2 Materials
2.1 Viral Antigen
Rotavirus antigens are expressed in Pichia pastoris.
2.2 Miniature
Columns
Miniature columns (0.2 and 0.6 mL) are purchased from Repligen
®
(Waltham, Massachusetts, USA). These are packed with the commercially available resins (Table 1). For custom designed resins,
bulk resin can be sent to Repligen, which can then be packed at a
desired bed volume in RoboColumns.
2.3 Automated
Workstation
The employed automated liquid handling station comprises of a
Tecan Freedom Evo 200 robot. The station is controlled by Freedom EVOware v2.6 (Tecan Group Ltd.) on an Intel i5 4670 CPU
machine with 4 GB of RAM running Windows 7 (Microsoft Corporation, WA, USA). The robot’s layout is depicted schematically
in Fig. 1a (see Notes 1–3). It includes an eight-channel liquid
handling (LiHa) arm, using short uncoated stainless-steel tips,
and an eccentric robot manipulator (RoMa) arm. Integrated
devices include the Te-Shuttle™ and Te-Chrom™ (fraction collection system) modules and an Infinite
® M200 Pro plate reader
(Tecan Group Ltd.), capable of UV/Vis and fluorescence measurements, and operated by i-control™ software (Tecan Group Ltd.).
An in-depth description of the layout is found in [10]. Briefly, all
carriers are supplied by Tecan and are responsible for holding plates
and troughs filled with buffers. Nine site hotel carriers are also
present and these are responsible for storing plates containing
collected fractions and newly prepared buffers. These are used to
blank correct the plate reader measurements of the collected fractions (see Note 4). All carrier and labware definitions are edited in
EVOware in order to implement robotic operations, including
plate transfers and liquid handling, robustly and reliably (see Note
5). Finally, the deployment of the robotic station made use of
standard liquid classes which also included those supplied with
Table 1
List of commercially available multimodal chromatography resins tested at 0.6 mL scale
Type
Resin
Manufacturer
Multimodal
Capto™ MMC
GE Healthcare
PPA Hypercel™
Pall
Nuvia™ cPrime™
Bio-Rad
CMM HyperCel™
Pall
Toyopearl
® MX-Trp-650M
Tosoh Bioscience
Eshmuno
® HCX
Merck KgaA
HTPD Purification of Viral Antigens
121
2.1 Viral Antigen
Rotavirus antigens are expressed in Pichia pastoris.
2.2 Miniature
Columns
Miniature columns (0.2 and 0.6 mL) are purchased from Repligen
®
(Waltham, Massachusetts, USA). These are packed with the commercially available resins (Table 1). For custom designed resins,
bulk resin can be sent to Repligen, which can then be packed at a
desired bed volume in RoboColumns.
2.3 Automated
Workstation
The employed automated liquid handling station comprises of a
Tecan Freedom Evo 200 robot. The station is controlled by Freedom EVOware v2.6 (Tecan Group Ltd.) on an Intel i5 4670 CPU
machine with 4 GB of RAM running Windows 7 (Microsoft Corporation, WA, USA). The robot’s layout is depicted schematically
in Fig. 1a (see Notes 1–3). It includes an eight-channel liquid
handling (LiHa) arm, using short uncoated stainless-steel tips,
and an eccentric robot manipulator (RoMa) arm. Integrated
devices include the Te-Shuttle™ and Te-Chrom™ (fraction collection system) modules and an Infinite
® M200 Pro plate reader
(Tecan Group Ltd.), capable of UV/Vis and fluorescence measurements, and operated by i-control™ software (Tecan Group Ltd.).
An in-depth description of the layout is found in [10]. Briefly, all
carriers are supplied by Tecan and are responsible for holding plates
and troughs filled with buffers. Nine site hotel carriers are also
present and these are responsible for storing plates containing
collected fractions and newly prepared buffers. These are used to
blank correct the plate reader measurements of the collected fractions (see Note 4). All carrier and labware definitions are edited in
EVOware in order to implement robotic operations, including
plate transfers and liquid handling, robustly and reliably (see Note
5). Finally, the deployment of the robotic station made use of
standard liquid classes which also included those supplied with
Table 1
List of commercially available multimodal chromatography resins tested at 0.6 mL scale
Type
Resin
Manufacturer
Multimodal
Capto™ MMC
GE Healthcare
PPA Hypercel™
Pall
Nuvia™ cPrime™
Bio-Rad
CMM HyperCel™
Pall
Toyopearl
® MX-Trp-650M
Tosoh Bioscience
Eshmuno
® HCX
Merck KgaA
HTPD Purification of Viral Antigens
121