69C93). using design of experiments. We used this strategy to develop and optimize built-in purification processes for any solitary\website antibody and a cytokine, obtaining yields of 88% and 86%, respectively, with process\ and product\related variants reduced to phase\appropriate levels for nonclinical material. (NRRL Y\11430) was revised to express G41, a solitary\website antibody, or G\CSF, a cytokine, as explained previously (Crowell et al.,?2018). The biophysical characteristics of each molecule can be found in Table?S1. Shake flask cultivations were conducted as explained MK-4305 (Suvorexant) previously (Timmick et al.,?2018), except rich defined press (Matthews, Kuo, et al.,?2017) was substituted for complex press. 4% glycerol was added for outgrowth and 5% methanol/30?g/L sorbitol was added for production. 0.1% CHAPs (3\[(3\cholamidopropyl)dimethylammonio]\1\propanesulfonate hydrate) was added to the medium MK-4305 (Suvorexant) for G\CSF cultivations. Additional supernatant was produced MK-4305 (Suvorexant) using the production module of the InSCyT system managed in perfusion mode (Crowell et al.,?2018). In the bioreactor, temp, pH, and dissolved oxygen were managed at 25C, 6.5, and 25%, respectively. All chemical reagents were purchased from Sigma\Aldrich. 2.2. Resin selection For G41, resins were selected based on our previously developed platform process for the purification PPP3CB of solitary\website antibodies (Crowell et al.,?2021). This platform process was based on the purification processes expected from our in silico tool for two different solitary\website antibodies (Timmick et al.,?2018). The selected resins included CMM HyperCel and HyperCel Celebrity AX (Pall Corporation). For G\CSF, our in silico tool was used exactly as explained in Timmick et al., and the selected resins included CaptoMMC ImpRes, HyperCel Celebrity AX, and MEP HyperCel (GE Healthcare Existence Sciences?and Pall Corporation). 2.3. Dedication of dynamic binding capacity A full factorial design of experiment (DoE) was designed to model dynamic binding capacity using JMP? Pro 14.0.0. (SAS Institute Inc.).?supernatant containing G41 was concentrated approximately 30\fold using Amicon? Ultra\15 Centrifugal Filter Devices with 3?kDa membranes (MilliporeSigma). The concentrated supernatant was then diluted 15\fold into the appropriate capture buffer. Nine experiments were conducted with capture buffers of 20?mM sodium citrate pH 4, 5, or 6 and conductivity 10, 20, or 30?mS/cm whatsoever permutations. Conductivity was modified using sodium chloride. All experiments were conducted on a Tecan Freedom EVO? 150, controlled by EVOware Standard version 2.7.30.0 (Tecan Trading AG). The system was equipped with an eight\channel liquid handling (LiHa) arm, an eccentric robot manipulator (RoMa) arm, 1?ml syringes, Te\Shuttle, and Te\Chrom modules. Absorbance was measured on a Tecan Infinite M200 Pro using 96\well UV transparent plates (Corning Inc.). Two hundred?microliter?prepacked OPUS? RoboColumns? were used (Repligen Corporation). Columns were equilibrated in capture buffer. The prepared supernatant was loaded up to 65 column quantities (CVs). The columns were then re\equilibrated in capture buffer and eluted with 20?mM sodium phosphate, pH 8.0, 300?mM NaCl. Two hundred?microliter?fractions were collected during the weight and elution methods and absorbance was measured at 280?nm and 260?nm. Absorbance measurements were corrected for liquid level using absorbance at A990\A900 (Diederich & Hubbuch,?2017). The dynamic binding capacity was determined as the amount of protein loaded onto the column when 20% breakthrough was reached. Protein concentration in the loaded supernatant was determined by size exclusion chromatography (SEC) (observe below). JMP? Pro 14.0.0 was used to model the DoE (SAS Institute Inc.). Dynamic binding capacity for G\CSF was modelled the same as.