October 18, 2020

What Is Miniaturized Purification and Why Is It the Future of Bioprocessing?

What Is Miniaturized Purification?

“Miniaturized Purification” describes the purification of target compounds on a miniature scale, using resin plates, miniature columns, or pipette tips. This technique requires lower sample and resin volumes, which reduces the resource demand and enables parallelization of experiments [e.g. 1].

Parallelization is especially beneficial when optimizing purification conditions with enhanced approaches such as Design of Experiments (DoE). A larger number of parameters can be studied at once, allowing for iterative optimization and shorter process development times [1].

Miniaturized Purification is part of High Throughput Process Development (HTPD) and relies on the use of automated liquid handlers [2]. Key benefits of automated miniaturized purification include:

High throughput - liquid handling robots can perform many runs in parallel and operate for hours on end without getting tired
Repeatability - the same protocol can be executed over and over again
Traceability - all experimental steps performed by robots are logged and stored for reference
Precision & accuracy - different robots execute the same experiment with equal precision and accuracy, removing human error and inter-operator variability

What Are Miniature Columns and What Are Their Applications?

Miniature columns are packed with 50-600 µL resins, and they are a scale-down model of regular column chromatography [3, 4]. While not exactly one-to-one, this scale-down model can be reliably scaled up to larger column volumes [5].

Atoll, later acquired by Repligen, was first to develop miniature columns, called OPUS® RoboColumn® pre-packed columns, and developed automated purification together with Tecan, a major supplier of lab automation equipment. This collaboration led to the development of the Te-Chrom® module, which supports the usage of miniature columns on the Tecan Freedom EVO® and Tecan Fluent® liquid handlers, equipped with fixed tips.

Tecan’s Te-Chrom® setup has recently been complemented with Synthace’s automated miniaturized purification protocol built using its software platform. This protocol increases flexibility and time savings of the purification process, and reduces errors by automatically structuring and analyzing resulting absorbance data. Results are also automatically visualized to provide immediate feedback to the user (for more information, see the technical note comparing Synthace and Tecan’s Freedom EVOware® and watch the video demonstration).

For the past decade, miniature columns have been used for antibody and other protein purification [4]. More recently, pharmaceutical process research and development (PRD) groups have started presenting successes in high-throughput purification of viral products. For instance, a team at UCL performed HTPD for the purification of rotavirus antigens [6] while Biogen performed HTPD for the purification of adeno-associated viral (AAV) vectors [7].

Which Vendors Supply Miniature Columns?

Vendors offering miniature columns (also called RoboColumns) include:

Repligen - OPUS® RoboColumn® miniaturized columns pre-packed with user-specified resins (e.g. CaptivA™)
Cytiva - PreDictor RoboColumn™ minicolumns pre-packed with various Capto, MabSelect, and Sepharose resins
Bio-Rad - Foresight™ RoboColumn™ microcolumns pre-packed with UNOsphere™, Nuvia™, CHT™, and MPC™ resins
Tosoh - ToyoScreen® RoboColumns® pre-packed with TOYOPEARL® resins
ThermoFisher - POROS™ RoboColumns™ containing POROS™ resins and CaptureSelect™ RoboColumns™ containing Human Albumin or FcXL Affinity Matrices
Sartorius - ScreenExpert RoboColumn™ miniaturized columns pre-packed with various chromatography sorbents upon request

Most RoboColumns are supported by Tecan Freedom EVO® and Fluent® liquid handlers but some can also be used with PerkinElmer JANUS G3 BioTX™ Pro robots.

What Are the Common Applications of Resin Plates?

Resin plates (also called filter plates) are 96-well plates with a filter membrane at the bottom. Eluents are typically collected using centrifugation or vacuum absorption after an incubation period on a shaker [2].

The 96-well format allows for many parameters to be studied at once. Large throughput can be achieved by integrating these plates with automated liquid handlers. Typically, a shaker and vacuum system or centrifuge are required to allow for automated operation.

Resin plates come in a variety of formats - depending on their application, they can be packed with:

different resins - to enable resin media screening
the same resin at the same volume - to study bind, wash, or elute conditions
the same resin at different volumes - to study adsorption isotherms

PreDictor Plates by Cytiva and Foresight™ Filter Plates by Bio-Rad are just two examples of commercially available resin plates.

The drawback of resin/filter plates is that these are not scale-down processes of bench, lab, or production chromatography runs. Typically, they are used to investigate initial conditions or to study binding conditions in detail [4, 8].

Why Is Miniaturized Purification the Future of Bioprocessing?

Miniaturized purification is an essential link in HTPD, which is an iterative process development chain that involves the integration of miniaturization, automation, and parallelization [4]. HTPD consists of:

Miniaturized bioreactors like Sartorius Ambr® 250 system or Applikon® micro-Matrix
Miniaturized purification as described above
Automated assays like ELISA and Bradford assay or analytics like electrophoresis, high-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS)

With the growing adoption of miniaturized, parallelized bioreactors and automated liquid handlers to enable complex DoE campaigns, miniaturized purification will become a standard protocol in all bioprocessing labs looking to achieve more robust bioprocesses in shorter development cycles with lower material costs.

The biggest benefit of HTPD is that production and purification parameters can be studied simultaneously, overcoming the traditional silos in bioprocessing [9]. Indeed, many parameters chosen to increase the efficiency of the cell-culture step upstream can have important consequences for the unit operations downstream [10].