Purpose <p>Structurally defined and pharmaceutically compliant insulin analogues are critical for ensuring consistent pharmacological performance. This study aimed to develop a robust and scalable downstream processing strategy for the efficient production and purification of recombinant Insulin Glargine Metabolite M1, a long-acting insulin analogue, with high purity and molecular integrity.</p> Methods <p>Recombinant <i>Escherichia coli</i> expressing Insulin Glargine Metabolite M1 was harvested post-fermentation by centrifugation. The target protein was recovered from inclusion bodies following cell lysis and solubilization. Sulphitolysis was performed to linearize the protein, followed by isopropyl alcohol (IPA) precipitation for concentration. Cyanogen bromide (CNBr) cleavage was used to remove the His-tag. The resulting pro-insulin intermediate was refolded to achieve its native conformation. Enzymatic conversion to mature Metabolite M1 was carried out using trypsin and carboxypeptidase B. Purification was achieved through anion exchange chromatography (A-IEX), hydrophobic interaction chromatography (HIC), and final polishing by reversed-phase chromatography (RPC).</p> Results <p>SDS–PAGE analysis confirmed successful maturation with the removal of high-molecular-weight impurities. Sequential chromatographic steps efficiently eliminated process-related contaminants, including misfolded species, aggregates, and residual enzymes. RPC yielded a single, sharp and symmetric peak, indicating high chemical purity of 99.5%. Electrospray ionization mass spectrometry verified the molecular identity and integrity of the product, with a mass of 5750 Da.</p> Conclusion <p>The developed integrated downstream process enables the production of highly pure and structurally homogeneous Insulin Glargine Metabolite M1, demonstrating its potential suitability for pharmaceutical applications and further biochemical characterization.</p> Graphical Abstract <p></p>