL)(ρg / mL)(U / mL)Fibrinogen50021.816.053172.01716.50.00097supernatantDrop-through54017.613.215655.5706.70.00025fraction% recovery in87.2%89.1%31.8%44.5%27.8%drop-through fraction

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Temperatureaaaaaaaaaa
Temperatureaaaaaaaaaa
Temperatureaaaaaaaaaa
Login to View More

Abstract

The present invention relates generally to a method of reducing the level of plasminogen and / or tissue plasminogen activator and / or other protease(s) in a solution comprising fibrinogen and / or Factor VIII and / or von Willebrand factor (VWF), the method comprising: (i) passing a feedstock comprising fibrinogen and / or Factor VIII and / or VWF through a hydrophobic charge-induction chromatographic resin under conditions selected such that the plasminogen and / or tissue plasminogen activator and / or other protease(s) is bound to the resin; and (ii) recovering the solution comprising fibrinogen and / or Factor VIII and / or VWF which passes through the resin; wherein the concentration of the plasminogen and / or tissue plasminogen activator and / or protease(s) in the recovered solution is reduced by at least 50% compared to the feedstock. Also provided are solutions and pharmaceutical formulations comprising the fibrinogen and / or Factor VIII and / or VWF recovered by such methods, and uses thereof.

Description

CROSS REFERENCE[0001]This application claims the benefit of U.S. Provisional Patent Application No. 61 / 733,761, filed Dec. 5, 2012, which is hereby incorporated by reference.TECHNICAL FIELD[0002]The present invention relates generally to a method of reducing the level of impurities in a solution containing at least one therapeutic protein and to the resultant therapeutic-protein containing solutions. More specifically, to a method of reducing the level of plasminogen and / or tissue plasminogen activator and / or other protease(s) in a feedstock comprising fibrinogen and / or Factor VIII and / or Von Willebrand factor (VWF). The present invention also relates generally to solutions and pharmaceutical formulations comprising the fibrinogen and / or Factor VIII and / or VWF recovered by such methods, and uses thereof.BACKGROUND[0003]Current methods of purifying a naturally-occurring or recombinant therapeutic protein from a solution comprising said protein usually carry at least some impurities i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K1/20A61K38/36A61K38/48C07K1/36
CPCC07K1/20C07K1/36A61K38/4833A61K38/363C07K14/745C07K1/18A61K38/36C07K14/75C07K14/755A61K38/00B01D15/363A61P7/04A61K38/37C07K1/16C07K1/22A61J1/05
Inventor PHAM, HUNGHEY, JEFFREY MICHAELNGUY, DARREN
Owner CSL BEHRING GMBH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products