Enhancement of CHO bioreactor performance has typically been derived from optimization of media formulations and feeding strategies, with advances in clone selection systems and cell engineering also playing an essential role. However, these breakthroughs in media development are usually not disclosed by the biopharmaceutical industry or media vendors due to commercial considerations. As a result, optimisation of CHO culture performance from the research sector is thus limited and timeconsuming with undesired and/or unexpected effects in essential steps (e. g. transfection) also observed. To address this deficit in information, in-house serum-free and chemically-defined media (SFM and CDM) were developed as working tools to study the effects of media additives in culture performance. Investigating the titer-enhancing effects of zinc, the specific productivity of DP12 and rCHO-K1 cell lines could be significantly increased. A correlated effect was also observed at transcriptional level, with increased oxidative respiration metabolism also associated with the zincsupplemented, higher-producing cultures. Building on from the knowledge gained, further investigation on essential additives for CHO survival was then performed, identifying putrescine as a vital supplement. Based on this phenotype, a novel auxotrophic-based selection system was designed. The method offers a drug-free, easy-to-apply and cost-effective system for cell line development, observed to successfully isolate hEPO- and GFP-expressing clones with stable production profiles for at least 42 generations. Further characterisation of the polyamine-dependent phenotype of CHO by gene expression microarray (Affymetrix) was then performed, suggesting an association between cessation of growth and increased G1/S transition but arrest at M/G1 checkpoint. Finally, to highlight the essential implications of media additives in other key steps for bioprocess optimisation, the effect of media additives in transfection was investigated. Assessing the efficiencies of liposome-, lipopolyplexes- and polymer-mediated transfections, an inhibitory role of ferric ammonium citrate was identified and a novel strategy to circumvent this inhibition was recommended