US 12,509,488 B2Granted

Preparative crystallization of recombinant human insulin

AssigneeBiocon Limited

InventorsSethumadhavan Sankaran, Sandeep Vishwanath Kamath, Qais Shabandri, Vibhava Shukla, Arul Marimuthu, Ankita Saikia, Sai Srikar Kandukuri

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Abstract

The present invention discloses a method for crystallizing recombinant Human Insulin at lab and manufacturing scale in the presence of zinc chloride and sodium chloride mixture, higher concentration of organic solvent (IPA-19 to 25 million) and adjusting the pH to 5.0 at a faster rate (≤5 minutes). The method further comprises adopting procedures wherein the settling time is reduced and the holding temperature is altered in order to facilitate consistent protein crystal formation between 15 μm-30 μm and to increase the robustness of the process.

Claims15

Dependent claims

A method for preparing a recombinant human insulin crystal comprising the steps of:

(a) dissolving a recombinant human insulin in a crystallization solution comprising an organic solvent and water;
(b) adding an aqueous solution comprising a mixture of zinc chloride and a salt to the crystallization solution at a final concentration of 0.3-0.5 ml per gram of recombinant human insulin;
(c) adjusting the pH of the crystallization solution to a range of 4.8 to 5.2 within 5 minutes following step (b);
(d) neat settling the crystallization solution at room temperature followed by a chilling neat settling at a chilling temperature to obtain a slurry containing the recombinant human insulin;
and
(e) freeze drying the slurry and thereby obtaining the recombinant human insulin crystal.

3 more elements

Dependencies are inferred from the claim text.