Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and suboptimal product quality. In addition, these variants may undergo immune reactions, henceforth need to be appropriately controlled to ensure consistent product quality and patient safety. Deamidation of insulin is the most common post‐translational modification occurring in insulin and insulin analogues. Asn^A21 desamido variant is also the most prominent product variant formed during human insulin manufacturing process and/or during the storage. Often, this deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of purified deamidated material is always being challenging due to highly similar mass, ionic, hydrophobic properties, and high structural similarity of the variant compared to the parent product. Present work demonstrates the simplified and efficient scalable process for generation of Asn^A21 deamidated variant in powder form with ~96% purity. The mixed-mode property of anion exchange resin PolyQuat was utilized to purify the deamidated impurity with high recovery. Subsequent reversed-phase high performance liquid chromatography (RP-HPLC) step was introduced for concentration of product in bind elute mode. Elution pool undergone isoelectric precipitation and lyophilisation. The lyophilized product allows users for convenient use of the deamidated impurity for intended purposes. Detailed characterization by Mass spectrometry revealed deamidation is at Asn^A21 and further confirmed that, structural and functional characterization as well as the biological activity of isolated variant is equivalent to insulin.

生物制药开发需要对产品相关的变体有适当的了解，这些变体是由于翻译后修饰和下游加工过程中形成的。这些变体可能导致产量低、生物活性降低和产品质量不佳。此外，这些变体可能会发生免疫反应，因此需要适当控制以确保一致的产品质量和患者安全。胰岛素脱酰胺是胰岛素和胰岛素类似物中最常见的翻译后修饰。ASN ^A21脱酰胺变体也是在人胰岛素制造过程和/或储存过程中形成的最突出的产品变体。通常，这种脱酰胺变体在 QC 系统的过程中和最终产品分析中用作杂质标准品。然而，由于与母体产品相比，变体具有高度相似的质量、离子、疏水特性和高度的结构相似性，大量纯化的脱酰胺材料的纯化始终具有挑战性。目前的工作展示了用于生成 Asn ^{A21的简化且高效的可扩展过程}粉末形式的脱酰胺变体，纯度约为 96%。利用阴离子交换树脂PolyQuat的混合模式特性对脱酰胺类杂质进行高回收率的纯化。随后的反相高效液相色谱 (RP-HPLC) 步骤被引入以结合洗脱模式浓缩产物。洗脱池经过等电沉淀和冻干。冻干产品允许用户方便地将脱酰胺杂质用于预期目的。质谱的详细表征显示脱酰胺作用在 Asn ^A21，并进一步证实，分离变体的结构和功能表征以及生物活性与胰岛素相当。