During initial Standard run, monitor run progress to ensure proper performance of the system (Figure?4C). j. After the run sequence is complete, use the Chromeleon analysis tool suite to quantify the area under the curve for pre-MP, MP and post-MP as a percent of total area for each sample. Expected outcomes This protocol describes a high-throughput method for cloning, expression, purification, and the initial evaluation of bispecific antibodies. The Golden Gate cloning method utilizes a Type IIS restriction enzyme (BsmBI in this protocol) and T4 DNA ligase to allow simultaneous and directional assembly of multiple DNA fragments. time and resources. Here, we describe a high-throughput protocol for cloning, expressing, purifying, Glutaminase-IN-1 and evaluating bispecific antibodies. This XCL1 protocol enables the rapid screening of large panels of bispecific molecules to identify top candidates for further development. Before you begin Experimental considerations Timing: 2 h 1. DNA fragments and construct design. Golden Gate Assembly provides a seamless and orderly strategy to clone multiple DNA fragments into a mammalian expression vector (Figure?1) (Engler et?al., 2008, 2009; Estes et?al., 2021; Gong et?al., 2021). The pTT5 vector is a suitable vector for both bacterial cloning as well as protein expression in mammalian hosts. It contains a CMV promoter to drive robust expression and an oriP DNA gyrase. HEK 293-6E suspension cells (National Research Council of Canada) are an ideal tool to transiently express recombinant protein in a short time frame (1?week) with minimal handling (Fang et?al., 2017; Vink et?al., 2014; J?ger et?al., 2015). Compared to Chinese hamster ovary (CHO) stable cell line expression, which often requires about one month, HEK 293-6E system offers a substantially reduced turnaround time. Though protein yields from a HEK 293-6E expression may be slightly lower than that from a CHO stable cell line, there is typically sufficient yield needed to perform the initial characterization and downstream analytics during early Glutaminase-IN-1 development (i.e., purity assessment, binding and functional analysis). Due to its reduced cycle time, the HEK 293-6E transient system is a preferred tool for high-throughput expression of bispecific antibodies. 3. Cell freezing, recovery and passaging.a. Prepare HEK 293-6E stocks.i. A cell stock could be obtained from a research cell bank (National Research Council of Canada). ii. Expand cell stock culture to 700?mL using cell culture medium, and centrifuge cells in the log growth phase (0.8C1.2??106 cells/mL) at 200??for 5?min at 20CC25C. Cell culture medium can be prepared using the table in the materials and equipment section.Typically, a 700?mL culture with a viable cell density (VCD) of 1 1.0??106 cells/mL can be expected to yield approximately 60C70 vials of cell stocks. iii. Resuspend cell pellets with 0.1 volume of freezing medium (90% v/v FreeStyle F-17 medium plus 10% v/v DMSO), and aliquot into cryogenic tubes. Each aliquot should contain 1??107 viable cells (in a volume of approximately 1?mL). iv. Freeze using a controlled-rate freezing apparatus (Thermo Scientific) and store at ?80C for at least 24 h. For long term storage, transfer cryovials to a liquid nitrogen tank (vapor phase). v. After two to three days, evaluate the viability of frozen cells by thawing a test vial via Glutaminase-IN-1 the procedure below. b. Recover cell stock.i. To recover cells from liquid nitrogen storage, thaw a cryovial in a 37C water bath, and thoroughly sanitize with 70% ethanol before opening. ii. In a biological safety cabinet, transfer cells into a 125?mL shake flask containing 19?mL of fresh cell culture medium (i.e., at an initial cell density of 5??105 cells/mL) and Glutaminase-IN-1 then place on a shaking platform set to 120 RPM in a humidified incubator controlled to 37C with 5% CO2. iii. Three days post-thawing, measure cell viability using the trypan blue method, using an automated analyzer (for example, the Vi-CELL XR automated cell viability analyzer (Beckman Coulter)), or using a hemocytometer and light microscope..