focus on Chromatography CaptureSMB for Effi cient Affi nity Purifi cation of Monoclonal Antibodies
Thomas Müller-Späth (a,b), Monica Angarita (a), Daniel Baur (a), Roel Lievrouw (c), Geert Lissens (c), Guido Ströhlein (b), Michael Bavand (b), Massimo Morbidelli (a) (a) Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zürich, Switzerland,
www.morbidelli-group.ethz.ch (b) ChromaCon AG, Zürich, Switzerland,
www.chromacon.ch (c) JSR Life Sciences, JSR Micro NV., Belgium
Corresponding author: Thomas Müller-Späth, ChromaCon AG, Technoparkstrasse 1, 8005 Zurich, Switzerland, email:
thomas.mueller-spaeth@
chromacon.ch.
A novel twin column counter-current chromatography process was used for the capture of monoclonal antibodies (mAbs) from clarifi ed cell culture harvest. The process features improved performance in terms of productivity, buffer consumption, product concentration and capacity utilisation compared to traditional batch chromatography. As presented in this case study, the advantages become more pronounced as loading fl ow velocities are increased. Due to its cyclic nature, the CaptureSMB process is very well suited for the integration with conventional batch-wise as well as with continuous processing.
Introduction
Affi nity chromatography stationary phase costs are one of the largest drivers of consumables costs in the downstream processing of biologics. However, in traditional batch chromatography, the stationary phases are not fully utilised due to the dynamic behaviour of column loading: As the feed is loaded an S-shaped internal product concentration front forms within the column as a result of isotherm and mass transfer effects. This leads to an early breakthrough and losses of the protein of interest before the full resin capacity has been utilised. Typically 30-50% of the maximum capacity, i.e. the static binding capacity, is unused in batch chromatography. After loading, the column is washed, the product is recovered and the column is cleaned. Thereby also the fraction of the resin that has not beed utilised is cleaned. Since cleaning is one the decisive drivers for resin degradation it is obvious that signifi cant cost savings can be obtained by improving the capacity utilisation of the chromatography step.
In the twin-column CaptureSMB process, this is achieved by loading one of the columns until a signifi cant amount of product has broken though and by simultaneously capturing the breakthrough in the interconnected second column (see Figure 1).
B – batch phase: The columns are disconnected and the previously upstream column is washed, eluted to recover the product, cleaned and re-equilibrated. In parallel, the column that has previously taken up the breakthrough from the upstream column is continued to be loaded at a lower fl ow rate.
The B phase is followed by another IC phase whereby the previously loaded column is placed in the upstream position and the equilibrated column is placed in the downstream position and the columns are loaded again in series. The IC phase is then followed by another B phase that is run as described above, just with the columns in opposite order. This completes one cycle and the CaptureSMB process can be carried out for as many cycles as desired.
Figure 1. Schematic of a breakthrough curve (BTC) (outlet concentration over elution volume) of column directly loaded with feed,. EV1, EVX: Elution volumes corresponding to 1% Feed concentration (1% DBC), and X% feed concentration, respectively (X% DBC). Area A corresponds to the maximum amount of product that can be loaded in a single column batch process, before product breakthrough. For two interconnected columns, areas A+B and D correspond to the amount of product that is bound on the upstream column and the downstream column, respectively, when the columns are loaded up to EVX. The area A+B+C corresponds to the static capacity.
The process comprises the following steps and is outlined in the schematic in Figure 2:
IC – interconnected phase: The two columns are connected in series and loading occurs through the upstream-column: The breakthrough of the fi rst column is captured in the second column. Once the upstream column has been loaded to high capacity utilisation, the loading is stopped and buffer is pumped through the two columns in series in order to fl ush unbound material from the upstream into the downstream column.
Figure 2. Schematic illustration of the twin-column CaptureSMB process. Performance Parameter Defi nition
CaptureSMB and batch processes are compared based on a number of performance parameters that are defi ned below.
The purity P is defi ned by the area ratio of the product peak and the total peak area of the analytical chromatograms and is given in percent [%]. This purity defi nition is typically applied, when calculating the purity with respect to product-related impurities such as aggregates or fragments. The areas are extracted from the analytical chromatograms of product fractions:
LAB ASIA - JANUARY/FEBRUARY 2014
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