Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

Abstract

Considerable effort is expended during the mammalian cell line construction process to find a stably-transfected clone capable of supporting the large-scale manufacture of a recombinant therapeutic protein. Such a clone must synthesise a sufficient volumetric concentration of the product with the correct biochemical characteristics (glycosylation, structural integrity, etc.). Furthermore, this performance must be maintained over the extended time period required to support a manufacturing campaign in 20,000 L bioreactors. However, a significant proportion of recombinant clonal cell lines show production instability over long-term sub-culture, where volumetric product yield and/or product quality are not maintained. This instability can potentially extend product development timelines and can affect the ability of a manufacturing process to meet market demand. In the worse-case scenario it can also jeopardise patient safety if product quality is impaired. In order to prevent this, industrial cell line construction processes include long-term stability studies where several candidate lead clones are serially sub-cultured and monitored for signs of instability before selecting the final production cell line. The roots of production instability are varied, but the epigenetic silencing of transgenes at sites of host cell chromosome integration, and the direct mutation or loss of transgenes are prominent molecular causes. Considerable research has been conducted by both industry and academic groups into the molecular mechanisms underpinning instability, with an emphasis on uncovering early predictive markers of incipient instability as well as preventing its occurrence. In this article we present a detailed overview of the industrial experience of production instability and its impact on the manufacturing of recombinant therapeutics. We also discuss our current understanding of the molecular causes of cell line instability and how this has been used to mitigate the impact of this phenomenon through novel vector redesigns and cell line screening.