Open-source milligram-scale, four channel, automated protein purification system

Abstract

Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, "one-protein-at-a-time" purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.

Fig 1. Protein purifier instrument.

A) Flow path diagram. Flow paths are colored by buffers (blue), load (orange), pre- and post-pump valves (gray), pre-column waste (purple), though column (red), post-column waste (purple), and fraction collector (pink). B) Front view of the front panel. The four-column configuration shown includes four 5 mL HisTrap^™ Excel columns, four 500 mL loads, and a fraction collector capable of holding 50 mL, 5 mL, and 1 mL centrifuge tubes. Buffer bottles have been partially cropped for clarity. C) Back view of the front panel, showing all the major components.

Fig 2. High level electronics diagram.

The protein purifier software is executed on a Raspberry Pi, which interfaces with all hardware peripherals through an I2C bus. While most peripherals are powered by a 24 V power supply, a secondary 5 V source generated by a voltage regulator on the Pi hat is used to power both the Raspberry Pi and the solenoid valve controller. The custom hat also uses a level shifter to convert the 3.3 V I2C signal of the Raspberry Pi into a 5 V signal for the attached peripherals.

Fig 3. System software diagram.

The software used by the system follows a client-server model. While the client interface can be generated by a GUI, interactive shell, or a script, the TCP data connections only allow for one client to interface with the system at a time. When there isn’t an active connection, the server will periodically broadcast its status on the UDP port. Once a client has connected, all data will flow to the server’s device interface, which is responsible for parsing commands to be executed and providing system updates to the client. After connecting, the client will select a hardware configuration to be used by the system, which is then forwarded to the hardware controller and subsequently processed by the hardware initializer. Afterwards, additional commands can be routed to the peripherals through the hardware controller.

Fig 4. Single-column flow rate stability.

Relative flow rates over time through 1 mL and 5mL HisTrap^™ Excel columns are shown. PBS was pumped through each column using the buffer and load flow paths for a duration of 120 minutes and measured every 10 minutes.

Fig 5. Parallelized elution profiles.

Fractions in units of column volumes collected over four 1 mL and 5 mL HisTrap^™ Excel columns in parallel are shown. PBS was pumped through the columns using both buffer and load flow paths, as indicated in the x-axis labels. Each color corresponds to one channel in each of the conditions.

Fig 6. Purity and oligomer analyses of parallel purified coronavirus Spike antigens and antibodies.

A) SARS-CoV-2 Spike RBD, 1 mL HisTrap Excel purification. 10 μL of Load (L) (identical for each channel), flowthrough (F) and elution fractions (1–5) were analyzed by reducing, Coomassie-stained SDS-PAGE. B) mAbs (Anti SARS-CoV-2 Spike and anti Influenza Hemagglutinin) and pAb (healthy serum), 1 mL HiTrap Protein A purification. 10 μL of elution fractions (1–3) were analyzed by Coomassie-stained SDS-PAGE in the presence (+) and in the absence (-) of reducing agent. C-F) Coronavirus Spike antigens, 5 mL HisTrap Excel purification. Parallel purified Coronavirus spike antigens, desalted and concentrated offline, were analyzed by reducing SDS-PAGE (5 μg protein loaded) and analytical SEC-MALS. The calculated molecular weights of the major peak are shown in red.