Method for purifying antibody having low isoelectric point
The present inventors discovered that additional aggregation of low-pI antibody can be suppressed by removing formed antibody aggregates after a certain period of time following Protein A column purification, acidic treatment, and neutralization. Furthermore, the present inventors found that efficient impurities removal for a low-pI antibody can be accomplished by using an anion exchange resin in the Bind/Elute mode and then a hydrophobic interaction chromatography or multimodal chromatography resin, compared with conventional methods.
1. A method for purifying a composition containing an antibody with a pI of 3.0 to 8.0, which comprises the steps of:
(a) treating the composition containing the antibody with a pI of 3.0 to 8.0 with an acidic condition;
(b) neutralizing the acidic composition obtained in step (a);
(c) holding the neutralized composition obtained in step (b) at least one hour; and
(d) removing aggregates from the neutralized composition after step (c), wherein step (a) is a virus-inactivating treatment step performed after Protein A column chromatography purification of the antibody with a pI of 3.0 to 8.0.
2. The method of claim 1 , wherein the aggregate removal is carried out by anion exchange chromatography, hydrophobic interaction chromatography, multimodal chromatography, or hydroxyapatite chromatography.
3. The method of claim 1 , wherein the pI of the antibody is 5.0 to 7.5.
4. The method of claim 1 , wherein the pI of the antibody is 5.0 to 6.5.
5. The method of claim 1 , wherein aggregates removal is carried out by a method for removing impurities from a composition containing an antibody with a pI of 3.0 to 8.0, which includes the steps of:
(i) loading the composition containing the antibody with a pI of 3.0 to 8.0 onto an anion exchange resin; and
(ii) eluting the antibody with a pI of 3.0 to 8.0 from the anion exchange resin with the Bind/Elute mode using an eluting solution that has a salt concentration higher than that of the composition of (i).
6. The method of claim 1 , wherein the antibody is a humanized antibody or a human antibody.
7. The method of claim 1 , wherein the antibody is an anti-IL-6 receptor antibody or an anti-IL-31 receptor antibody.
8. A method for producing a composition containing an antibody with a pI of 3.0 to 8.0, in which the content ratio of the antibody aggregates are 3% or less, by performing the method of claim 1 .
9. A method for producing a pharmaceutical composition containing an antibody with a pI of 3.0 to 8.0, which includes the steps of:
(a) purifying an antibody with a pI of 3.0 to 8.0 by the steps of:
(i) treating the composition containing the antibody with a pI of 3.0 to 8.0 with an acidic condition;
(ii) neutralizing the acidic composition obtained in step (i);
(iii) holding the neutralized composition obtained in step (ii) at least one hour; and
(iv) removing aggregates from the neutralized composition after step (iii); wherein step (i) is a virus-inactivating treatment step performed after Protein A column chromatography purification of the antibody with a pI of 3.0 to 8.0; and
b) mixing the antibody with a pI of 3.0 to 8.0 from step a) with a pharmaceutically acceptable carrier and/or additive, thereby producing the pharmaceutical composition containing an antibody with a pI of 3.0 to 8.0.
10. A method for removing an antibody aggregate from a composition containing an antibody with a pI of 3.0 to 8.0, which includes the steps of:
(a) treating the composition containing an antibody with a pI of 3.0 to 8.0 with an acidic condition;
(b) neutralizing the acidic composition obtained in step (a);
(c) holding the neutralized composition obtained in step (b) at least one hour; and
(d) removing aggregates from the neutralized composition.
11. A method for purifying a composition containing an antibody with a pI of 3.0 to 8.0, which includes the steps of:
(a) treating the composition containing an antibody with a pI of 3.0 to 8.0 with an acidic condition;
(b) neutralizing the acidic composition obtained in step (a); and
(c) holding the neutralized composition obtained in step (b) at least one hour; and
(d) removing aggregates from the neutralized composition step (c).