Chapter 9
High-Throughput Process Development
for the Chromatographic Purification of Viral Antigens
Shaleem I. Jacob, Spyridon Konstantinidis, and Daniel G. Bracewell
Abstract
Chromatography is a widely used method in the biotechnology industry and functions to separate the
desired product from process and product related impurities. There is a multitude of resins available based
on different modalities (such as charge, hydrophobicity, and affinity) to provide a spectrum of approaches to
meet the separation challenges of the diverse products. The challenge of developing viral antigen purification processes is addressed in this method. A unique feature of this product class is that in order to protect
against more than one strain of an antigen, vaccines are often multivalent. This entails multiple production
processes for each antigen, all of which will require separate development and validation. Ideally, a universal
purification method is sought, but differences in the protein subunits (frequently used as the antigens)
make this challenging and often-bespoke purification steps are required. This means process development
for the chromatographic stages of these products can be particularly challenging and labour intensive. With
the numerous choices available, making critical process decisions that are usually unique to each product,
process, and strain, can be costly and time-consuming. To address this, scale down purification at <1.0 mL
column volume and automation approaches are increasingly applied to increase throughput. In this work, a
method is described wherein a Tecan Freedom EVO
® automated liquid handler is deployed for the
evaluation of different resin chemistries and buffer conditions to find a suitable purification strategy. This
method allows for the rapid evaluation of the separation viral antigens where limited information on
chromatography behavior is known at the early stages of process development. Here, we demonstrate the
methodology firstly by explaining the automated purification script and secondly by applying the script for
an efficient purification development for different serotypes of rotavirus antigens.
Key words Process development, High-throughput process development (HTPD), Rotavirus antigens, Purification, Chromatography
1 Introduction
High-throughput (HT) development activities are currently performed in both upstream and downstream unit operations and
benefit by the advancements of automated liquid handlers [1–
3]. Such screening activities, even from the early stages of process
development, can lead to optimal or near optimal conditions in a
systematic and efficient fashion [4–7]. In downstream applications,
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
119
High-Throughput Process Development
for the Chromatographic Purification of Viral Antigens
Shaleem I. Jacob, Spyridon Konstantinidis, and Daniel G. Bracewell
Abstract
Chromatography is a widely used method in the biotechnology industry and functions to separate the
desired product from process and product related impurities. There is a multitude of resins available based
on different modalities (such as charge, hydrophobicity, and affinity) to provide a spectrum of approaches to
meet the separation challenges of the diverse products. The challenge of developing viral antigen purification processes is addressed in this method. A unique feature of this product class is that in order to protect
against more than one strain of an antigen, vaccines are often multivalent. This entails multiple production
processes for each antigen, all of which will require separate development and validation. Ideally, a universal
purification method is sought, but differences in the protein subunits (frequently used as the antigens)
make this challenging and often-bespoke purification steps are required. This means process development
for the chromatographic stages of these products can be particularly challenging and labour intensive. With
the numerous choices available, making critical process decisions that are usually unique to each product,
process, and strain, can be costly and time-consuming. To address this, scale down purification at <1.0 mL
column volume and automation approaches are increasingly applied to increase throughput. In this work, a
method is described wherein a Tecan Freedom EVO
® automated liquid handler is deployed for the
evaluation of different resin chemistries and buffer conditions to find a suitable purification strategy. This
method allows for the rapid evaluation of the separation viral antigens where limited information on
chromatography behavior is known at the early stages of process development. Here, we demonstrate the
methodology firstly by explaining the automated purification script and secondly by applying the script for
an efficient purification development for different serotypes of rotavirus antigens.
Key words Process development, High-throughput process development (HTPD), Rotavirus antigens, Purification, Chromatography
1 Introduction
High-throughput (HT) development activities are currently performed in both upstream and downstream unit operations and
benefit by the advancements of automated liquid handlers [1–
3]. Such screening activities, even from the early stages of process
development, can lead to optimal or near optimal conditions in a
systematic and efficient fashion [4–7]. In downstream applications,
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
119