An in vivo platform to select and evolve aggregation-resistant proteins

Abstract

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.

Aggregation Time Machine: A Platform for the Prediction and Optimization of Long-Term Antibody Stability Using Short-Term Kinetic Analysis

“…It is well established that the thermodynamic stability is an important factor in reducing mAbs aggregation. 20 , 43 , 47 , 48 In contrast, several studies could not confirm the general assumption that the stabilization of the native state of mAbs can effectively slow down aggregation that occurs through the (partially) unfolded state. 14 , 24 , 26 , 27 , 33 , 36 , 49 , 50 These contradictory results can be explained using the aggregation phase space shown in Figure 4 .…”

Discovery-stage identification of drug-like antibodies using emerging experimental and computational methods

“…For evaluation of native antibody aggregation, the tripartite TEM β-lactamase enzyme assay was adapted to link aggregation to E. coli cell growth. 100 For this assay, scFvs were inserted between two domains of the β-lactamase enzyme. Expression of the resulting tripartite fusion protein resulted in antibiotic resistance if the scFv remained soluble and did not disrupt folding of the enzyme.…”

Population Modelling in Affective Disorders

“…It is possible to conceptualise drug discovery in terms of an evolutionary process in which a number of candidate compounds compete for treatment efficacy and the winner takes all [ 35 ]. In the past decade, evolutionary approaches which are shown to speed up drug discovery [ 36 ••] were successfully implemented, for example, to identify antifungal compounds which can be repurposed to target carcinogenic tumours [ 37 ]. Below, I will argue that using an evolutionary computational framework can also speed up the discovery of novel behavioural interventions.…”