High throughput protein production from transient transfection of mammalian cells is used in multiple facets of research and development studies. Commonly used formats for these high number expressions are 12-, 24- and 96-well plates at various volumes. However there are no published examples of a 96-deep well plate microscale (1,000L) suspension process for mammalian transient expression. For this reason, we aimed to determine the optimal operating conditions for a high producing, microscale HEK293 transient system. We evaluated the hydrodynamic flow and measured the oxygen transfer rate (OTR) and transient protein expression for 96-deep well plates of different well geometries filled at 600-1,000L working volumes and agitated at various speeds and orbital diameters. Ultimately, a round well-round bottom (RR) 96-deep well plate with a working volume of 1,000 mu L agitated at 1,000RPM and a 3mm orbital diameter yielded the highest and most consistent total transient protein production. As plate cultures are subject to evaporation, water loss from different plate seals was measured to identify an optimal plate sealing method. Finally, to enable higher capacity protein production, both expression and purification processes were automated. Functionality of this end-to-end automation workflow was demonstrated with the generation of high levels of human IgG1 antibodies (360 mu g/mL) with reproducible productivity, product quality and 78% purification recovery. Biotechnol. Bioeng. 2015;112: 1832-1842. (c) 2015 Wiley Periodicals, Inc.