An Alternative to Process Integration: Clarification and First Step of Monoclonal Antibodies Purification Using Flocculation, Adsorption and Filtration

Research Article

Austin J Clin Immunol. 2021; 7(1):1041.

An Alternative to Process Integration: Clarification and First Step of Monoclonal Antibodies Purification Using Flocculation, Adsorption and Filtration

Carvalho RJ*

Federal University of Rio de Janeiro (UFRJ), Chemical Engineering Program, COPPE, Brazil

*Corresponding author: Rimenys J Carvalho, Federal Rural University of Rio de Janeiro (UFRRJ), Chemical Engineering Department, BR 465, Km 7, Seropédica/RJ 23897-000, Brazil

Received: March 06, 2021; Accepted: March 27, 2021; Published: April 03, 2021


Background: New strategies for up and downstream integration process are being required for end-to-end continuous process for recombinant protein. Single-use materials as well as low cost and easy processing are very welcome for this development. Monoclonal antibodies are largely produced in biopharmaceutical industry what makes it a good protein model to be used in this process.

Objective: A new strategy for integrating up and downstream processes for monoclonal antibodies was developed as a three-step process: flocculation of the cell culture harvest (batch mode); followed by anion-exchange chromatography for impurities adsorption in a slurry, and single-pass tangential flow filtration (both semi-continuous mode).

Methodology: To develop this integrated process, separated studies of flocculation and adsorptions conditions with anion-exchange beads were performed. After defining the optimal conditions, flocculation was performed, and the supernatant was pumped in a vessel along with beads suspension for adsorption. The adsorption was carried out in a residence time determined in the previous studies. Then, the suspension with supernatant was filtered where the antibodies were recovered in the permeate.

Results: Under the adsorption conditions applied (pH 6.5 and 250 mM NaCl), the purer antibodies were recovered in the permeate, whereas the beads with adsorbed impurities remained in the retentate. Steady state profile was obtained during adsorption and filtration for all conditions studied, where no loss of product was obtained. Differently, when the overall process was considered, global yields varied between 61% and 90% due to the void volumes of the runs. Additionally, higher concentration of beads (sample/beads ratio of 41) enabled high amount of impurities removal: 98.9% of DNA and approximately 70% of host cell proteins. Regarding the retention devices studied, depth filter yielded lower void volumes when compared to lamella settler (higher than 5-fold), begetting a global antibodies recovery of 90.4% in 20% higher productivity.

Conclusion: Combining both clarification and impurities removal protocols into a single one proved to be a simple, efficient and fast alternative, which improvement could be obtained by its fully automatization.

Keywords: Anion exchange chromatography; Flocculation; Integration process; Monoclonal antibodies; Protein purification; Single-pass tangential flow filtration; Semi-continuous process


Currently, Monoclonal Antibodies (mAbs) are the most produced biopharmaceuticals, given their various therapeutic applications, including several types of cancer, autoimmune diseases and arthrosis [1]. Indeed, more than 50% of the biopharmaceuticals approved in the market today are mAbs or mAb-related products – fragments or fusion proteins with partial mAb structure [1,2]. The growing number of mAb derivatives represents an important share in the market, which reached more than US$ 123.03 billion on sales in 2019- an 14% increase is expected through to 2027 [3]. In view of this, mAbs have attracted a great deal of attention from industries and research groups all over the world seeking to improve production processes.

In the last 20 years, significant improvements in upstream mAbs production processes led to high titers of these products, reaching values up to 25g/L [4]. Consequently, the bottleneck of the production shifted to the downstream processes. Indeed, in the last years, several improvements on stationary phases and developments on new processes have emerged in order to overcome this specific downstream issue [5]. Innovation on process development has brought up the new paradigm of “continuous process” (both up and downstream), notion which has attracted the attention of traditional biopharmaceutical producers due to advantages such as: higher productivity, higher flexibility, lower footprint, lower work volume and higher automatization of the process [6,7].

The so-called continuous upstream process, which has first been applied to unstable proteins, had its mode of operation extended to more stable proteins such as mAbs, due to several advantages entailed by this process [7]. In contrast, continuous downstream processes had a late development because of their complexity (resulting from the several purification steps required in it) [5,7]. Most of the continuous downstream methods currently developed comprise one purification step only. Periodic Countercurrent Chromatography (PCC) [8] and Countercurrent Tangential Chromatography (CCTC) [9], for instance, illustrate one-step-continuous-purification processes. Yet, both allow for combination or integration into other processes such as Tangential Flow Filtration (TFF) and negative mode chromatography. Furthermore, combining continuous and periodic processes among themselves is equally possible [7].

Continuous or not, the production process requires an efficient system to integrate both up and downstream process; to avoid product loss and to clarify the supernatant as much as possible [7,10]. Filtration has successfully replaced centrifugation over the years given its lower cost, highly effective and flexible technique. In addition, several options on single-use products are currently available in the market, and some types of filtration, such as TFF, are easily adapted to continuous or straight-through processes [11-13]. New techniques exploring TFF have emerged, aiming to employ a continuous purification mode that easily integrates with upstream process, e.g. Countercurrent Tangential Chromatography (CCTC) [9,14]. In CCTC-where chromatographic particles flow through sequential hollow fibers-adsorption, washing and elution steps take place just as in an ordinary chromatographic column, with the advantage of no need for packing [7,9]. Thus, clarification and capture purification steps would be performed in a single step. Regardless of the filter’s high efficiency, their capacity might be drastically reduced when large cells concentration is obtained from cell cultures. An alternative to this problem consists in the prior flocculation of the cell culture, whereby the presence of particles is largely reduced given the fast sedimentation of the formed flocs-strategy which has been extensively demonstrated in the literature [15,16].

Indeed, several studies can be found in the literature on mammalian cells’ flocculation at the clarification stage by using different flocculating agents, such as the cationic polymers PDADMAC and chitosan [17,18], caprylic acid [19], and calcium phosphate [20]. Flocculation presents clear advantages besides being a low cost and efficient method: it not only removes cells and debris but also impurities such as host cell DNA, Host Cell Proteins (HCP) and viruses [18,21,22].

In this work, a new process to integrate clarification with first purification step for mAbs was developed by combining flocculation, anion exchange chromatography in a slurry, and single-pass tangential flow filtration techniques; all performed in a sequential process in which the first part (flocculation) is performed in a batch mode, whereas the second one (adsorption and tangential flow filtration) is as a semi-continuous mode. In this proof-of-concept work, a steady state profile was attempted during adsorption and filtration steps of the system.

Materials and Methods


The cells used for production were CHO (Chinese Hamster Ovary Cells) DP12, producing humanized IgG1 anti-IL8 monoclonal antibodies. The base medium TC-LECC and the feed medium TCX7D used were both from Xell (Germany). With regard to the salts used, PBS tablet, sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic and sodium hydroxide were purchased from Sigma-Aldrich (USA). Q-Sepharose™ Fast Flow was purchased from GE Healthcare (USA). All the ultrapure water used was purified by the system Milli-Q from Merck Milllipore (USA). Polydiallyldimethylammonium Chloride (PDADMAC) was purchased from Merck (USA), and chitosan was purchased from Sigma (USA).

The tangential filtration system applied was QuixStand™ benchtop system from GE Healthcare (Sweden) with a hollow fiber CFP-2-G-4X2MA from GE Healthcare (USA) of 0.2μm cut-off, 60cm high, featuring 650cm2 of membrane surface area, 1.75mm lumen, supported pressure in the feed of 25psig, and Transmembrane Pressure (TMP) of 15psig. The transmembrane pressure was obtained through pressures gauges measurements of this system.

The High-Performance Liquid Chromatography (HPLC) system, used for analytical purposes, was Shimadzu Prominence (USA), equipped with three pumps, column oven, automatic injector with temperature control, and UV 280nm detector.


Cells production: CHO Productions were performed using Erlenmeyer flasks at 37ºC and 180rpm and incubated in a 5% CO2 environment using base medium TC-LECC. The cultures were usually harvested in the 5th day after inoculation. In order to reach higher cell densities, a manual feeding to the flask was generated daily, based on cell exponential growth, using the concentrated feed medium TCX7D. Cells counting and viability were determined by the trypan blue method using Vi-Cell XR automated counter from Beckman Coulter (USA).

Flocculation studies: Previous flocculation studies have been carried out with the use of two polymers as flocculation agents, namely PDADMAC and chitosan [18]. In this study, protocols were applied by considering the best conditions observed in a previous study: 5pg/ TC chitosan flocculating agent applied to the suspension at pH 6.5 (cell broth plus flocculation agents), then further agitated at 100rpm at room temperature (approximately 25ºC) for 30min. Finally, once the agitation was over, the resulting flocs were let to settle down prior to pumping the supernatant into the system.

Anion exchange adsorption studies: The Anion Exchange (AEX) adsorption studies were carried out using Q-Sepharose™ Fast Flow as adsorption beads in suspension. The kinetics studies were performed in 1.5mL tubes using a ratio of 1:10 particles: sample volume. Agitation was performed at 1200rpm using a Thermo Mixer from Eppendorf (USA) at room temperature.

Samples from Flocculated Supernatant (FSN) were taken after 0, 0.5, 1, 2, 5, 10, 30 and 60 min of adsorption.

Moreover, adsorption studies were performed at pH 6.5, under different NaCl concentrations of 50, 150, 250 and 350 mM, by using a flocculated supernatant diluted twice in Phosphate Buffered Saline (PBS) with 10mM phosphate + 150mM NaCl plus the supplementation with NaCl. In all adsorption studies, the particles were first equilibrated with their respective buffer three times at 1200rpm at room temperature. The ratio of particles to buffer was the same one applied to the samples.

With regard to sample preparation, the supplementation of PBS with NaCl was doubled in concentration. The agitation and temperature conditions were the same as in previous studies.

Sequential clarification and AEX adsorption system: The sequential process was performed in three different steps further to cell cultivation: (a) flocculation for cell separation and impurities removal (batch mode), (b) AEX adsorption in a slurry as a first purification step, and (c) single-pass Tangential Flow Filtration (TFF) for clarification (semi-continuous mode – b and c), (Figure 1).