Separation among product variants is one of the few remaining challenges in well established antibody downstream platform. While monomeric variants are often monitored for consistency, the uncertain safety profile of High Molecular Weight Species (HMW) requires a stringent clearance criteria (e.g. &lt;1% in drug substance). A large portion of development effort is dedicated to address this issue in many projects. HMW can come from cell culture where intra- and inter-molecular disulfide bond shuffling is possible in intracellular redox environment. HMW can also come from downstream where chemical (e.g. low pH) and mechanical (e.g. shear) stress are common. Together they present a heterogeneous distribution in both size and surface properties. The resolution between HMW (mostly in the form of dimer) and monomer in downstream platform relies on the subtle difference in local surface charge and/or hydrophobicity instead of size. Here we present several cases of monoclonal antibody (mAb) and fusion protein purification where significant HMW heterogeneity exists. Some HMWs are separated by ion-exchange while others require hydrophobic interaction or mixed mode chromatography for clearance. Selective precipitation can also serve as an additional mean if included in platform. Overall, nonnative HMWs formed in downstream processing are relatively easy to remove, while HMWs originated from upstream with more native structures are often more difficult to separate. Outstanding challenge has been observed where heterogeneity exhibits in both monomer and HMWs and all species drive a complex competition in purification behaviors. In some cases, transient conformational change occurred at air-water (mixing) and solid-water (adsorption) interface, and resulted in less consistent clearance. This situation requires in-process stability control which is difficult for manufacture operation. The experiences across multiple molecules reinforce the understanding that process capability is limited by the exposed surface structural signatures of the HWMs rather than their absolute quantity. Therefore a more comprehensive strategy is desirable in both upstream and downstream development in order to safeguard the platform implementation.

Heterogeneous high molecular weight species in purification platform

249th National Meeting (2015)

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