Elsevier

Journal of Chromatography A

Volume 1389, 10 April 2015, Pages 85-95
Journal of Chromatography A

Twin-column CaptureSMB: A novel cyclic process for protein A affinity chromatography

https://doi.org/10.1016/j.chroma.2015.02.046Get rights and content

Highlights

  • CaptureSMB improves the utilization of affinity stationary phases.

  • By applying a dual-flow rate strategy CaptureSMB requires only two columns.

  • The performance of CaptureSMB and batch chromatography was compared.

  • CaptureSMB outperforms in terms of load, productivity and buffer consumption.

Abstract

A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10–30% depending on the manufacturing scenario.

Introduction

In the last two decades the sales of monoclonal antibodies (mAbs) and other Fc fusion therapeutic proteins have significantly increased [1]. Additionally, a higher number of candidates have been approved to enter clinical trials for many applications including autoimmune disease, transplant rejection and cancer [1], [2], [3], [4], [5], [6].

Most mAbs are currently produced in recombinant cell culture in batch or fed-batch mode [4], [6], [7]. However, process intensification toward continuous fermentation is being increasingly investigated in the pharmaceutical industry [1], [8], [9].

The advantages of continuous manufacturing over batch processes include steady-state operation, small equipment size, high volumetric productivity, streamlined process flow, low cycle times and reduced capital cost [9]. Moreover, continuous manufacturing has proven to be critical for the production of labile proteins [8], [9].

The fact that several mAbs are about to lose patent exclusivity over the next eight years [1], an increased demand on recombinant proteins, advances in fermentation techniques, characterized by higher titers [6], [7], [10], [11] and the evolving competitive business, are driving the biopharmaceutical industry toward more efficient and cost-effective downstream processes that meet those requirements.

Protein A affinity chromatography is often employed for capture of mAbs and Fc fusion proteins from cell culture supernatants due to its capability of delivering product with high purity, yield and throughput [2], [3], [5], [6], [7], [12], [13]. Although its price is almost one order of magnitude higher compared to non-affinity materials [12], no alternative processes that reach comparable purity levels have been established in industry. Besides, the selective interaction of protein A with the Fc region of numerous types of mAbs and fusion proteins, allows the development of platform or template processes for process harmonization of multi-product manufacturing [2], [3], [5], [6], [7], [12].

Commonly, the capture step has a transit time in the range of one to two days. This time restriction along with the dynamic binding capacity (DBC) of the resin employed, and the volume and amount of material to be purified, determine the amount of resin and therefore the column dimensions required for the capture step. The DBC depends on the binding conditions, the target protein itself (size and biochemical properties), the feed concentration, the loading flow rate, the resin properties (bead size, pore structure and phase ratio), and the functional ligand properties (type and density). All these factors influence the thermodynamics and mass transfer kinetics of the protein–resin interaction, eventually determining the shape of the breakthrough curve (BTC) [8], [10], [11], [12], [14], [15].

The value of the DBC at a particular residence time is determined by the feed volume (i.e. protein mass) that can be loaded onto the column until a certain percentage of the feed concentration is reached at the column outlet, typically 1%, i.e. DBC1%.

Batch mode capture operations are usually loaded up to 80–90% of DBC1% in order to avoid product losses and to account for resin ligand density variability, packing variability, and capacity losses of the resin with time, which are caused by extensive column cleaning (ligand leaching) [1], [16], or irreversible adsorption of impurities. The elution volume (EV) required to reach a certain value of DBC decreases with the flow rate and titer concentration while it increases with the column length [10], [15]. Therefore, increasing the throughput leads to higher unused capacity of the column, which is a disadvantage especially in the case of expensive resins such as protein A. Depending on the shape of the BTC, typically 30–50% of the maximum binding capacity, i.e. the static binding capacity, remain unused. In addition, washing and regeneration of the unsaturated column portion leads to excessive buffer consumption [16].

Continuous counter-current chromatography processes, where the counter-current movement of the liquid and stationary phases is simulated by switching valves, such in the case of Simulated Moving Bed (SMB), were already developed in the 50s for the purification of small molecules [17]. The concept of SMB proved to be very efficient for binary separations using 3–4 columns, especially in the case of difficult separations where the components have a selectivity close to 1, showing a much better performance than batch chromatography [18]. Other continuous processes combining the advantages of both batch chromatography (gradient elution) and SMB, such as the Multicolumn Counter-current Solvent Gradient Purification (MCSGP) were developed for ternary or more complex separations [19], [20], [21]. This process has proven to be especially advantageous in the case of polishing steps, where the characteristic yield-purity tradeoff of batch chromatography can be circumvented using non-affinity resins.

Combining the advantages of affinity chromatography with counter-current sequential loading processes, gave rise to the CaptureSMB process described in this work. By overloading a first column far beyond its DBC1% and capturing the product that breaks through on a second interconnected column, the utilization of the stationary phase is improved. In this way, the first column is loaded much closer to its static binding capacity. By using only two columns, a superior performance of the capture step in terms of load, productivity, and buffer consumption is achieved compared to batch chromatography.

The concept of twin-column CaptureSMB is shown schematically in Fig. 1. The area A in Fig. 1 represents the mass loaded on a single column, e.g. during a typical batch process, whereas areas A + B correspond to the mass loaded to the same column during CaptureSMB, while the breakthrough (BT) (area C) is captured by the second column.

A number of processes based on the concept of sequential loading for affinity chromatography have been evaluated for recombinant protein purification, among these, periodic counter-current chromatography (PCC) processes using 3-, 4- and up to 12-column configurations [1], [8], [14], [16], [22]. So far, no route to experimental implementation of a two column process has been presented for protein A capture of mAb together with specific adaptations that need to be made to the loading flow rate.

This work describes the design and operation of the twin-column CaptureSMB process. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch runs were compared. A systematic study was performed in order to evaluate the effect of the flow rate, the column length and the titer concentration on the process performance and the product quality.

Section snippets

CaptureSMB process description

A cycle of the process in steady state using two identical columns is shown schematically in Fig. 2. A cycle is completed when a column has gone through all the operations (loading, washing, elution and regeneration) and returned to its initial state. Each cycle comprises two “switches” with an interconnected (IC) and a batch step. During the IC step, the columns are connected in series and loaded up to a value X% of the DBC of the first column, while the breakthrough is captured by the second

Monoclonal antibody

Recombinant IgG1 expressed in Chinese hamster ovary (CHO) cells was provided by JSR Life Sciences (JSR Micro N.V., Leuven, Belgium) as clarified cell culture supernatant (cCCS) containing monoclonal antibody (mAb) at a concentration of 1.2 g/L and pH 7.2. The material was stored at −80 °C and was freshly thawed before experiments. Prior to use, the cCCS was filtered using 0.2 μm cut-off sterile Filtermax vacuum filters (TPP, Trasadingen, Switzerland) and 0.5 g/L NaN3 was added in order to avoid

Adsorption isotherms

The adsorption behavior of IgG1 to the protein A affinity resins Amsphere™ and MabSelect Sure was determined from batch adsorption experiments (data are shown in the supplementary material). The best fit values for the saturation capacity, Qsat, and the adsorption coefficient H assuming a Langmuir isotherm model are shown in Table 1. Both resins showed a very favorable isotherm under the conditions evaluated, with higher Qsat and H values for MabSelect Sure than Amsphere™. The high salt

Conclusions

We have described for the first time the design and implementation of a periodic counter-current chromatography process using only two columns for the affinity capture of mAbs using protein A resins. By applying a dual-flow rate strategy the advantages of sequential loading were exploited while using the minimum required number of columns. In this way, the resin volume required, the column dimensions and the buffer consumption were significantly reduced in comparison to batch chromatography.

In

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgments

We gratefully acknowledge the project OPTICO for “Model-based Optimization and Control for Process-Intensification in Chemical and Biopharmaceutical Processes” (Grant No. 280813) for partial financial support.

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