Process Chromatography Selection for Downstream Processing Applications

Published on: 
BioPharm International, BioPharm International-09-01-2016, Volume 29, Issue 9
Pages: 28–33

Industry experts discuss best practices for selecting a separation technology.

Process chromatography has changed significantly since it was first introduced to the biopharmaceutical industry. Important factors such as column design, resin development, and automation have all played a role in simplifying the purification process, in some cases reducing it from five steps to three (1). Selecting the appropriate separation technology plays a big role in maximizing productivity in any downstream processing application. Some factors companies may consider when selecting a separation technology include the stability of the biologic, buffer compatibility, and ease of scale-up. In this roundtable discussion, industry experts Tomas Björkman, senior scientist, R&D at GE Healthcare Life Sciences; Alejandro Becerra-Arteaga, PhD, purification team manager, MSAT at MilliporeSigma; and Peter Levison, PhD, senior marketing director of Downstream Processes at Pall Life Sciences discuss important considerations in process chromatography selection.

Separation Technologies

BioPharm: What are the most commonly used separation technologies for biologics that are not monoclonal antibodies (mAbs)? 

Björkman (GE Healthcare): If one excludes mAbs, then the molecules of interest are generally peptides, recombinant proteins, or vaccines. For these molecules, chromatographic techniques such as ion-exchange, hydrophobic interaction chromatography, and multimodal resins are common. Affinity resins like Capto L (antigen-binding fragments [Fabs], domain antibodies [dAbs], etc.), heparin, and blue dye-based resins are also used. For peptides, reversed-phase chromatography is another possibility. By contrast, immobilized metal affinity chromatography is not often used because tagged proteins are uncommon in process scale.

Becerra-Arteaga (MilliporeSigma): Ion-exchange chromatography is most commonly used to separate product- and process-related impurities in the purification of biologics that are not mAbs. In cases where ion exchangers cannot provide the required separation, hydrophobic interaction and mixed-mode adsorbents are generally used, although in some cases, these may require more effort to optimize compared to ion exchangers. In the case of Fc-fusion proteins, Protein A can still be utilized as a capture step.

Levison (Pall): [Some commonly used separation technologies include] ion-exchange, mixed-mode, hydrophobic interaction, and size-exclusion chromatography.

BioPharm: What are some new separation technologies available for downstream processing?

Becerra-Arteaga (MilliporeSigma): In the harvest steps, the use of precipitation and flocculation techniques has recently proven to be an alternative for improved filter capacity in the clarification of high cell density cultures, as well as a means of reducing some process impurities in the initial purification step.

The use of activated carbon as an adsorbent in polishing steps is gaining attention in the industry. This technology can provide good host cell protein removal within a relatively broad operating window of pH and conductivity, since the binding of impurities is primarily due to van der Waals interactions as well as size exclusion.
Membrane adsorbers have been available for more than a decade, and while their acceptance has increased in the past few years, their use may be more applicable for early-stage molecules and in the production of small batches.

Levison (Pall): With the introduction of continuous chromatography and optimization of flow through approaches to membrane chromatography, novel ligands, and base matrices may gain broader application as the demands on the chromatographic processes change. For example, selectivity of chromatographic matrices has been enhanced over the past five years with the introduction of novel mixed-mode moieties such as CMM HyperCel, and these facilitate improved separation compared to classical ion exchange and hydrophobic interaction chromatography. This minimizes use of lyotropes and enables chromatography at higher salt concentrations thereby improving product economics. Furthermore, when used in a continuous mode, the effective ligand/ligate contact time is increased and so base matrices that allow maximal binding of target molecules to the sorbents are highly desirable to improve productivity of the chromatographic process.

Björkman (GE Healthcare): Lately, large efforts have been made in configuration of the chromatography separation steps, [such as] set-ups where more than one column or mode of separation are operated simultaneously aiming at a continuous process. Periodic counter current chromatography (PCC) allows overloading of columns and continuous loading at the same time. Arranging two different modes of chromatography in series, operated with or without intermediate buffer conditioning is another setup known as straight through processing.

Also, the ability to use concentrated buffer components and mixing them in-line under controlled conditions (in-line conditioning) has simplified the chromatographic operation. Other simplifications are single-use systems where critical systems components (columns, flow kit etc.) are used for one campaign only, omitting the cleaning steps and the need for cleaning validation. There is a continuous development of new chromatography resins combining separation modes. One example of this is the diffusion restricted Capto Core resin combining size exclusion with a functionalized core.

Buffer Selection

BioPharm: What are some considerations when selecting the appropriate buffer?

Levison (Pall): It is important to consider the suitability of a buffer in any given stage of operation for direct-use in the subsequent downstream processing step. If the buffer composition is unsuitable, then you need to consider additional unit operations including diafiltration, dilution, etc. However, these can affect volume, buffer and water storage, utilization, and disposal.

Björkman (GE Healthcare): One must consider the stability of the buffer molecule and the product of interest as well as the suitability of the buffer for use in biotherapeutic applications (e.g., no toxic buffers). At the same time, one must choose a solution with the correct buffering capability (e.g., buffer capacity and charge). Ideally, the chosen buffer should be affordable, have minimal environmental impact, and enable process optimization (e.g., by minimizing buffer exchange steps).

Becerra-Arteaga (MilliporeSigma): The buffering capacity within the target pH range for the downstream step is an obvious consideration. In addition to the buffer pKa, another consideration is the potential use of the same buffer in multiple steps in the process. For example, acetate is commonly used as an elution buffer on Protein A capture as well as a bind-and-elute buffer in the cation-exchange step of a mAb process. Additional factors include cost, ease of use (e.g., mixing), and disposal requirements, particularly at large scale.

Chromatography considerations

BioPharm: What are some alternatives to Protein A chromatography?

Becerra-Arteaga (MilliporeSigma): It is challenging to compete with a technology able to provide >95% yield and purity with minimal process development required. However, there are mAb purification processes that utilize cation exchange (CEX) chromatography as the capture step instead of Protein A. For example, Tao et al. (2) evaluated several high-capacity CEX resins and showed [some] of these media also provided sufficient capacity and host cell protein clearance to replace Protein A in a three-step chromatography process. Other alternatives include mixed-mode resins with cation and hydrophobic ligands, but the optimization of the elution and cleaning steps of these media can be challenging.


Levison (Pall): Protein A is the dominant affinity capture step for mAbs. There are alternatives that can be used but they need to be part of a multistage chromatography sequence; these include mixed mode sorbents, or sorbents functionalized with synthetic moieties that mimic elements of the Protein A structure.

Björkman (GE Healthcare): Several attempts have been made to use alternatives [to Protein A chromatography] (precipitation, ion exchangers, or multimodals/mimetics), and all of these have been shown to work to varying extents, but Protein A is outstanding. It is difficult to achieve similar purity, robustness, and high yield under generic conditions. Protein A enables a platform process for mAbs thus enabling higher throughput of projects through process development and manufacturing.

BioPharm: What are some common chromatography column issues one might experience? How can these issues be addressed?

Björkman (GE Healthcare): Some common chromatography column issues include:

  • Column packing. Chromatography requires that one packs a stable bed with good flow properties. Earlier column packing was an art, today automatic packing solutions, such as the AxiChrom columns, are available and making this step much more reliable. Bioburden may occur and its origin varies. It can be from previous steps or introduced during packing (slurry handling). In order to address bioburden, one needs good sanitization methods and knowledge about likely species present in the local environment. Techniques such as closed column packing and/or the use of pre-sanitized single use columns and flow kits can help to minimize bioburden outbreaks.

  • Resin fouling. [Avoiding resin fouling] requires good strip and clean-in-place (CIP) methods.

  • Column net fouling. Good cleaning methods are critical [for avoiding column net fouling].

  • Issues related to strategy. For example, one needs a good control strategy to ensure reproducibility between batches. Also, scale-up strategy is important to ensure that changes in scale do not affect the process outcome. One must also consider strategies for storing column/resins between campaigns.

Levison (Pall): In traditional methods, column packing tends to be problematic and affect performance during reuse. Operator involvement can also introduce new risks. However, with a robust sorbent, column packing concerns can be reduced. Newer chromatography solutions, such as the Pall Life Sciences Resolute chromatography columns with AutoPak technology, support extremely compressible or extremely incompressible resins automatically while eliminating almost all operator involvement.

Becerra-Arteaga (MilliporeSigma): Changes in the process performance, such as pressure increase or broadening of the elution peak, can occur after multiple column reuses if the resin is not cleaned properly. The effectiveness of a cleaning regime can be demonstrated with blank runs every few cycles to ensure minimal carry-over. It is important to also consider that changes in the upstream steps may impact the useful lifetime of the resin.

The process performance, for example aggregate removal on a cation exchanger, may be significantly impacted by minor changes in the conductivity or pH of the buffers. It is important to consider these buffer specifications at large scale may be +/- 1 mS/cm and +/- 0.1 pH unit. Thus, during process development it is important to ensure the process is robust within these typical ranges, otherwise a tighter specification may be required but not necessarily easy to implement at large scale.


Process Scale-Up

BioPharm: What are some challenges companies may face when scaling-up process chromatography for downstream processing? How might you address these challenges?

Levison (Pall): Scaling up chromatography is relatively straightforward--provided constant bed height and constant linear velocity are maintained.

The only cautionary note is not to develop methods in small laboratory columns of < 10 cm diameter at excessive flow rates, because the wall effect becomes much less of a factor at > 10 cm diameter. Such high flow rates may not be sustainable or cause overcompression of the bed, which will have an impact on performance.

Becerra-Arteaga (MilliporeSigma): The process developed at laboratory scale may not completely fit in the facility where the scaled-up process will be transferred. For example, the tanks available may not be of appropriate size to collect the elution pools, particularly if dilutions or pH adjustments need to be made before the next step. The column sizes available may not be large enough and can result in an increase in the number of cycles and overall processing time. Similarly, if the pump capacity of the chromatography systems is insufficient, a smaller column or a change in the process flow rates may be required. Some of these challenges may be addressed through paper exercises and early communication during process development with manufacturing teams.

Robust column packing and long-term bed stability may be a challenge for some chromatography media, particularly at pilot and large scale. The column packing depends on the media characteristics, the packing method and the type of hardware. Vendors can generally provide best practices and recommendations for packing their resins in different types of columns. Even if a packed column meets the acceptance criteria for height equivalent to a theoretical plate and asymmetry, the long-term stability of the bed may be compromised if the packing is not optimal, or if a critical operating pressure is exceeded.

This issue could be the result of under- or over-compression of the packed bed and/or increased pressure on large diameter columns (i.e., > 45 cm) due to reduced wall support. An accurate measurement of the resin slurry concentration, and resin volume is critical to ensure the bed is packed to an optimal compression. The maximum operating pressure of a packed bed can be determined by generating pressure versus flow curves after a column is packed.

It is important to note that the ionic strength and viscosity of the mobile phase changes the slope of these pressure-flow curves. Thus, it is common to observe a higher pressure during the elution step compared to other steps at the same flow rate due to the increase in viscosity.

Björkman (GE Healthcare): Scale-up strategy is very important. In general, we recommend keeping bed-heights and residence times throughout scales. Standardization of hardware platforms, chromatography columns, and systems also helps to ensure seamless scale-up. Improvements in upstream processing have increased titers, shifting bottlenecks downstream from being a volumetric issue to where it is mainly the binding capacity of the resin that is limiting.

Thus continuous techniques like periodic counter current chromatography take better advantage of the resin’s fully capacity by enabling overloading. Other overloading techniques or flow through techniques are also interesting as it enables handling of more sample per time unit.

Increases in mass demands also mean that storage and buffer tanks can create bottlenecks, thus inline conditioning systems, preparing buffers in-line from stock solutions, become attractive. For the same reasons, processing in which multiple unit operations are connected without any hold tanks can help to alleviate storage issues.

Companies must plan for liquid consumption and waste. Large columns at high flowrates need adequate piping and both pure water and wastewater systems must be sized to handle the demand. If onsite systems cannot meet the needs for buffer and cell-culture media preparation, these can be bought from sub-suppliers rather than prepared onsite.

As facility utilization continues to be the number one driver in terms of cost of manufacturing, time aspects, work scheduling, cleaning validation, and campaign switching, [the management of] multipurpose facilities is becoming more important. Good process modeling is therefore becoming increasingly critical in order to enable accurate scenario planning and effective facility utilization.


1. S. Haigney, BioPharm International 28 (11) 2015.
2. Tao et al., Biotechnol. Bioeng. online, DOI: 10.1002/bit.25192

Article Details

BioPharm International
Vol. 29, No. 9
Pages: 28–33


When referring to this article, please cite as C. Hroncich, " Process Chromatography Selection for Downstream Processing," BioPharm International 29 (9) 2016.