Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.

生物治疗药物可能包含多种不同的翻译后修饰 (PTM)，需要对其进行评估，并可能进行监测和控制，以确保可重复的产品质量。在生物治疗的早期开发过程中，可以通过计算机识别和表征以及进一步的分子工程来防止意外的 PTM 。人 IgG1 (mAb1) 和双特异性 IgG-配体融合蛋白 (BsAbA) 的质量测定表明存在异常 PTM，分别导致主要 +80 Da 和 +16/+32 Da 链变体。对于mAb1，分析型阳离子交换色谱显示存在占20%的酸性峰。A + 79.957 Da 修饰位于轻链互补决定区 2 内，并根据精确质量、同位素分布和碰撞诱导解离时的完全中性丢失反应，被鉴定为硫酸化。修饰和未修饰 Fab 的自上而下超高分辨率 MALDI-ISD FT-ICR MS 允许将硫酸化分配到特定的 Tyr 残基。发现相对于受影响的 Tyr 的氨基末端位置 3 的天冬氨酸在确定硫酸化方面发挥着关键作用。对于 BsAbA，观察到 +15.995 Da 修饰并定位于三个特定 Pro 残基，解释了 +16 Da 链 A 以及 +16 Da 和 +32 Da 链 B 变体。通过与含有两种异构体形式的合成肽的共色谱法，在胰蛋白酶肽图谱中，BsAbA 修饰被验证为 4-羟基脯氨酸，而不是 3-羟基脯氨酸。最后，介绍了我们基于内部计算机预测器的警报系统方法。该系统旨在通过早期生物治疗开发过程中的分子设计和工程来防止这些 PTM。