Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production

Max Hebditch,Ryan Kean,Jim Warwicker

doi:10.1016/j.csbj.2020.03.002

Max Hebditch, Ryan Kean + Show 1 more

Open Access

https://doi.org/10.1016/j.csbj.2020.03.002

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Abstract

Engineered proteins are increasingly being required to function or pass through environmental stresses for which the underlying protein has not evolved. A major example in health are antibody therapeutics, where a low pH step is used for purification and viral inactivation. In order to develop a computational model for analysis of pH-stability, predictions are compared with experimental data for the relative pH-sensitivities of antibody domains. The model is then applied to proteases that have evolved to be functional in an acid environment, showing a clear signature for low pH-dependence of stability in the neutral to acidic pH region, largely through reduction of salt-bridges. Interestingly, an extensively acidic protein surface can maintain contribution to structural stabilisation at acidic pH through replacement of basic sidechains with polar, hydrogen-bonding groups. These observations form a design principle for engineering acid-stable proteins.

Highlights

The promise of therapeutic antibodies relies on the ability of the pharmaceutical industry to develop large scale manufacturing processes that can produce safe, economic and reproducible formulations
Following protein A purification, antibodies expressed from mammalian sources require a low pH hold for viral inactivation which can lead to further aggregation [7]
We report that the CH1 domain appears to be the least destabilised by acid titration, potentially due to its IDP-like characteristics, but more importantly our calculations suggest that the CH2 domain is the most destabilised at low pH, due to a large loss of ionisable interactions which are stabilising at neutral pH, but destabilising at low pH

Summary

Introduction

The promise of therapeutic antibodies relies on the ability of the pharmaceutical industry to develop large scale manufacturing processes that can produce safe, economic and reproducible formulations. These biopharmaceutical production processes are limited by non-specific interactions between the proteins, which can lead to reversible or irreversible association. Protein A chromatography was one of the first protein purification processes developed [2], and is still the state of the art for the manufacture of biotherapeutic antibodies [3,4] as it is highly effective, resulting in > 98% purity in a single step [5] It is highly stable, retaining specificity over multiple uses [6]. It has been observed that exposure to acidic pH can cause polyreactivity, which may be part of the natural immune response, but is an issue for developing targeted therapeutics [10,11]

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