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European Biopharmaceutical Review

Flexibility in Bioprocessing

Traditionally, bioprocessing of protein therapeutics was a cumbersome, rigid operation, beginning with mammalian or microbial culture in glass and then steel tank bioreactors of increasing size. Following clarification of the culture, the protein of interest was purified via a series of glass or steel chromatographic columns containing one of a limited series of resins, as well as ultrafiltration/diafiltration and microfiltration to separate the protein from smaller components. This model succeeded in developing and producing many of the early protein therapeutics, but was inherently inefficient and inflexible.

Today, thanks to advances in cell culture, equipment, separation technologies and applications for plastics, bioprocessing has made great strides towards becoming a flexible, adaptable technology for the production of a wide range of protein therapeutics.

Flexibility in Bioreactors

Perhaps no other area of bioprocessing has received as much attention from single-use technologies as bioreactors, which has resulted in an enormous flexibility of options. Single-use has been driven by a number of factors, including the high capital cost and long installation time of hard-piped, stainless steel systems. Of course, disposables present the highly attractive feature of eliminating bioreactor cleaning, which is labour-intensive, time-consuming and energetically expensive due to the large volumes of water for injection typically used for cleaning (1).

All manufacturers of single-use bioreactors have employed a type of multi-laminate, sterile plastic bag to line a structural vessel to support it. However, that is where the similarities stop. Many have used impellers in a cylindrical tank: some top-mounted (Thermo Fisher/HyClone, Sartorius) and others bottom-mounted (Xcellerex/GE, EMD Millipore); some of these with off-centered and others centered impellers.

Then there are manufacturers that have taken a more radical approach, with rocker-type mixing of pillow-shaped bags (Wave/GE, Applikon, Sartorius, Pall), or an orbitally rotated, shakeflask design (Kuhner), or a paddle in a squared tank (ATMI). One of the most novel designs is from PBS Biotech, which uses a large air-driven wheel inside a drum-shaped bag. Clearly, there is a lot of flexibility in bioreactors stemming from all these options.

Combined Approaches

However, with such an array of designs, can one move a process from one platform to another? In theory, yes, provided that the cells are well-nourished, suspended, temperatureand pH-controlled, and have good control of oxygen and carbon dioxide.

In practice, a combination of theoretical and empirical approaches is used. For example, mixing must be sufficient to provide good suspension of cells, gas and nutrient transfer, but not so vigorous as to damage cells. The cell growth stage also matters – for instance, many in the biopharma industry use the rocker-type bioreactors for scale-up of mammalian cells, passaging them every two or three days into a larger vessel. During the final production stage, however, the cells are typically transferred to some sort of stirred tank – in effect, changing the type of bioreactor.

We have seen comparable bioreactor profiles of cultures producing recombinant proteins from either stainless or singleuse bioreactors. Growth and titer were comparable in these runs. Note that this was for a heavily (30 per cent) glycosylated cytokine, with significant sensitivity to post-translational modification. Yet product profile, including glycosylation, was similar whether the bioreactor was of stainless steel or single use.

Even stainless steel bioreactors can be made more flexible through modular approaches. For example, working with a combination of one to four 500l stainless steel bioreactors, each on wheels with quick-connect gas and liquid connections, Gallus has been able to adjust total bioreactor volume, as well as produce continuously via perfusion cultures.

Downstream Benefits

Flexibility in the purification of the protein of interest has also increased enormously from advances both in single-use technologies and separation science. Rather than the traditional glass and stainless columns, chromatographers can now choose from an array of pre-packed polymer-walled columns containing a variety of resins. These are conveniently supplied with column packing test data (Asymmetry, HETP) and can offer either single or multiple uses, depending on the application and economics.

Moving beyond columns to membrane supports brings the advantage of higher flow rates and therefore shorter processing times. The trade-off for these higher flow rates has reduced binding capacity, but this is much less important in polishing chromatographic steps. Most protein purification processes begin with a primary capture step, such as Protein A affinity for Immunoglobulin G antibodies and ion-exchange for other proteins. One or more separation steps follow this initial capture as polishing steps for the removal of host-cell proteins (HCP) and other residual impurities.

Natrix Separations has developed a hybrid approach, where a membrane framework structure contains a hydrogel matrix more like a traditional chromatographic support. This results in much higher capacities than standard membranes, but allows for the faster flow rates that membranes provide. An assessment shows this could be a promising alternative to more conventional membranes, showing comparable reduction of DNA and HCP across a two-log scale-up.

Enhanced Operations

Another area of enhanced flexibility has resulted from newer, salt-tolerant ion-exchange chromatography membranes and resins (Sartorius, Pall). Binding is generally minimally affected by salt concentrations, resulting in the removal of impurities across a wide range of salt concentrations. This allows the user to avoid dilution of the feedstock with water, often required to reduce salt levels to allow good binding on the ion-exchange support.

The result is elimination of a unit operation (dilution) plus a reduced feedstock volume and therefore shorter processing times. This type of chromatography, however, is not intended for capture or bind-and-elute processes, since bound materials are very difficult to remove. The feature also recommends single-use applications because cleaning of the support is challenging.

Multimodal Support

Flexibility has also resulted from new combinations of separation techniques in one chromatographic operation, through the use of multi-mode or mixed-mode supports. These additional chromatographic supports offer new power in separations, by exploiting a combination of protein properties interacting with the support. For example, a combination of hydrophobic interaction and ion-exchange is presented in supports such as Capto-Adhere and Capto- MMC (GE), Nuvia cPrime (Bio-Rad) and MEP- and PPAHyperCel (Pall).

Another multi-modal support, Capto Core-700 (GE), adds the additional modality of size-exclusion to this mix. New applications are also being found for the classic multi-modal support of hydroxylapatite (CHT™, Bio-Rad). These rely both on this support's unique combination of specific ion-binding for phosphate and calcium, and ion-exchange properties. These types of chromatographic support have enabled purification scientists to solve very challenging separations, such as fusion proteins and Immunoglobulin M antibodies (3,4).

Aseptic Filling

Nearly all protein pharmaceuticals are given by injection or parenterally, and due to their sensitive natures must be filled into patient doses aseptically without terminal sterilisation. This requires stringent aseptic techniques, specialised equipment and processing.

Single-use or disposable technologies have added to the flexibility of such operations. For example, Gallus replaced a stainless steel filling machine with a unit whose entire flow-path is composed of single-use materials. The heart of this system is a unique linear peristaltic pump, which allows a three-log range of filling volumes, based on simple replacement of the flow tubing with different diameter hoses. The pump’s stroke length is also adjustable to vary the volume dispensed. The combination of these two orthogonal controls, tubing diameter and stroke length, allows filling of volumes from 0.1-100ml.

Other modalities contributing to flexibility include prefilled syringes, which are becoming more popular even for clinicalphase products, and the use of clear plastic instead of glass vials. Such plastic vials are part of the innovative technology of filling sealed vials using a unique puncture and laser-reseal operation (Aseptic Technologies). This method avoids working with open vials, thereby removing the risk of contamination.

Environmental Modules

Entire upstream and downstream operations can be built in a flexible fashion using closed system, bioreactors and self-contained environmental modules around each downstream unit operation. This concept, pioneered by Xcellerex/GE, has been put into place at several facilities.

Controlled environmental modules (CEM) serve as a mechanism to separate unit operations around each chromatographic or filtration skid, with ports for operators’ gloved hands to enter and control equipment. As such, it largely separates the operator from the equipment, allowing a single, ballroom suite approach to the different steps of the process. This provides flexibility in the facility, as separate rooms for each operation are no longer necessary. Environmental monitoring, both in the ballroom and the CEM units, shows good control, with environmental specifications being met.

Name of the Game

The wide array of choices available to the bioprocessors, whether in upstream, downstream, filling operations or facilities, provides welcome flexibility. Furthermore, many of these options allow more rapid processing, faster turnaround between runs, smaller footprints, lower capital costs, and more powerful and rapid separations. Flexibility is the name of the game in bioprocessing.


1. Sinclair A et al, The environmental impact of disposables, Biopharm International supplement, November 2008
2. Ultee M and Grégoire A, Presentation at the BioProduction Conference, Berlin, 25th October 2012
3. Rea DW, Ultee ME, Chen SX and Loisel TP, Solutions for purification of Fc-fusion molecules, Biopharm International supplement, pp20-25, March 2008
4. Valasek C et al, Production and purification of a PER.C6- expressed IgM antibody therapeutic, Bioprocess International, December 2011

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Michiel E Ultee, PhD, is Chief Scientific Officer at Gallus BioPharmaceuticals, serving as a scientific resource for projects, as well as a liaison with clients and the biopharma community. He has over 30 years of experience in the industry and has worked on the development of antibodies since 1975. Michiel earned his graduate and undergraduate Biochemistry degrees from Northwestern University and Dartmouth College. He holds four US patents and has published numerous scientific articles. Prior experience includes postdoctoral research in immunology at NYU Medical School and various positions at Laureate BioPharma.
Michiel E Ultee
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