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.</p

Purification of SARS-CoV-2 Spike using a commercial instrument.

SARS-CoV-2 Spike was purified using a 5 mL HisTrap Excel column on a commercial chromatography instrument (ӒKTA Pure), desalted and concentrated offline, and analyzed by reducing SDS-PAGE (2.5 μg protein loaded) and analytical SEC-MALS. The calculated molecular weight of the major peak is shown in red. (TIF)</p

Build guide.

A detailed guide on how to build the automated protein purifier. (PDF)</p

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.</p

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.</p

Bill of materials.

A list of all the materials/components needed to build the automated protein purifier. (XLSX)</p

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.</p

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.</p

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.</p