Method of separating hemoglobin A2

Details Method of separating hemoglobin A2 Method of separating hemoglobin A2

2001-04-18

2002-12-03

Therkorn, Ernest G. (Department: 1723)

Liquid purification or separation

Processes

Chromatography

C210S635000, C210S198200, C530S385000, C530S416000, C436S066000

Reexamination Certificate

active

06488857

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a method for separating hemoglobin A
2
by cation exchange liquid chromatography.
BACKGROUND ART
Hemoglobin A
2
is composed of &agr; and &dgr; chains, and is used, like hemoglobin F, as a diagnostic indicator of Mediterranean anemia (thalassemia). For testing of Mediterranean anemia, cation exchange liquid chromatography is generally employed to separate from a hemoglobin mixture and quantitate hemoglobin A
2
(hereinafter referred to as HbA
2
).
Separation of a hemoglobin mixture, i.e., a hemolyzed sample, with the use of cation exchange liqud chromatography generally results in the appearance of peaks of hemoglobin A
1a
(hereinafter referred to as HbA
1a
) and hemoglobin A
1b
(hereinafter referred to as HbA
1b
), hemoglobin F (hereinafter referred to as HbF), labile hemoglobin A
1c
, (hereinafter referred to as labile HbA
1c
), stable hemoglobin A
1c
(hereinafter referred to as stable HbA
1c
), hemoglobin A
0
(hereinafter referred to as HbA
0
) and the like. On this occasion, an extended time has been required to achieve complete separation of HbA
0
and HbA
2
from each other since they show the similar retention behaviors with respect to a cation exchange column and their peaks occur in close proximity to each other.
One method of separating HbA
2
from a hemoglobin (hereinafter referred to as Hb) mixture is disclosed, for example, in U.S. Pat. No. 4,810,391 which utilizes a weak cation exchange packing material and is characterized by the use of three types of buffering eluents having different phosphate concentrations and pH's. A first eluent is a phosphate buffer maintained at a concentration of 1-20 mm and at a pH of 6.5-6.9 for use in elution of HbA
1a
and HbA
1b
. A second eluent is a phosphate buffer maintained at a concentration of 15-55 mM and at a pH of 6.4-6.8 for use in elution of HbA
1c
and HbA
0
. A third eluent is a phosphate buffer maintained at a concentration of 60-100 mM and at a pH of 6.4-6.8 for use in elution of HbA
2
.
EP-0315187 discloses a method for quantification of HbA
2
wherein HbA
1a
and HbA
1b
are eluted by a first eluent, HbA
1c
and HbA
0
by a second eluent and HbA
2
by a third eluent.
However, in the above-described methods, the eluents are distinguished from each other substantially by the salt concentration only. This extends a measurement time and prevents quick separation and measurement of a lot of specimen, which have been problems. Another problem arises when HbA
2
and HbA
1c
are concurrently separated and measured, i.e., precise measurement of HbA
1c
is disturbed significantly by the insufficient separation of HbA
1c
Also, the pH's of the eluents are lower than an isoelectric point of hemoglobin. This creates another problem, i.e., increases the tendency of Hb and other blood components to be adsorbed on packing material to thereby shorten a column service life.
In view of the above-described problems encountered in conventional methods of separating HbA
2
from a Hb mixture, an object of the present invention is to provide a method which enables quicker and more precise separation of HbA
2
than conventional methods.
DISCLOSURE OF THE INVENTION
A first invention of the present application is a method for separating hemoglobin A
2
from a hemoglobin mixture by cation exchange chromatography, characterized in that at least two types of eluents are used including an eluent (eluent A) for elution of faster hemoglobins than hemoglobin A
0
and an eluent (eluent B) for elution of hemoglobin A
0
and slower hemoglobins than hemoglobin A
0
, and that the eluent A has a pH of 4.0-6.0 and the eluent B is maintained at a pH of at least 0.5 higher than that of the eluent A.
In a particular aspect of the first invention, the eluent B is maintained at a salt concentration lower than that of the eluent A.
In a more limited aspect of the first invention, an eluent (eluent D) is further used for elution of at least hemoglobin A
2
. The eluent D is maintained at a pH of at least 0.5 lower than that of the eluent B.
In a more limited aspect of the first invention, a salt concentration of the eluent B is rendered lower than that of the eluent A and a salt concentration of the eluent D is rendered higher than that of the eluent B.
In a further particular aspect of the first invention, an eluent (eluent C) is further used having an elution power weaker than those of the eluents B and D. Those eluents are delivered in the sequence of B, C and D.
In a further particular aspect of the first invention, an eluent (eluent H) at a pH of 6.8-12 is delivered to follow the eluent B, or alternatively, to follow the eluent D when the eluents B and D are used in combination. The eluent H preferably contains a chaotropic ion.
A second invention of the present application is a method for separating hemoglobin A
2
from a hemoglobin mixture by cation exchange chromatography, characterized by the use of at least two types of eluents including an eluent (hereinafter referred to as “eluent E”) which contains a chaotropic ion and also contains an inorganic acid, organic acid and/or any salt thereof having a buffer capacity at a pH of 4.0-6.8 and an eluent (hereinafter referred to as “eluent F”) which contains an inorganic acid, organic acid and/or any salt thereof having a buffer capacity at a pH of 6.5-8.0.
In a particular aspect of the second invention, an eluent (hereinafter referred to as “eluent G”) is further used having a lower pH and a higher salt concentration than the eluent F. The eluent G is delivered to follow the eluent F.
In a further aspect of the second invention, an eluent (eluent H) at a pH of 6.8-12 is delivered to follow the eluent F or G. Preferably, the eluent H contains a chaotropic ion.
In another particular aspect of the first or second invention, an azide ion is incorporated in at least one type of eluent and/or a hemolyzing solution.
In a further particular aspect of the first or second invention, a packing material is employed having at least one type of strong cation exchange group.
The first invention is now described.
In the first invention, “an eluent (referred to in this specification as “eluent A”) used to elute faster hemoglobins than hemoglobin A
0
” refers to the eluent used to elute Hb's that exhibit shorter retention time than HbA
0
while the hemoglobin mixture is subjected to separation by cation exchange liquid chromatography. General examples of Hb's to be eluted by the eluent A include HbA
1a
, HbA
1b
, HbF, labile HbA
1c
, stable HbA
1c
and the like. It should be understood that not all of the above-listed Hb's but at least one type thereamong is needed to be eluted by the eluent A. The first invention, while its primary object is to separate HbA
2
, also enables the separation and quantitation of stable HbA
1c
by performing elution of HbA
1a
, HbA
1b
, HbF, labile HbA
1c
and stable HbA
1c
with the aid of the eluent A.
The eluent A has a pH of 4.0-6.0, preferably of 4.5-5.8. If a pH of the eluent A is below 4.0, denaturation of hemoglobin may be caused to occur. If a pH of the eluent A exceeds 6.0, hemoglobins may be charged less positively to reduce its tendency to be retained by the cation exchange group, resulting in its reduced resolution. The eluent A is not necessarily a single type of eluent, and may be an eluent group A consisting of plural types of eluents having differing compositions and elution powers (in the case where the eluent A consists of n types of eluents, they are hereafter designated “eluent A1, eluent A2, . . . , eluent An”), so long as the eluent group A satisfies the above-specified conditions required for the eluent A. When desired to separate and quantitate stable HbA
1c
, as well as HbA
2
, within a shorter period of time, the eluent components A1, A2, A3 and the like may be delivered in a sequence of increasing elution power according to a linear gradient elution technique, a stepwise gradient elution technique or a combination thereof.
In the first invention, “an eluent (referred to in this specification

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