Continuous Bioseparations: Fitting the Pieces Together

Published on: 
BioPharm International, BioPharm International-05-01-2017, Volume 30, Issue 5
Pages: 26-29

Continuous downstream bioprocessing is proving its worth, but connecting different operations and integrating upstream remains a challenge.

Today, downstream bioprocessing facilities are taxed as never before. Growth in demand for monoclonal antibodies (mAbs), expanded biosimilars research, and upstream productivity advances have meant larger numbers of smaller batches and much more product to purify.

Factor in the impact of patient-friendly formulas that require smaller, less frequent doses of more potent drugs, and the need to work with new materials with different impurities (e.g., antibody fragments and cell therapies), and many companies are questioning just how well, and how long, their batch-based bioseparation operations can keep up.

Adding motivation for continuous bioseparation is public outcry at drug pricing and the movement to reduce the cost of goods. Downstream batch bioprocessing, which relies heavily on multistep processes, large volumes of buffers, and tools such as Protein A resins, which can cost $15,000/L, contributes significantly to cost.

If a Toyota-trained engineer were to examine downstream batch-based bioseparation, it would be easy to find examples of waste. For instance, chromatography resins’ binding capabilities are never fully used, hold times are long, and equipment only runs a small percentage of the time, Michael Egholm, president of biopharmaceuticals at Pall Life Sciences, noted in a 2015 presentation at BPI Boston (1).

Process intensification

Vendors and manufacturers are addressing these problems via process intensification, processing smaller volumes of material semicontinuously or continuously. “Process intensification with connected purification steps, and semi-continuous chromatography systems, is being tested and scaled up to pilot and production scales,” says Kevin Tolley, senior applications scientist with Thermo Fisher. “As additional product and process information is gathered and system complexities are better understood, the momentum in this space will increase to support much wider adoption,” he adds.

Single-use systems are playing a more prominent role

At the same time, single-use product development is progressing in continuous downstream separations. “When we look at how best to facilitate process intensification, continuous technologies designed as single-use systems show the most potential, while reducing set-up times by eliminating the need for cleaning and cleaning validation,” says Peter Levison, PhD, Pall’s senior marketing director of downstream processing.

New equipment and materials (e.g., resins, filters, and membranes) that are being tailored specifically for continuous downstream bioprocessing include:

  • Continuous depth and tangential flow filtration via single-use membrane filtration systems such as Pall’s Stax and Millipore’s Clarisolve.

  • Periodic countercurrent chromatography (e.g., GE ÄKTA’s PCC)

  • Simulated moving bed (SMB) chromatography, such as Pall Corp.’s Cadence BioSMB; Semba Biosciences’ ProGMP; Sanofi’s Accelerated seamless antibody purification (ASAP); and ChromaCon’s CaptureSMB, the first GMP-scale version of its ContiChrom lab-scale systems, which is manufactured by Lewa Process Technologies and was commercialized in June 2016 (2)

  • Sequential multicolumn chromatography (e.g., Novasep’s BioSC )

  • Sequential continuous countercurrent tangential chromatography, such as Chromatan’s ICS

  • Single-use chromatography membranes such as Natrix Separation’s HD

  • Monoliths and novel adsorbents, including BIA Separations’ CIM, BioRad’s CHT, and Purifidify’s FibroSelect, which is being evaluated by GlaxoSmithKline (3)

  • Expanded bed adsorption (EBA), as applied in DSM’s Rhobust platform (4).

“Innovators are still working hard to create large-scale purification or separation technologies that can be adopted in mainstream bioprocessing,” says Eric Langer, principal with BioPlan Associates. “It’s not problems with specific existing equipment or resins, but the lack of adaptable technologies that appears to be the challenge,” he says.

“Many unit operations need to work together if a continuous downstream biopharmaceutical processing strategy is to succeed. Incremental process intensification may be helpful, but won’t radically change manufacturing paradigms in the near-term,” says Langer.

Others see industry and regulator conservatism as the greatest challenge. “Continuous methodology is based on proven, existing batch methods, so there is not an introduction of anything ‘new’ per se. The real obstacles are more intangible. They come in the form of an industry that is not only very resistant to change, but also highly regulated, often with established platform processes,” says Levison. New facilities and new product introductions currently offer the greatest opportunities for continuous bioseparations, he says.

Biopharmaceutical manufacturers and technology providers are evaluating continuous processes and combining different solutions, a growing number of which are single-use, to reduce the overall number of downstream processing steps needed.

Reducing the need for centrifugation steps

Among the solutions are acoustic wave separation, which Pall developed to reduce the need for centrifugation and filtration steps in clarification; membrane separation; and DSM’s EBA, which is said to reduce filtration, centrifugation, and chromatography to a single step.  Adoption of multicolumn chromatography and SMB is also moving forward, says Langer. “These technologies have been around for at least a decade, but now omplexities and problems of multi-channel operations are being addressed, and scales are increasing,” he notes.

Vendors have performed extensive studies on how continuous downstream platforms might reduce overall biopharmaceutical costs and timelines. In late 2015, Pall introduced its Cadence portfolio, with products designed to enable scalable continuous bioprocesses from process development through GMP production, says Levison. It included the Acoustic Separator to remove debris from harvested cell culture fluid and reduce the need for traditional centrifugation; BioSMB PD and Process platform of single-use multi-column chromatography solutions for bind/elute (using Protein A affinity, ion exchange, mixed mode, and hydrophobic interaction) and flow through methods such as size exclusion or ion exchange polishing; and the Inline Concentrator, which enables single-pass tangential flow filtration (SPTFF) technology for process intensification.

In 2016, Pall researchers evaluated the impact of this platform with anion exchange (AEX) membrane adsorber and mixed-mode cation exchange chromatography (5). They found that the productivity of Protein A separation improved by 74% with the continuous process. Productivity of the cation exchange step increased six fold, contaminant removal improved by 4.5 log; and soluble aggregates were reduced by 50%. In addition, the volume of Protein A resin required was reduced by 95%, and buffer, by 44%.

In 2015, studies by EMD Millipore (6) evaluated a bioseparation platform for mAbs based on capture chromatography using continuous multicolumn processes and incompressible Protein A, as well as precipitation and single-use depth filtration, anion, and cation exchange. Not only did the researchers find that the platform would reduce equipment costs by more than 55%, footprint by 32%, water use by 85%, buffer requirements by 58%, and overall time by 45%, but that it reduced overall unit operation costs by 21% at the 3-kg level, and by 29% at the 15-kg level (7), suggesting continuous downstream processes’ potential.

Integrating up and downstream

Some research projects have the ambitious goal of integrating upstream with downstream continuous biopharmaceutical processing. In December 2016, for example, Univercells, a vaccine and drug development company, received a $12-million grant from the Bill and Melinda Gates Foundation to develop a continuous biopharmaceutical platform that would incorporate continuous processing and process intensification, both up and downstream.

Using perfusion upstream upstream and Natrix Separation’s single-use Protein A capture chromatography membrane downstream, the company plans to develop a portable micro-manufacturing cabinet that could be rapidly deployed wherever and whenever needed (8).


Membrane separation systems under development

Downstream, the Natrix membrane is being designed to offer a 30-fold increase in productivity compared with Protein A resin, at a significantly lower cost. For antibody production, the company claims, it allows capture, virus deactivation, polishing, and sterile filtration to be accomplished in one cycle and within 24 hours.

Merck is also working with Natrix, EMD Millipore, and Novasep on a broad-based project, COMPAC2T, for Continuous Mode Purification and Cell Culture Technology, with the goal of establishing an integrated biopharmaceutical manufacturing process that would use continuous manufacturing both upstream and downstream. Xavier Le Saôut, associate manager of biotech process sciences technology and innovation at Merck Serono, described the project in a 2016 webcast (9).

It would use continuous downstream processing, operate on a single skid, and incorporate monitoring and process analytical technology. Studies so far have suggested that use of the integrated approach would allow working load capacity to be increased by 40%, so that 4.3 g of mAb could be purified in 48 h, and Protein A resin could be cycled 65 times, recovering more of its value.

The company has been evaluating both resins and membranes to compare costs and productivity, and planned to evaluate both fed batch and perfusion materials. So far, research suggests that global yield improvement could exceed 75%.

Another continuous downstream chromatography platform, ASAP, developed by researchers at Sanofi Pharmaceuticals, combines three connected operations: capture chromatography, ion exchange, and anion exchange polishing. In tests, all three were run simultaneously on a single skid, requiring only four (rather than the usual nine) buffers. The process was evaluated using single-use Sartobind membranes from Sartorius Stedim, and reportedly allowed purification to take place in 2.6 fours without any operator intervention, improving productivity by a factor of 50, allowing the processing of 125 g/L/h (10).

Improving capture

Despite the high cost of Protein A resins, traditional capture chromatography will remain a downstream focus, particularly when used in continuous multicolumn systems that leverage more of the resin’s untapped value. Improving capture chromatography, whether with Protein A or other new affinity resins, is a top priority, says Tolley. “An effectively designed and efficiently operated capture step can reduce the burden on other purification steps and greatly improve the overall purification process,” he says.

Thermo Fisher has been addressing the need for consistent pressure/flow characteristics and high resolution required for semicontinuous chromatography applications, with new grades of POROS resins, Tolley says. An example is MabCapture A Select, which offers a lower cost Protein A alternative that can maintain binding capacity at higher processing flow rates, he says, allowing significant reduction in processing time, better resin utilization, and enhanced process efficiency.

Diversifying resins

Within the past year, the company has introduced resins specifically for the gene-therapy market, such as CaptureSelect AAV8 and AAV9 affinity chromatography resins, which offer high binding capacity and target specificity, permitting fewer unit operations and significant improvement in overall process recovery, Tolley says.  Pall has diversified its resin portfolio with bulk, pre-packed, and custom solutions available for batch, continuous or semi-continuous operations, says Levison.

Multicolumn systems reduce resin and buffer consumption

Research has already shown the potential for multicolumn continuous chromatography to reduce the cost of resin and buffer. A few years ago, Genentech studied use of GE ÄKTA’s PCC system, and found that it could reduce resin and buffer consumption by approximately 40% (11).

In 2016, researchers at Merck and the University of Lorraine, France, compared the performance of five different commercial Protein A resins with Novasep’s BioSC continuous downstream technology (12). Although tests were primarily driven to compare resins, researchers found that use of the technology reduced the volume of resin required by 20-40%, and the need for buffer by 20%.

Pall is now exploring opportunities for a single-use perfusion application, Levison says. The company is also developing enabling technologies for continuous virus inactivation and filtration, as well as in-line diafiltration, he adds.

Incremental downstream efficiency improvements can add up. “Every step that we can streamline [downstream] improves the consistency and quality of the process, and, ultimately, the product,” he says.


1. M. Engholm, “Game-Changing Advances in Continuous Processing,” presentation at BPI Boston 2015,
2. “Chromacon’s Partner Lewa Ships the First Twin Column Chromatography Unit for GMP Production,” Press Release, July 29, 2016.
3. “Puridify and GSK Extend Evaluation of FibroSelect,” Press Release,, May 12, 2016.
4. G. Zijlstra, “Continuous XD Cell Cultures Coupled to the Rhobust EBA,” a presentation at the Engineering Conferences International annual meeting, 2013,
5. X. Gjoka et al., Journal of Biotechnology, pp. 11-18, 242 (2017).
6. C. Gillespie et al., “New Downstream Processing Platforms for MAbs,” in Continuous Processing in Pharmaceutical Manufacturing, First Edition, G. Subramanian, editor (John Wiley and Sons, 2015).
7. A New Model for Continuous Processing in Downstream Purification,
8. R. Jacquemart et al., Computational and Structural Biotechnology Journal 14 (2016) 309-318.
9. Informa,, “Implementation of Continuous Downstream Processing,” a Knect365Bioprocessing Webinar, February 24, 2016.
10. B. Mathes et al., “Accelerated Seamless Antibody Purification Process Intensification With Continuous Disposable Technology,”, May 11, 2016.
11. E. Mahajan et al., Journal of Chromatography, 1227 pp. 154-162 (2012).
12. N. Hillbold et al., Biotechnology Progress, preprint article posted on April 3, 2017.

Article Details

BioPharm International
Volume 30, Number 5
May 2017
Pages: 26–29


When referring to this article, please cite it as A. Shanley, "Continuous Bioseparations: Fitting the Pieces Together," BioPharm International 30 (5) 2017.