3 Methods
3.1 Miniature
Column
Chromatography
The implementation of the miniature column technique on the
aforementioned robotic station follows closely the operation of
bench/large scale chromatography throughout eight main steps
(i.e., (1) removal of storage solution, (2) equilibration, (3) loading,
(4) wash, (5) elution, (6) strip, (7) regeneration, and (8) storage).
Hardware differences between HT RoboColumn and conventional
chromatography means that analogies need to be made. In HT
chromatography, solutions are transferred to the RoboColumns
discretely, as opposed to continuously, and they are aspirated across
different locations within and between labware. In the case of
gradient based separations, a gradient is first broken into a series
of small steps with each step being a buffer with a given composition (see Note 9). Hence, all buffers in HT column experiments
need to be prepared in advance at the correct volume and composition and be placed in specific labware and locations within the
robotic station. Since a robot is usually equipped with eight channels, and it is not possible to mix liquids continuously, each of these
channels play the role of a simple inlet/outlet pump delivering
liquids to, up to eight columns in parallel. Moreover, in HT column
chromatography, the role of a fractionator is fulfilled by the
Te-Shuttle module which collects effluent, or fractions, from each
RoboColumn to different wells in 96 well collection plates. Finally,
the plate reader integrated with the robot, plays the role of the
detector since it reads the plates containing the collected fractions
at particular wavelengths and modes compatible with the capabilities of the reader (see Notes 10 and 11). Figure 2 details how the
different components of the robotic station are used during a
typical experiment with eight RoboColumns, whereas Fig. 3
depicts an illustrative example of collecting the first 12 elution
fractions in a collection plate.
3.2 Robotic Buffer
Preparation
Buffer preparation is implemented on the aforementioned robotic
station and it includes both stock preparation and elution buffer
preparation in the case of HT column experiments employing
gradient elution. This employs custom written MATLAB (The
MathWorks) codes, compiled into executables, which are launched
through in-house developed VBA tools providing an interface for
end-user input definition. For buffer stock preparation, the tools
require the specification of their conjugate acid–base pairs and salt,
along with their desired composition (e.g., buffer concentration,
pH and salt concentration), and generate robotic instructions that
are loaded and executed in generic Tecan Freedom EVOware
scripts and result in the preparation of stocks in troughs (i.e.,
100 mL). The same tools are implemented to prepare buffers
corresponding to the steps in elution gradients in multiple
HTPD Purification of Viral Antigens
123
3.1 Miniature
Column
Chromatography
The implementation of the miniature column technique on the
aforementioned robotic station follows closely the operation of
bench/large scale chromatography throughout eight main steps
(i.e., (1) removal of storage solution, (2) equilibration, (3) loading,
(4) wash, (5) elution, (6) strip, (7) regeneration, and (8) storage).
Hardware differences between HT RoboColumn and conventional
chromatography means that analogies need to be made. In HT
chromatography, solutions are transferred to the RoboColumns
discretely, as opposed to continuously, and they are aspirated across
different locations within and between labware. In the case of
gradient based separations, a gradient is first broken into a series
of small steps with each step being a buffer with a given composition (see Note 9). Hence, all buffers in HT column experiments
need to be prepared in advance at the correct volume and composition and be placed in specific labware and locations within the
robotic station. Since a robot is usually equipped with eight channels, and it is not possible to mix liquids continuously, each of these
channels play the role of a simple inlet/outlet pump delivering
liquids to, up to eight columns in parallel. Moreover, in HT column
chromatography, the role of a fractionator is fulfilled by the
Te-Shuttle module which collects effluent, or fractions, from each
RoboColumn to different wells in 96 well collection plates. Finally,
the plate reader integrated with the robot, plays the role of the
detector since it reads the plates containing the collected fractions
at particular wavelengths and modes compatible with the capabilities of the reader (see Notes 10 and 11). Figure 2 details how the
different components of the robotic station are used during a
typical experiment with eight RoboColumns, whereas Fig. 3
depicts an illustrative example of collecting the first 12 elution
fractions in a collection plate.
3.2 Robotic Buffer
Preparation
Buffer preparation is implemented on the aforementioned robotic
station and it includes both stock preparation and elution buffer
preparation in the case of HT column experiments employing
gradient elution. This employs custom written MATLAB (The
MathWorks) codes, compiled into executables, which are launched
through in-house developed VBA tools providing an interface for
end-user input definition. For buffer stock preparation, the tools
require the specification of their conjugate acid–base pairs and salt,
along with their desired composition (e.g., buffer concentration,
pH and salt concentration), and generate robotic instructions that
are loaded and executed in generic Tecan Freedom EVOware
scripts and result in the preparation of stocks in troughs (i.e.,
100 mL). The same tools are implemented to prepare buffers
corresponding to the steps in elution gradients in multiple
HTPD Purification of Viral Antigens
123