Preparation and composition of inter-alpha proteins from blood
Inventors
Lim, Yow-Pin • Sirya, Edward S. • Brne, Peter
Assignees
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Publication Number
US-9139641-B2
Publication Date
2015-09-22
Expiration Date
Abstract
The present invention generally provides processes for purification of Inter-alpha inhibitor proteins (IαIp) and compositions thereof from blood.
Core Innovation
The invention concerns purification of inter-alpha inhibitor proteins (IαIp proteins) in which IαIp proteins comprise inter-alpha inhibitor (IαI) having two heavy chains (H1 and H2) and a light chain (L), and pre-alpha inhibitor (PαI) having a heavy chain (H3) and a light chain (L). The core approach performs a wash step after the IαIp proteins are bound to a chromatography support, and the wash step exposes bound IαIp proteins to a wash buffer having a pH of about 4.0 or lower.
The invention maintains a controlled loss of IαIp proteins from the support during the wash step, with the wash resulting in a loss of 20% or less of the IαIp proteins from the support. In documented examples, low-pH wash conditions are used while maintaining high binding/yield and improved purity.
A further documented aspect combines different wash strategies, including a high-salt wash together with a subsequent very low-pH wash. The purified IαIp proteins are presented for therapeutic use and as compositions and kits, including therapeutic and analytical kits that contain IαIp purified under low-pH wash conditions.
Claims Coverage
The partial content provides two independent claims. Across these claims, the inventive coverage centers on purifying IαIp proteins by using a low-pH wash during chromatography while limiting protein loss from the chromatography support to 20% or less.
Low-pH wash chromatography for purified IαIp proteins with limited support loss
A method for purifying IαIp proteins, wherein the IαIp proteins comprise IαI with heavy chains (H1 and H2) and light chain (L), and PαI with heavy chain (H3) and light chain (L), the method comprising exposing IαIp proteins bound to a chromatography support to a wash buffer having a pH of about 4.0 or lower, wherein the wash step results in a loss of 20% or less of the IαIp proteins from the support.
Placing blood or plasma fractions on chromatography support and low-pH wash for purified IαIp proteins
A method for purifying IαIp proteins, the method comprising placing blood, a blood plasma fraction, or an intermediate plasma fraction on a chromatography support, and subjecting the support to a wash buffer with a pH of about 4.0 or lower, wherein the wash step results in a loss of 20% or less of the IαIp proteins from the support.
Both independent claims require chromatography with a low-pH wash (about 4.0 or lower) and limit wash-associated loss of IαIp proteins from the chromatography support to 20% or less; the second claim further specifies the use of blood or blood/plasma fractions as the starting material.
Stated Advantages
Improved purity while maintaining high binding/yield during low-pH wash purification.
Enhanced competitive ELISA signal without loss of trypsin inhibitory activity.
Documented Applications
Therapeutic use for sepsis, systemic inflammatory response syndrome (SIRS), septic shock, and other inflammatory/infectious conditions.
Use in connection with rheumatoid arthritis.
Use in connection with cancer/cancer metastasis.
Use in connection with preterm labor.
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