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Article history: Gene technology has facilitated the biologization of manufacturing, i.e. the use and production of complex biolog-
Received 1 February 2017 ical molecules and systems at an industrial scale. Monoclonal antibodies (mAbs) are currently the major class of
Received in revised form 20 March 2017 biopharmaceutical products, but they are typically used to treat specific diseases which individually have compa-
Accepted 23 March 2017
rably low incidences. The therapeutic potential of mAbs could also be used for more prevalent diseases, but this
Available online 25 March 2017
would require a massive increase in production capacity that could not be met by traditional fermenter systems.
Keywords:
Here we outline the potential of plants to be used for the very-large-scale (VLS) production of biopharmaceutical
Biologization proteins such as mAbs. We discuss the potential market sizes and their corresponding production capacities. We
Molecular farming then consider available process technologies and scale-down models and how these can be used to develop VLS
Monoclonal antibodies processes. Finally, we discuss which adaptations will likely be required for VLS production, lessons learned from
Plant-based production existing cell culture-based processes and the food industry, and practical requirements for the implementation of
Process economy a VLS process.
Very-large-scale production © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
2. Potential future applications and market size for monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3. Plant cultivation strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
4. Process schemes for monoclonal antibody production in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
4.1. Clarification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
4.2. Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
5. Scalable process models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
6. Process adaptations for VLS requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
7. Translation into VLS applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
Abbreviations: ATPS, aqueous two-phase extraction; cGMP, current good manufacturing practice; CHO, Chinese hamster ovary; HCP, host cell protein; HIV/AIDS, human
immunodeficiency virus/acquired immunodeficiency syndrome; mAb, monoclonal antibody; SMB, simulated moving bed; UF/DF, ultrafiltration/diafiltration; VFU, vertical farming
unit; VLS, very large scale.
⁎ Corresponding author at: Institute for Molecular Biotechnology, Worringerweg 1, RWTH Aachen University, 52074 Aachen, Germany.
E-mail addresses: johannes.buyel@rwth-aachen.de (J.F. Buyel), richard@twymanrm.com (R.M. Twyman), fischer@molbiotech.rwth-aachen.de (R. Fischer).
1
Current address: Indiana Biosciences Research Institute, 1345 W. 16th St. Suite 300, Indianapolis, IN 46202, USA.
http://dx.doi.org/10.1016/j.biotechadv.2017.03.011
0734-9750/© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
J.F. Buyel et al. / Biotechnology Advances 35 (2017) 458–465 459
over cell culture platforms for upstream production, whereas the down- or VFUs therefore appear to be the most likely scenarios for the VLS pro-
stream capture and polishing steps would be similar to those already duction of mAbs in plants.
used for the purification of mAbs from CHO cells, with similar cost impli-
cations (Ma et al., 2015; The CMC Biotech Working Group, 2009). Howev- 4. Process schemes for monoclonal antibody production in plants
er, the extraction and clarification steps between upstream production
and downstream capture/polishing differ substantially between plant- One key characteristic of plant-based manufacturing processes is the
based and fermentation-based processes. The major difference is that broad spectrum of techniques initially used by academic and industrial
mAbs are generally secreted by cultured cells and recovered from the me- development teams to facilitate extraction, clarification and product re-
dium, whereas they must be released from intact plants by some form of covery (Fig. 1). These techniques have recently been reviewed (Buyel et
mechanical tissue and cell disruption which also releases a large number al., 2015b) and continuous operations, such as screw-press extraction,
of dispersed particles and soluble impurities that must be removed prior are likely to be the most beneficial for VLS applications (Buyel and
to chromatography (Buyel et al., 2015b). These issues require cost-effec- Fischer, 2014c). Typically, large numbers of dispersed particles and
tive and scalable solutions before VLS mAb production in plants becomes large quantities of host cell protein (HCP) are released during product
commercially viable, as discussed in more detail below. extraction, which can be addressed by using flocculants or other filter
Previous studies have indicated that the production of mAbs in aids, as well as various conditioning steps such as a temperature or pH
plants is not competitive at low expression levels in the 0.02– shift. These measures can increase the capacity of filters to
0.05 g kg− 1 range because the costs per gram of purified product ~1000 L m−2 and remove N90% of the HCP (Buyel and Fischer, 2014b;
would reach €10,000–20,000 (Buyel and Fischer, 2012, 2014c) com- Menzel et al., 2016).
pared to ~€200 g−1 for mAbs produced in CHO cells (Lim et al., 2010).
However, feeding the same cost models with data from recent reports 4.1. Clarification
in which mAb yields in plants reached 2.0 mg kg−1 (Zischewski et al.,
2015) and in which filter capacities for the processing of plant extracts The most common unit operations for clarification are centrifugation
reached 100–1000 L m−2 (Buyel et al., 2014b) showed that production and/or filtration. The former can be difficult to scale up due to the changes
costs for plant-derived mAbs can fall substantially below €100 g−1 (our in the apparatus geometry, and it may be necessary to reduce the flow
unpublished data). Therefore, the production of mAbs in plants per se rate by N50% to maintain clarification capacity or settling area (Roush
can be economically competitive with CHO cells. Even if the investment and Lu, 2008). Combinations of filters are therefore preferred for the clar-
costs for the production facilities are similar, the operating costs for ification of plant extracts because they can also remove pigments, HCP
plant-based processes will be lower especially due to the simple cultiva- and DNA (Kandula et al., 2009). However, the filter must be chosen care-
tion of intact plants compared to the cost-intensive sterile requirements fully because protein products can adsorb to the diatomite contained in
for cell suspension cultures. most filters (Buyel et al., 2015a). The optimal combination of filters in
terms of capacity, clarification and product recovery must be determined
3. Plant cultivation strategies for each process depending on the particle size distribution, e.g. using bag
filters, depth filters and/or pre-coat filters.
There are several types of plant-based expression systems that re-
quire cultivation techniques similar to those used with mammalian cell 4.2. Purification
cultures, e.g. aquatic plants, plant cell suspension cultures, and plant tis-
sue cultures such as hairy roots (Xu et al., 2012). Even though these sys- The purification of mAbs typically involves an initial capture step
tems can benefit from simple nutrient requirements, the absence of using a Protein A resin (The CMC Biotech Working Group, 2009). There-
animal components and pathogens, they will be subject to the same fore, despite the different upstream production and extraction/clarifica-
scale-up limitations as CHO cells as they require sterilizable fermenters. tion procedures associated with fermenters and plants, most mAb
Thus, they are ultimately unsuitable for the VLS production of mAbs. In production processes converge at this process step. The pH and temper-
contrast, there are two major expression strategies using intact plants ature shifts discussed above may also facilitate mAb purification be-
that have the potential to reduce the operating costs for upstream pro- cause both conditioning steps may inactive proteases that could harm
duction substantially compared to cell cultures. In the transient expres- the product and/or the Protein A resin. This is particularly important
sion approach, wild-type plants are vacuum infiltrated with in the context of the resin life cycle because Protein A resins are one of
Agrobacterium tumefaciens carrying a vector encoding the mAb heavy the largest capital investment costs, i.e. ~$US 11 million for the produc-
and light chains, allowing large quantities of mAb to accumulate within tion of 10 t of mAb using CHO cells (Kelley, 2007). In contrast to the pro-
5–8 days (Shoji et al., 2012). Although this method is rapid, its potential teases mentioned above, mAbs typically remain stable at pH ~3.5 and
drawback for VLS production is that each plant in every batch must be in- ~70 °C (Rouet et al., 2014; Woodard et al., 2009).
filtrated with bacteria, which merely transfers the fermentation costs to Regardless of the conditioning, the concentrations of mAbs in plant
the production of bacteria (Houdelet et al., 2017). This is not the case for extracts are generally in the 0.1–0.2 g L−1 range and thus 50–100-fold
transgenic plants, where well-defined master and working seed banks lower compared to processes based on optimized CHO cells, although
can be established and used for consecutive batches without the need yields of N 2 g kg−1 have also been reported for plants, corresponding
for additional manipulation (Sack et al., 2015). Therefore, transgenic to ~ 0.5 g L−1 (Zischewski et al., 2015). Accordingly, instead of the
plants appear to be the most suitable plant-based format for VLS produc- high binding capacities (~ 50 g L− 1) of packed-bed columns
tion. There are three options to cultivate such plants: under open field (Thommes and Etzel, 2007), the faster flow rates of membrane ad-
conditions, in conventional greenhouses or in VFUs. Open fields offer vir- sorbers (up to 18 m h−1) help to reduce the process time (Varadaraju
tually unlimited scaling potential but no containment, so it is unlikely et al., 2011). Alternatively, an ultrafiltration/diafiltration (UF/DF) step
that this approach will be used for pharmaceutical products (Fischer et can be integrated prior to Protein A column chromatography to reduce
al., 2012). In contrast, VFUs provide a high degree of containment and the process volume and accelerate product recovery (Lightfoot and
can designed to comply with cGMP as described above. However, this Cockrem, 2013) particularly if the mAb concentrations are higher (e.g.
benefit comes at the cost of greater capital investment for the vertical N0.5 g L−1). Cost-effective replacements for the Protein A ligand, e.g.
cultivation racks and automation equipment. Greenhouses offer a com- mixed-mode resins such as MEP HyperCel that are designed specifically
promise between capital investment and ease of scale-up. The plants to bind mAbs, will also be valuable for VLS mAb manufacturing
we used to produce the first cGMP-compliant batch of mAb used in clin- (Jackewitz, 2012), especially if the nonspecific binding of HCPs is
ical trials were cultivated in a greenhouse (Ma et al., 2015). Greenhouses negligible because they have been removed by pre-conditioning
J.F. Buyel et al. / Biotechnology Advances 35 (2017) 458–465 461
Fig. 1. Comparison of process schemes for the large-scale production of mAbs. Production in CHO cells (A; (Kelley, 2007)) is compared to a reported tobacco-based platform (B; (Ma et al.,
2015)) and its recent improvement (C; Fraunhofer IME unpublished data) as well as a hypothetical continuous very-large-scale process (D). API – active pharmaceutical ingredient.
462 J.F. Buyel et al. / Biotechnology Advances 35 (2017) 458–465
steps such as low-pH incubation or heat treatment (Buyel et al., More than one process condition may achieve the same overall desir-
2014a; Hassan et al., 2008). ability, e.g. as a tradeoff between productivity and resin capacity utiliza-
The polishing steps that follow the Protein A capture step are sim- tion, a situation described as Pareto optimality, hence the final choice of
ilar for all mAb manufacturing platforms, typically involving ion ex- process may depend on manufacturer preferences. A set of the most de-
change or hydrophobic interaction chromatography (Ma et al., 2015; sirable combinations of operations can be selected, e.g. based on mAb
The CMC Biotech Working Group, 2009). A design-of-experiments yield. However, before this presumably optimal process is scaled up,
approach is often recommended for the optimization of these steps its potential benefits must be compared with the cost of implementa-
(Buyel and Fischer, 2013) because this exploits the individual prop- tion at the production scale.
erties of each mAb product and complies with quality-by-design
guidelines (Rathore, 2009). 6. Process adaptations for VLS requirements
Virus filtration is another cost driver for mAb production, accounting
for up to 12% of the cost of goods for processes based on CHO cells The performance of optimal processes (e.g. the highest mAb
(Kelley, 2007). This step may not be necessary for plant-based produc- yield) in small-scale experiments must be confirmed at the pilot or
tion processes because plants do not support the growth of human process scale to ensure that the selected operation conditions
viral pathogens (Fischer et al., 2012). To comply with industry standards match with the corresponding VLS equipment. However, process
(Shukla and Gottschalk, 2013), a virus-filtration step was incorporated scale-up must be preceded by the detailed cost–benefit analysis of
as part of the manufacturing process for the HIV-neutralizing mAb each step, taking into account all aspects that may be negligible during
2G12 produced in tobacco (Ma et al., 2015). However, this accounted screening or small-scale evaluation, but that can become cost drivers in
for b5% of the consumables costs and b 3% of the predicted cost of VLS processes. For example, flocculation was found to increase depth
goods (our unpublished data). filter capacity during the clarification of plant extracts by a factor of
Selecting the sequence of unit operations to achieve the most cost- five, reducing the cost of filters accordingly. However, implementing
effective purification becomes more challenging when using plants for flocculation at the process scale also introduces additional costs that
VLS mAb production. It is prohibitive to perform VLS process optimiza- are not evident at the laboratory scale (e.g. an additional hold tank to
tion ‘at scale’ due to the high costs of consumables and labor. Instead, accommodate the flocculation process, which as a consequence be-
the rapid testing of different process setups (including cost estimates comes discontinuous or semi-continuous at best if the process cycles
for scaled-up production) is facilitated by a rational model-based ap- among several hold tanks). Furthermore, pumps are required to add
proach. This requires the availability of models that adequately describe the flocculant, which must be pharmaceutical grade in terms of purity
the different unit operations, as discussed in more detail below. and must be held with a sufficient inventory. Finally, the flocculant
may interfere with subsequent chromatography steps (Buyel and
5. Scalable process models Fischer, 2014a) and adequate quality controls may be required to dem-
onstrate that the flocculant is not present in the final mAb formulation.
The choice of unit operations and devices for a given mAb produc- The different operations available for extraction, clarification, HCP
tion process should be based on the recovery, yield, purity and per- removal and purification are summarized in Table 1 in terms of their
formance that can be achieved at production scale. Small-scale benefits and drawbacks for the VLS production of mAbs, and these
models can be used to assess these quality indicators in a rapid and must be balanced according to their effects on the cost of goods. Here,
cost-effective manner if they provide scalable information about the overall effect on process or product costs must be considered rather
the various devices, facilitating cost estimates for different process than the impact on a particular process step. For example, if UF/DF is
setups (Buyel et al., 2015a; Chhatre et al., 2011). Two constraints used to reduce the process volume before Protein A capture, the isolated
apply to the sample size used in these approaches. Smaller sample evaluation of this step would indicate an increase in costs. However, if
or aliquot sizes, e.g. 50–2000 μL in 96- or 384-well plate formats, the entire process is taken into account, the benefits of process volume
are typically associated with lower costs and higher throughput, reduction achieved by UF/DF would include the use of a smaller Protein
and thus facilitate the screening of a broader set of process condi- A column and smaller downstream storage/hold tanks, thus reducing
tions to increase the likelihood of identifying optimal parameter both the capital and consumables costs overall. These savings should
combinations, e.g. achieving the highest yield, lowest costs or fastest then be offset against the increased costs of implementing the UF/DF
processing. However, sample volumes must be large enough to en- step to determine whether there is an overall benefit.
sure that equipment with properties comparable to process-scale One trend that is apparent for the large-scale manufacturing of mAbs
devices can be used, which may require N0.2 L per run (Buyel and using mammalian cells is the increasing number of continuous process-
Fischer, 2014d; Roush and Lu, 2008). Recent developments have re- es, either in part (such as cultivation and chromatography) or even
duced the sample volumes required for flocculation testing to completely integrated continuous production (Klutz et al., 2015;
b1.0 mL, and authentic process-scale geometries have been introduced Xenopoulos, 2015). The benefits of continuous production include the
into small filtration devices (Buyel et al., 2015a; Espuny Garcia Del Real higher space–time yield of manufacturing facilities, smaller footprints
et al., 2014). Screw-press and blender-based extraction have been and lower investment costs. Therefore, plant-based production facilities
shown to scale effectively from 150 g to N 200 kg biomass (Buyel and should follow this lead if possible, adjusting for the discrete growth
Fischer, 2014b), and rapid, scalable screening formats are also available phases of plants by staggered cultivation and automated harvesting
for chromatography-based purification steps (Chollangi et al., 2014; and extraction. In this context, regulatory issues may arise from contin-
Keller et al., 2015). If scalable equipment is not available for screening, uous manufacturing in terms of batch definition (Hertrampf et al.,
a two-tier approach can be applied, in which the first dataset is gathered 2015), and technical issues may arise due to the need for some equip-
in a screening format that yields qualitative results, and the most prom- ment redundancy to insure against device failure.
ising steps are then verified using scalable equipment that provides
quantitative data and estimates the production costs. Combining the 7. Translation into VLS applications
data for different process steps allows the overall performance of the
process to be evaluated, so that different setups can be compared in To our knowledge, the largest cGMP-compliant plant-based produc-
small-scale experiments. tion process for mAbs has been established by the Fraunhofer IME (Aa-
Several functions have been developed to optimize dependent vari- chen, Germany) for the manufacture of 2G12. This facility can process
ables (also known as “responses”) simultaneously, thus maximizing the 250 kg of tobacco leaf biomass per batch, corresponding to approxi-
desirability of a process outcome (Anderson and Whitcomb, 2005). mately 900 L of bulk extract (Ma et al., 2015). The original downstream
J.F. Buyel et al. / Biotechnology Advances 35 (2017) 458–465 463
Table 1
Benefits and drawbacks of downstream processing operations for the very-large-scale (VLS) production of mAbs.
Extraction Blade-based Applicable to all types of proteins Discontinuous Alternating operation of several devices
homogenizer Well established at kg scale Limited scalability due to can facilitate semi-continuous
volume/area ratio and power input processing
Infiltration centrifugation Reduced HCP extraction Discontinuous
Reduced particle burden Limited scalability due to g-force
requirements
Limited to secreted proteins
Technically demanding
Rhizosecretion Continuous Dilute target protein Hydroponic cultivation is required
Reduced hcp extraction Large process volumes
Reduced particle burden Limited to secreted proteins
Screw-press Applicable to all types of proteins May interfere with target proteins Extraction in the absence of buffer
Continuous Sensitive to low pH
Reduced particle burden
Well established at tonne scale
Clarification Centrifugation Low consumables costs Discontinuous
Well established at L scale High energy demand
Limited scalability due to g-force
requirements
Filtration Continuous High consumables costs
Established at m3 scale May bind target protein
Linear scalability
DNA/HCPb reduction
Filter aids Established at m3 scale Increased investment and
Increased filter capacity Consumables costs
May bind target protein
Flocculation/filter aids Established at m3 scale Increased investment and Special assays confirming the absence
Increased filter capacity consumables costs of flocculants in the final product can
May bind target protein be required
May interfere with subsequent
process steps
Conditioning/HCP ATPSb Increased purity Increased investment and Continuous operation has been
removal Process volume reduction consumables costs reported
Reduced particle burden Increased disposal costs
Heat treatment Continuous High energy demand
Established at m3 scale Increased investment costs
Increased purity May interfere with target protein
Low-pH precipitation Increased purity Discontinuous Continuous operation may be possible
Increased investment and via in-line mixing
consumables costs
May interfere with target protein
UF/DFb Process volume reduction Increased investment costs Continuous operation possible
Established at m3 scale Increased purity for certain target
proteins
Purification Membrane adsorbers High flow rates Discontinuous Supply may be difficult
Low binding capacity
Packed-bed High binding capacity Discontinuous
chromatography Low flow rates
SMBb chromatography Continuous Sophisticated control system Applicable to adsorbers and
Reduced investment and consumables required packed-bed columns
costs
a
Discontinuous operations may be converted to semi-continuous operations by a combination of numbering-up and alternating use.
b
Abbreviations: ATPS - aqueous two-phase extraction, HCP - host cell protein, SMB - simulated moving bed, UF/DF - ultrafiltration/diafiltration.
process incorporated alternating blade-based homogenizers, five filtra- the VLS production of mAbs should therefore be designed with the
tion steps for extract clarification, a Protein A membrane adsorber for long-term stability of raw material supplies in mind.
mAb capture and initial purification, a subsequent ceramic hydroxyap- Using the expression levels and product recoveries discussed above,
atite polishing step followed by nanofiltration, UF/DF, and a final 0.2- the 250-kg Fraunhofer IME process would yield 0.0002–0.0005 t of mAb
μm sterile filtration step (Fig. 1). Since 2009, this process has been mod- per batch, which corresponds to an annual output of 0.005–0.016 t as-
ified to accommodate a packed-bed Protein A column to replace the suming 45 batches per year. This falls short of the anticipated top-end
membrane adsorbers that are no longer available from the manufactur- demand of 50 t per year by a factor of at least 3000. Accordingly, the cur-
er. Therefore, an additional UF/DF step was introduced before the cap- rent “large-scale” plant-based mAb production process can be regarded
ture chromatography step to concentrate the extract by a factor of 15, as “small scale” in the context of VLS mAb manufacturing. The current
reducing the time required for loading the Protein A column by the process equipment is therefore unlikely to be suitable if the process is
same factor but otherwise maintaining the overall process time. Fur- scaled up by three orders of magnitude.
thermore, the small-scale screening of new depth filters resulted in an Instead of trying to develop entirely new solutions for this scale-up
optimized clarification procedure, reducing the number of steps from problem, the plant biotechnology community can learn and benefit
five to three and the cost of goods by 40% (our unpublished data). from the expertise developed during the optimization of CHO cells, par-
These modifications highlight how cost–benefit analysis can be affected ticularly with regard to chromatography (Fig. 2). Examples include the
by the changing consumables market, i.e. in this case discontinued potential integration of simulated moving bed chromatography (Baur
membrane adsorbers and the availability of new filters. Processes for et al., 2015) and continuous processing operations to reduce the size
464 J.F. Buyel et al. / Biotechnology Advances 35 (2017) 458–465
Fig. 2. Comparison of devices and equipment currently used in pilot-scale production and potential alternatives for VLS production.
and footprint of the equipment, increasing the space–time yield as de- substantial investment, and it remains to be seen which incentives and
scribed for the single-use continuous manufacturing of mAbs (Klutz et motivations will be required to encourage public and private stake-
al., 2015). Another benefit of smaller equipment is the reduction in cap- holders to tackle this challenge.
ital costs, which is particularly striking for the Protein A resin, resulting
in cost reductions of 10–30% (Angarita et al., 2015).
The benefits of VLS processes are likely to be even greater if we Acknowledgements
also embrace the technologies, experiences and procedures that
have been gathered during almost 200 years of industrialized food This work was funded by the Fraunhofer-Gesellschaft Internal Pro-
processing (Fig. 2) (Goody, 1997). The food-processing industry grams under Grant No. Attract 125-600164 and the European Research
often handles multi-tonne quantities of plant biomass, which is Council Advanced Grant “Future-Pharma”, proposlal number 269110.
processed by extraction, blanching, filtration and separation, under The authors have no conflict of interest to declare.
highly-regulated hygienic conditions designed to minimize process-
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