Membrane Adsorber for the Fast Purification of a Monoclonal Antibody Using Protein A Chromatography

Abstract

Monoclonal antibodies are conquering the biopharmaceutical market because they can be used to treat a variety of diseases. Therefore, it is very important to establish robust and optimized processes for their production. In this article, the first step of chromatography (Protein A chromatography) in monoclonal antibody purification was optimized with a focus on the critical elution step. Therefore, different buffers (citrate, glycine, acetate) were tested for chromatographic performance and product quality. Membrane chromatography was evaluated because it promises high throughputs and short cycle times. The membrane adsorber Sartobind^® Protein A 2 mL was used to accelerate the purification procedure and was further used to perform a continuous chromatographic run with a four-membrane adsorber-periodic counter-current chromatography (4MA-PCCC) system. It was found that citrate buffer at pH 3.5 and 0.15 M NaCl enabled the highest recovery of >95% and lowest total aggregate content of 0.26%. In the continuous process, the capacity utilization of the membrane adsorber was increased by 20%.

Keywords: membrane adsorber; monoclonal antibody; periodic counter-current chromatography; protein A chromatography.

Figure A1

Calibration data of the mAb at the 4MA-PCCC system with a 2 mm flow through cuvette. Purified mAb solution was measured in the UV cuvettes. Small deviations occurred due to the use of optical fibers. Limit of detection (LOD) and limit of quantification (LOQ) were determined individually and the averages were calculated: LOD = 0.009 g/L and LOQ = 0.029 g/L.

Figure A2

Results of the citrate buffer screening with chromatography experiments (Recovery and Peak height) and stability testing (Monomeric antibody and Aggregate content). The range for the next optimization step is marked with a box as well as the final operation point with a “×”.

Figure A3

Results of the citrate buffer optimization with chromatography experiments (Recovery and Peak height) and stability testing (Monomeric antibody and Quality measured with Tycho NT.6). The final operation point is marked with a “×”.

Figure 1

Process steps in the downstream processing of a monoclonal antibody (summarized and modified from [12]).

Figure 2

Chromatography (left), aggregation and stability (right) results of the DoE screening experiments with three elution buffers (a): citrate buffer pH 2.5–4, 0–0.5 M NaCl; (b): glycine buffer pH 2.5–4, 0–0.5M NaCl; (c): acetate buffer pH 3.5–4, 0–0.5 M NaCl). Aggregation and stability were measured after one day at 4 °C. Citrate buffer showed the best performance during chromatography runs as peaks were high and sharp (R = Recovery). The worst runs are marked with arrows. The aggregation was low for acetate buffer and increased at low pH and with salt (citrate and glycine buffer). The highest aggregation was measured with glycine buffer and is marked with an arrow. Stability experiments showed a clear trend: with increasing pH, the stability increases for citrate and glycine buffer. The models are shown with a surface diagram and experimental data was added.

Figure 3

Optimization of Protein A chromatography elution with citrate buffer (a) and robustness testing (b). By increasing the pH, the performance of the chromatography decreased and the peaks became flatter. During the robustness testing, the result of the chromatography was not influenced by small fluctuations in the buffer system.

Figure 4

Determination of a suitable flow rate for loading of the Sartobind^® Protein A membrane adsorber for periodic counter-current chromatography (PCCC) operation. By increasing the flow rate, the double breakthrough curve got steeper. 1.5 mL/min was chosen as suitable flow rate.

Figure 5

Chromatogram and reducing SDS-PAGE of two cycles with the 4MA-PCCC using Sartobind^® Protein A for the purification of a monoclonal antibody. Two PCCC cycles are shown in the chromatogram. A cycle consists of the loading, elution and regeneration of the four membrane adsorbers. The product peaks are marked with black arrows. Throughout the PCCC run, a trend was observed in that the peaks decrease during the cycles. The reason for this is the complex system setup.