The increasing prevalence of low pI non-mAb therapeutics as well as current challenges in mAb-aggregate separations and low recoveries motivate further development in the multimodal anion exchange (MM AEX) space. In this work, linear salt gradient experiments at pH 7 were used to evaluate the retention of model proteins (with pI from 3.4 to 6.8) in 17 novel MM AEX prototype systems. The ligands were organized into three series. Series 1 extended previous work in multimodal ligand design and included a hydroxyl variant and linker length variants. Series 2 and 3 investigated the nature of hydrophobicity in MM AEX systems by adding hydrophobic (series 2) or fluorine (series 3) substituents to a solvent exposed phenyl ring. Compared to the commercial resin Capto Adhere, the series 1 and 3 ligands exhibited weaker binding, while some of the series 2 aliphatic prototypes showed dramatically increased retention and unique selectivities. Within series 1, the model proteins eluted earlier in the gradient as the charge-hydrophobic group distance on the ligand was increased from 4.9 Å to 8.5 Å. For the aliphatic variants in series 2, proteins that eluted early in the salt gradient were not affected by the increase in ligand hydrophobicity, while the later eluting proteins bound stronger as the length of the aliphatic substituent increased. The series 3 variants indicated that phenyl ring fluorination created subtle changes in protein elution in these MM AEX systems. Retention data from the three series was used to generate a partial least squares QSAR model based on both protein and ligand descriptors which accurately predicted protein retention with a training R² of 0.81 and a test R² of 0.76. The retention characteristics of some prototypes such as the earlier elution and unique selectivities compared to Capto Adhere suggest that they could potentially provide unique selectivities and increased recovery for the downstream processing of both mAb and non-mAb biotherapeutics.

低pI非mAb疗法的普及率不断提高，以及mAb聚集体分离和低回收率方面的当前挑战，促使多峰阴离子交换（MM AEX）空间的进一步发展。在这项工作中，使用pH为7的线性盐梯度实验来评估模型蛋白在17种新型MM AEX原型系统中的保留（pI从3.4到6.8）。配体分为三个系列。系列1扩展了先前在多峰配体设计中的工作，并包括羟基变体和接头长度变体。系列2和3通过向溶剂暴露的苯环中添加疏水性（系列2）或氟（系列3）取代基来研究MM AEX系统中疏水性的性质。与商品树脂Capto Adhere相比，系列1和3的配体显示出较弱的结合力，而2系列脂族原型中的某些原型则显示出显着提高的保留率和独特的选择性。在系列1中，随着配体上电荷-疏水基团距离从4.9到8.5的增加，模型蛋白质在梯度中更早地洗脱。对于系列2中的脂族变异体，在盐梯度中较早洗脱的蛋白质不受配体疏水性增加的影响，而后来洗脱的蛋白质随着脂族取代基长度的增加而结合得更牢固。系列3的变体表明，在这些MM AEX系统中，苯环氟化在蛋白质洗脱中产生了细微的变化。来自三个系列的保留数据用于基于蛋白质和配体描述符生成偏最小二乘QSAR模型，该模型可通过训练R准确预测蛋白质保留²为0.81，测试R ²为0.76。与Capto Adhere相比，某些原型的保留特性（如更早的洗脱和独特的选择性）表明，它们可能为mAb和非mAb生物治疗剂的下游加工提供独特的选择性并提高回收率。