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Methods of Removing Contaminants

A precipitate and solution technology, applied in the field of ApoA-I preparations, can solve the problems of low virus retention, long filtration time, filter fouling to reduce capacity and processing capacity, etc.

Active Publication Date: 2019-03-19
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 high viral clearance
In particular, chaotropic substances that may be used to stabilize apolipoproteins can alter the filterability of pathogens (such as viruses) and possibly filter membranes
As a result, the presence of certain concentrations of chaotropic substances may also allow penetration of undesired viruses through the filter membrane, thereby negating the usefulness of the step of processing the apolipoprotein-containing solution

Method used

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  • Methods of Removing Contaminants
  • Methods of Removing Contaminants
  • Methods of Removing Contaminants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] The following example describes the sequential arrangement of the 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] Apo A-I protein concentration

[0142] The Apo A-I protein concentration is generally measured by measuring absorbance at 280 nm using WFI (water for injection) as a diluent, and is usually 5 to 30 g / L. The protein is calculated as follows:

[0143]

[0144] Alternatively, Apo A-I protein concentration can be determined using turbidimetry or high performance capillary electrophoresis (Hewlett Packard 3D CE, Agilent Technology). Briefly, for high performance capillary electrophoresis, t...

Embodiment 2

[0167] Filtration was compared using BioEx filters in the presence of different GuHCl concentrations (1.7M & 3.2M) and Apo A-I concentrations (5.8, 8.9, 19.7 g / 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 in this system. These results underscore the benefits of optimizing the concentration level of GuHCl to facilitate viral filtration.

Embodiment 3

[0170] Apo A-I samples were spiked with MVM at a ratio of 1:1000. The spiked samples were filtered at a protein concentration of 10 to 12 g / L through a Planova BioEX virus removal filter in dead end mode at a pressure of approximately 2.4 bar. A sample of the filtrate was taken and determined for residual viral infectivity (Table 1). The results confirmed that complete virus retention was achieved over the range of GuHCl concentrations. Lower GuHCl concentrations resulted in an increase in the log reduction value (LRV) of MVM.

[0171] Table 1

[0172]

[0173] In yet another virus filtration study conducted 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 removal of viruses (eg, parvoviruses) with a diameter of about 20 nm using a virus removal filter (such as BioEx) in the manufacture of Apo A-I preparations.

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Abstract

The present invention provides a method for purifying Apo A-I comprising providing a solution comprising Apo A-I and guanidine hydrochloride and filtering the solution through a filter having a pore size ranging from 15nm to 35nm thereby reducing Apo A-I steps of virus contamination. The present invention provides an Apo A-f preparation having an LRV (log reduction value) of at least 12 log against parvoviruses; and / or having an LRV of at least 9 log against non-enveloped viruses; and / or against lipid envelopes Viruses have an LRV of at least 8.5 log. The present invention also provides pharmaceutical compositions and reconstituted HDL preparations comprising Apo A-I and methods for treating diseases, disorders or conditions.

Description

technical field [0001] The present invention relates to a method for purifying apolipoproteins, in particular for removing viral pathogens from solutions containing apolipoprotein A-I (ApoA-I), and to providing an Apo A-I preparation. Background technique [0002] Apolipoprotein is the major protein component in 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 evolved from a common ancestral gene that are structurally similar. These protein molecules generally contain a series of 22-amino acid tandem repeats, often separated by proline residues. The repeating 22-amino acid segment forms an amphiphilic alpha-helix capable of binding to both lipid and water surfaces. In the case of human Apo A-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, 183-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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