An integrated automated pipeline for cloning, transient expression, and purification augmenting the plant cell pack platform for biopharmaceutical screening.

Biopharmaceuticals, particularly monoclonal antibodies (mAbs), are a rapidly expanding class of therapeutics and have benefitted from high-throughput screening (HTS) strategies for protein engineering. Obtaining such complex biopharmaceuticals from plants might be advantageous because transient expression can yield grams of product within a week. For HTS, this system was scaled down as plant cell pack (PCP) technology to microtiter plate format and automated on a liquid-handling station, but is limited by manual cloning and protein purification. Therefore, we first generated a modular vector library comprising 15 pTRAc backbone constructs differing in regulatory gene expression and protein targeting elements, enabling rapid integration of any target gene via restriction-ligation cloning. Building on this, we developed an automated, seamless cloning workflow prior to transient expression and integrated a magnetic bead-based downstream purification process for single-chain fragment variable (scFv) mAbs. This automated workflow enables sequential cloning, expression, and purification of up to 375 protein variants simultaneously, each with 10 PCP replicates, within 9 days on a single platform. In contrast, manual processing allows only 15 variants with 10 replicates in the same period - a 25-fold increase in throughput, while costs decreased by 30%, from 79 € to 61 € per variant. Using a recombinant immunotoxin against acute myeloid leukemia based on the anti-CD64 H22 scFv as case study, a recovery of 63% and purity > 95% were obtained, suitable for in vitro assays. Thus, integrating our cloning and purification workflows into the PCP platform establishes an automated pipeline that accelerates early biopharmaceutical development.