Contaminant removal method

A drug and composition technology, applied in the field of ApoA-I preparations, can solve the problems of low protein recovery, low virus retention, high virus clearance is not optimal, etc.

Active Publication Date: 2016-03-30
JET
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result of filter fouling, there may be a drop in filtration selectivity which results in lower protein recovery and/or lower virus retention
Additionally, filter fouling reduces capacity and throughput, which results in longer filtration times and/or requires increased filter area
In addition, operating conditions that are optimal for maintaining apolipoprotein solubility and preventing aggregation may not be optimal for ensuring hi

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] The following example describes a sequence of method steps of a preferred embodiment of the method according to the invention. The process involves the fraction IV precipitate-derived feedstock being dissolved in the presence of GuHCl, filtered to remove filter aids, followed by pH adjustment, heat treatment, dilution and virus filtration. Alternatively, heat treatment can also be performed after the virus filtration step.

[0140] Methods and Materials

[0141] ApoA-I protein concentration

[0142] ApoA-I protein concentration is generally measured by measuring absorbance at 280 nm using WFI (Water for Injection) as a diluent, and it is usually 5 to 30 g / L. Protein is calculated as follows:

[0143]

[0144] Alternatively, ApoA-I protein concentration can be determined using nephelometry or high performance capillary electrophoresis (Hewlett Packard 3DCE, Agilent Technology). Briefly, for high-efficiency capillary electrophoresis, the method involves the followi...

Embodiment 2

[0167] Filtration was compared using BioEx filters in the presence of different GuHCl concentrations (1.7M & 3.2M) and ApoA-I concentrations (5.8, 8.9, 19.7g / L) ( Figures 1 to 3 ). The virus filtration process was performed in a similar manner as described in Example 1 above.

[0168] Additional filtration studies confirmed that lower concentrations of GuHCl (1.0M & 1.3M) could lead to solution instability which caused filter plugging of the system. These results highlight the benefit of optimizing the concentration level of GuHCl to facilitate virus filtration.

Embodiment 3

[0170] ApoA-I samples were spiked with MVM at a ratio of 1:1000. Fortified samples were filtered through PlanovaBioEX virus removal filters in dead-end mode at a pressure of approximately 2.4 bar at a protein concentration of 10 to 12 g / L. Samples of the filtrate were taken and assayed for residual virus infectivity (Table 1). The results confirmed that complete viral retention was achieved over a range of GuHCl concentrations. Lower GuHCl concentrations resulted in increased log reduction values ​​(LRV) of MVM.

[0171] Table 1

[0172]

[0173] In yet another virus filtration study performed under similar conditions, MVM virus penetration was observed in the presence of 3.4M GuHCl. Therefore, concentrations below about 3.0 M GuHCl are believed to improve the removal of viruses with a diameter of about 20 nm (eg, parvovirus) in the manufacture of ApoA-I preparations using virus removal filters such as BioEx.

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Abstract

A method for purifying Apo A-l is provided including the steps of providing a solution comprising Apo A-l and guanidine hydrochloride and filtering the solution through a filter having a pore size in a range from 15 nrn to 35 nm to thereby reduce viral contamination of the Apo A-l. An Apo A-f preparation is provided having at least a 12 log LRV (log reduction value) for a parvovirus; and/or at least 9 log LRV for a non-enveloped virus; and/or at least 8.5 log LRV for a lipid enveloped virus. Also provided are pharmaceutical compositions and reconstituted high density lipoprotein formulation comprising Apo A-l and methods of treating diseases disorders or conditions.

Description

technical field [0001] The present invention relates to a method for purifying apolipoprotein, in particular for removing viral pathogens from a solution containing apolipoprotein A-I (ApoA-I), and to providing an ApoA-I preparation. Background technique [0002] Apolipoproteins are the major protein component of soluble lipoprotein complexes, and apolipoprotein A-I (ApoA-I) is the major protein component of high-density lipoprotein (HDL) particles. [0003] The A, C, and E families of apolipoproteins have evolved from a common ancestral gene, and they are structurally similar. These protein molecules typically contain a series of 22-amino acid tandem repeats, often separated by proline residues. The repeating 22-amino acid segment forms an amphipathic α-helix capable of binding both lipids and water surfaces. In the case of human ApoA-I (243 amino acids; 28.1 kDa), there are eight 22-mer and two 11-mer amphipathic helices (Lund-Katz & Phillips, 2010, Subcell Biochem. 51, ...

Claims

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Application Information

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IPC IPC(8): C07K14/775C07K1/34C07K1/36A61K38/16
CPCA61K38/00C07K14/775A61P3/06A61P9/00A61P9/10A61P3/10Y02A50/30C07K1/34
Inventor G·瓦仁Y·卢西亚C·凯姆佩M·斯图克基
Owner JET
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