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BIOPROCESSING
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and optimization of cell culture processes as well as in troubleshooting cell culture problems, and their use will continue to increase.
Development and Optimization of Downstream Processes
High-throughput methods can be used to improve the speed and range of process development studies performed by studying a range of unit operations and process parameters with far less sample than traditional development normally requires. These high-throughput approaches rely on very small-scale chromatography columns or batch adsorption to evaluate chromatography resins, determine static and dynamic binding capacity, and evaluate a large variety of process parameters to aid in process optimization and the mapping of a purifi cation design space.17
As shown in Table 1, many of the operating parameters of process scale columns can be investigated in parallel using either batch experiments in microtiter plates or micro-columns. For example, equilibrium constants and maximum (static) binding capacities of diff erent chromatography media under diff erent binding or elution conditions can be readily measured using microtiter plate-based high- throughput methods. For such batch adsorption studies, microtiter plates fi lled with chromatography media are used in conjunction with instrumentation to control the addition and removal of liquid from the wells. These plates can either be purchased already containing chromatography media from vendors such as GE Healthcare, Thermo Scientifi c, and Bio-Rad, but these suppliers only produce plates containing their chromatography media. As an alternative, empty microtiter plates can be fi lled in-house with any chromatography media but care must be taken and a reliable method must be used to ensure uniform distribution of the media and reproducibility.18 Microtiter plates are also available with ion exchange membranes from
Table 1. Parameters to be Considered in Chromatography Step Development
Process Parameter or Feature
Equilibrium constant for interaction between resin and sample constituents at diff erent process conditions
Maximum binding capacity for sample constituents at diff erent process conditions
Dynamic binding capacity for sample constituents at diff erent process conditions
Feed composition on the capacity and strength of interaction
Buff er composition including additives, pH, and ionic strength on binding, wash, and elution effi ciency
Gradient strength and composition
Packing quality and its eff ect on column performance Resin lifetime due to physical fouling Resin lifetime due to chemical fouling Cleaning effi ciency and its eff ect on lifetime Mechanical stability of a resin
Compatible with High- Throughput Screening?
Yes Yes Yes Yes Yes
Yes No No Yes Yes No
Table 2. Comparison of High-Throughput Methods for the Development of Chromatographic Separations23
Attribute Micro-pipette Microtiter plates Micro-columns Binding capacities Dynamic
Static
Automation Flexibility Cost
Easy High High
Diffi cult
Very High Very Low
Dynamic Easy High
Very High
Pall Corporation and with ion exchange and hydrophobic membranes from Sartorius Stedim Biotech.
In general, information from batch adsorption studies is used to establish the media to be used for purifi cation and the general binding and elution conditions for each chromatography. These broad design parameters can then be refi ned using micro-columns to fully defi ne and characterize each unit operation. Once these have been established, the purifi cation design and operating space can be verifi ed using a qualifi ed or validated manufacturing model. Atoll off ers micro-columns packed with any commercially available chromatography media in sizes ranging from 50 μL to 200 μL packed with any resin. PhyNexus off ers pipette tips in sizes of 10 μL to 500 μL pre-fi lled with a variety of chromatography media which can be used like micro-columns. Bio-Rad also off ers 10-μL to 500-μL micro-columns. However, these columns are only available packed with Bio-Rad chromatography media. Any of these micro-columns can be connected to systems such as the Freedom EVO® system from Tecan19 or the PerkinElmer Janus® BioTX Pro, which allow several micro-columns, typically multiples of 8, to be run in parallel.
A qualitative comparison of the capabilities and attributes of diff erent high-throughput formats is shown in Table 2.20
While micro-
columns provide a more representative scale-down model for certain chromatography operations, microtiter plates have greater fl exibility and lower cost for developing a chromatography unit operation.
T e Application of High-T roughput Technologies in Downstream Processing
Many examples of successful application of high-throughput techniques in downstream process development have been reported. These include investigations of a second step in the MAb purifi cation process by cation exchange,21 exchange,23
weak partitioning,22
of purifi cation conditions,25 adsorption system,26
or hydrophobic interaction chromatography,24 purifi cation of virus -like particles.28
High-throughput methods have been by a number of investigators for the development of chromatography steps for the MAb purifi cation processes,29-35
separations such as two-phase aqueous systems.36 Lacki et al35
as well as the development of non-chromatographic For example,
describe the use of microtiter plates to test diff erent
multimodal anion screening
estimation of dynamic binding capacities,27
characterization of a multi-component and
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