A process for isolating properly folded insulin or insulin analog from an aqueous mixture comprising the properly folded insulin or insulin analog and process related impurities, wherein the process comprises: subjecting the aqueous mixture to two orthogonal chromatography purification steps in tandem to separate the isolated properly folded insulin or insulin analog from the related process impurities; wherein the two orthogonal chromatography purification steps in tandem comprise a first chromatography step that separates molecules based upon charge and a second chromatography step that separates molecules based upon hydrophobic interactions; wherein the first chromatography step that separates molecules based upon charge is selected from anion exchange chromatography and mixed mode anion exchange chromatography; wherein the second chromatography step that separates molecules based upon hydrophobic interactions is selected from reverse phase high pressure liquid chromatography (RP-HPLC) and hydrophobic interaction chromatography; wherein the MMA chromatography is performed with a resin comprising cross-linked agarose conjugated to N-benzyl-N-methyl ethanolamine; wherein the AEX chromatography is performed with a resin comprising agarose conjugated to DEAE; wherein the MMA or AEX chromatography comprises; (i) applying to an MMA or AEX chromatography matrix an aqueous solution in which properly folded precursor insulin or insulin analog has been enzymatically digested to produce the properly folded insulin or insulin analog; (ii) washing the MMA or AEX chromatography matrix with a first wash solution comprising a buffer at a basic pH and then washing the MMA or AEX chromatography matrix with a second wash solution comprising a buffer at an acidic pH greater than or higher than the pH capable of eluting the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix; and (iii) eluting the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix with an eluting solution comprising a buffer at an acidic pH sufficient to elute the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix to provide a mixture of the properly folded insulin or insulin analog for the second chromatography step. wherein the two orthogonal chromatography purification steps in tandem comprise a first chromatography step that separates molecules based upon charge and a second chromatography step that separates molecules based upon hydrophobic interactions; wherein the first chromatography step that separates molecules based upon charge is selected from anion exchange chromatography and mixed mode anion exchange chromatography; wherein the second chromatography step that separates molecules based upon hydrophobic interactions is selected from reverse phase high pressure liquid chromatography (RP-HPLC) and hydrophobic interaction chromatography; wherein the MMA chromatography is performed with a resin comprising cross-linked agarose conjugated to N-benzyl-N-methyl ethanolamine; wherein the AEX chromatography is performed with a resin comprising agarose conjugated to DEAE; wherein the MMA or AEX chromatography comprises; (i) applying to an MMA or AEX chromatography matrix an aqueous solution in which properly folded precursor insulin or insulin analog has been enzymatically digested to produce the properly folded insulin or insulin analog; (ii) washing the MMA or AEX chromatography matrix with a first wash solution comprising a buffer at a basic pH and then washing the MMA or AEX chromatography matrix with a second wash solution comprising a buffer at an acidic pH greater than or higher than the pH capable of eluting the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix; and (iii) eluting the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix with an eluting solution comprising a buffer at an acidic pH sufficient to elute the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix to provide a mixture of the properly folded insulin or insulin analog for the second chromatography step. (i) applying to an MMA or AEX chromatography matrix an aqueous solution in which properly folded precursor insulin or insulin analog has been enzymatically digested to produce the properly folded insulin or insulin analog; (ii) washing the MMA or AEX chromatography matrix with a first wash solution comprising a buffer at a basic pH and then washing the MMA or AEX chromatography matrix with a second wash solution comprising a buffer at an acidic pH greater than or higher than the pH capable of eluting the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix; and (iii) eluting the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix with an eluting solution comprising a buffer at an acidic pH sufficient to elute the properly folded insulin or insulin analog from the MMA or AEX chromatography matrix to provide a mixture of the properly folded insulin or insulin analog for the second chromatography step.