Transient Gene Expression‐Based Protein Production in Recombinant Mammalian Cells

Expression of mammalian proteins for diagnostics and therapeutics: a review

“…In antibody production via transgenic cell lines, these proteins can be expressed transiently or stably. Transient expression has a relatively short period for protein harvesting but generates a lower yield as while they can express the foreign gene, it is not integrated into the genome and therefore is not replicated and lost through time [ 15 ]. Therefore, transient expression systems are only for short-term usage [ 16 ].…”

“…The expression vector can be designed to increase expression with high expressing promoters such as pCMV or pCNA [ 23 ]. Cell culture temperature, which is slightly hypothermic was proven to cause a rise in transgene expression [ 15 ]. Furthermore, hyperosmolality which can be achieved with either continued supplementation of feeds and build-up of metabolic waste or salt supplementation can increase gene expression levels [ 24 ].…”

“…Thereby, causing proportions to vary from batch-to-batch and because of this, the culture findings are less predictable and there is a threat of contamination whether in form of bacteria, viruses or prions which also impedes further optimisation and standardisation of systems [ 83 ]. This unfortunately limits its use for therapeutic protein production as serum content increases the risk of contamination such as pathogen transmission and additionally, its high protein content can interfere with product purification during the downstream process [ 11 , 15 ]. On the bright side, this media is customizable to increase cell proliferation for a broad variety of mammalian cell types since serum contains a large number of active substances required for animal cell survival and development [ 84 ].…”

“…Transiently transfected cells generate a lower yield. Transient expression is based on transgene-carrying plasmids transfected into the cell nuclei are transcribed as non-integrated plasmids rather than being permanently incorporated into the genome and thus the foreign gene is not replicated and gets diluted over time as it is lost through cell division, degradation or other factors [ 15 ]. Transient gene expression is most frequently and successfully employed technique for functional studies such as biochemical and preclinical assessments as well as initial screening for new expression strategy, especially when examining the design of the vector construct and molecular candidates [ 16 ].…”

“…Besides improving the assembly and secretion rates of the expressed protein, lower temperatures reduce product aggregation due to improved endoplasmic reticulum machinery [ 107 ]. Hypothermia causes a rise in transgene expression, as well as an accumulation of cells in the G1 process and a decrease in cellular metabolism [ 15 ]. However, while reducing culture temperature does not impact protein function or activity, it decreases cell growth rate and impedes cell division [ 108 ].…”