Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 °C.
Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.
Modern healthcare systems increasingly depend on biopharmaceutical proteins 1. Producing these proteins in plants is advantageous due to the low pathogen burden and greater scalability compared to conventional expression systems 2-4. However, the downstream processing (DSP) of plant-derived pharmaceuticals can be challenging because the disruptive extraction procedures result in a high particle burden, with turbidities exceeding 5,000 nephelometric turbidity units (NTUs), and host cell protein (HCP) concentrations often exceeding 95% [m/m] 5,6.
Elaborate clarification procedures are required to remove dispersed particles 7-9, but chromatography equipment is less expensive to operate in bind-and-elute mode during initial product recovery if there is an earlier step for the efficient removal of HCPs 10,11. This can be achieved either by precipitating the target protein using flocculants 12 or low pH 13,14, as well as by causing the HCPs to aggregate. The selective aggregation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant HCP in green plants such as tobacco (Nicotiana tabacum), can be promoted by adding polyethylene glycol 15, but this is expensive and incompatible with large-scale manufacturing. Heat treatment has been shown to denature and precipitate more than 95% of tobacco HCPs, while protein malaria vaccine candidates such as Vax8 remain stable in solution 16-18.
Three different approaches were used to achieve the heat-induced precipitation of tobacco HCPs: (i) blanching, i.e., the immersion of intact leaves in hot liquid, (ii) a temperature-controlled stirred vessel, and (iii) a heat exchanger (Figure 1) 16. For intact leaves, blanching achieved the rapid and efficient precipitation of HCPs and was also easy to scale up and compatible with existing large-scale manufacturing processes that include an initial step to wash the plant biomass 19. In contrast, temperature-controlled vessels are already available in some processes and can be used for the thermal treatment of plant extracts 20, but their scalability and energy transfer rate are limited because the surface-to-volume ratio of the tanks is progressively reduced and becomes unsuitable at process scale. A heat exchanger is a technically well-defined alternative to heated stirred vessels but requires an abundant supply of heating and cooling media, e.g., steam and cold water, as well as a tightly controlled volumetric flow rate that is adapted to the heat exchanger geometry and media properties, e.g., the specific heat capacity. This article shows how all three methods can be used for the heat-induced precipitation of tobacco HCPs, and plant HCPs in general. The establishment and operation of each method in a laboratory setting can be used to evaluate their suitability for larger-scale processes. The major challenge is to identify adequate scale-down models and running conditions for each operation that resemble the devices and conditions used during process-scale manufacturing. The data presented here refer to experiments conducted with transgenic tobacco plants expressing the malaria vaccine candidate Vax8 and fluorescent protein DsRed 16, but the method has also been successfully applied to N. benthamiana plants transiently expressing other biopharmaceutical proteins 21.
A design-of-experiments (DoE) approach 22 can facilitate process development, and flocculants 23 can also be beneficial in this context as previously described 8. The main difference between blanching, heated vessels and heat exchangers is that blanching is applied to intact leaves early in the process whereas the others are applied to plant extracts (Figure 1).
Figure 1: Process Flow Scheme Illustrating the Implementation of Three Different Methods for Tobacco HCP Heat Precipitation. The plant material is washed and homogenized before clarification and purification. The equipment for the blanching step (red) can easily be added to the existing machinery. In contrast, using a stirred vessel (orange) and especially a heat exchanger (blue) requires one or several additional devices and tubing. Please click here to view a larger version of this figure.
1. Cultivate the Tobacco Plants
2. Optional: Heat Precipitation by Blanching
NOTE: Carry out the steps described in steps 2.1 to 2.12 in order to precipitate tobacco HCPs by blanching. Skip the entire section 2, if the HCPs will be precipitated in a heated vessel (section 4) or using a heat exchanger (section 5).
3. Protein Extraction from Tobacco Leaves
CAUTION: The next steps involve a blender with rotating blades. Do not work in the blender bucket while it is mounted on the blender motor.
4. Optional: Heat Precipitation in a Stirred Vessel
NOTE: Conduct the steps described in sections 4.2 to 4.11 in order to precipitate tobacco HCPs in a stirred vessel. Skip the entire section 4, if HCPs have been precipitated by blanching (section 2) or will be precipitated using a heat exchanger (section 5).
5. Optional: Heat Precipitation in a Heat Exchanger
NOTE: Conduct the steps described in sections 5.2 to 5.12 in order to precipitate tobacco HCPs using a heat exchanger. Skip the entire section 5, if HCPs have been precipitated by blanching (section 2) or in a heated vessel (section 4).
6. Bag Filtration of the Plant Extract
7. Sample Analysis
Heat precipitation of tobacco host cell proteins by blanching
The blanching procedure described in section 2. was successfully used to precipitate HCPs from tobacco leaves with 70 °C, reducing the TSP by 96 ± 1% (n = 3) while recovering up to 51% of the Vax8 target protein, thus increasing its purity from 0.1% to 1.2% before chromatographic separation 16. It was also possible to recover 83 ± 1% (n =3) of the fluorescent protein DsRed, increasing its purity from 3.3% to 64.1%. The blanching procedure was easily integrated into a standard extraction and clarification scheme consisting of biomass washing, homogenization, bag filtration and depth filtration (Figure 1) 7. Preparing the blanching equipment (Figure 2) added about 5 min to the set-up time for the clarification devices, which routinely takes 20 min. Another 7 min was required to perform the blanching of intact leaves in addition to the typical extraction and clarification time of 45 min. However, only 2 min of the additional 7 min was actual "hands-on" time. Additionally, short incubation times of less than 1 min are possible, reducing the blanching time from 7 min to approximately 3 min. Therefore, blanching not only increases the initial purity of a product in crude plant extracts, but is also rapidly completed with no additional process equipment, thus offering the potential to replace at least an initial chromatography step. The blanching bath temperature remained constant, i.e., < 0.2 °C fluctuation, during all experiments even immediately after the addition of harvested leaves which were at ambient temperature. This ensured the process was repeatable, i.e., an average coefficient of variation of 17% (n = 24), in terms of TSP reduction, product yields and filter capacity in the subsequent clarification steps (Figure 3). However, the filter capacity declined at blanching temperatures > 63 °C, potentially increasing the costs of filter consumables. This can be addressed by adding flocculants after protein extraction 8 and filter aids after bag filtration 9, which can restore or even increase filter capacities. A temperature of 60 °C was sufficient to remove 80 ± 3% (n = 3) of HCPs and increase Vax8 purity 2.6 ± 0.1-fold (n = 3) without affecting filter capacity. Therefore, HCP removal by blanching is compatible with target proteins that have moderate heat stability, i.e., a melting temperature below 70 °C 27,28. However, increasing the temperature to 70 °C or more may result in an HCP removal of over 95% (Figure 3). It was useful to conduct the corresponding set of experiments in a well-designed manner using a statistical approach 22 because this allowed the rapid identification of the most relevant process parameters, i.e., heating time and temperature. At the same time, the DoE method generated a predictive model to facilitate process optimization 16.
Figure 2: Schematic Setup of Three Methods to Precipitate Tobacco HCPs in Intact Leaves or Extracts Thereof. A. Blanching was carried out in a water bath heated with a thermostat (T) into which a basket containing intact leaves was submerged. The water bath was agitated to ensure a homogenous and constant temperature. B. A vessel containing a magnetic stir bar and leaf extract was submerged into a water bath. The temperature in the extract was monitored to ensure that the required temperature was achieved. C. A heat exchanger (H) was connected to a pump (P) and a thermally insulated storage vessel containing the plant extract. The heat exchanger was submerged in a water bath and the temperature of the heated extract was monitored. Please click here to view a larger version of this figure.
Figure 3: Comparison of Three Heat Precipitation Methods Showing Their Effect on Process Performance and the Purification of Two Target Proteins. A. Conditions supporting the removal of more than 90% of HCPs were identified for blanching, a stirred vessel and a heat exchanger setup, all of which increased the purity of the target proteins Vax8 and DsRed by a minimum of 2.5-fold and a maximum of 19-fold, with blanching performing best. In contrast, only the stirred vessel setup increased the capacity of the subsequent depth filtration step used for clarification of the heat treated plants or extracts. Error bars indicate the standard deviation (n = 3). B. The HCP content of samples after different heat treatment conditions can be analyzed and compared using Coomassie-stained polyacrylamide gels. RuBisCO (green arrows) is removed along with other HCPs as the temperature during heat treatment increases, whereas DsRed (red arrow) remains in solution. * indicates a samples that was exposed to heat for 0.5 min, all other samples were treated for 3.0 min or more. Re-print with permission from 16. Please click here to view a larger version of this figure.
Heat precipitation of HCPs in a stirred vessel
A stirred vessel for heat precipitation removed a maximum of 84 ± 1% (n = 3) HCPs, achieving a purity of 0.33 ± 0.02% (n = 3) for Vax8 and 20.2 ± 1.4% (n = 3) for DsRed (Figure 3). The heat treatment was carried out in a vessel separate from the homogenizer to prevent delays reflecting device occupancy when processing multiple samples in series. The handling effort required for the laboratory-scale process was similar to that for blanching but an additional stainless steel vessel and a dedicated cooling step were required. Furthermore, heat transfer to the extract was slower than during blanching, with incubation times of at least 5 min, i.e., 10 times longer than for blanching. The delayed heating was caused by the vessel, which posed an additional barrier to heat transfer, and the ~ 300% greater mass that was heated in the vessel due to the presence of extraction buffer in addition to the plant biomass. Precipitating proteins also adhered to the walls of the vessel, gradually building up an additional heat transfer barrier and increasing the effort required for subsequent cleaning. Setting the water bath temperature 8 °C above the temperature used for heat precipitation compensated for energy losses in the partially open system and achieved the desired extract temperature. In contrast to blanching (section 2) and the heat exchanger setup (section 5), HCP precipitation in a vessel increased the capacity of downstream depth filtration by 2.5-fold, reflecting the lower sheer forces in the vessel compared to pumping extract through the heat exchanger or homogenization after blanching, probably resulting in larger aggregates that were easier to remove in the bag filtration step.
Heat precipitation of HCPs in a heat exchanger
Approximately 88.3 ± 0.7% (n = 12) of the HCP content was consistently removed from the extract using a heat exchanger within the temperature range 60 – 70°C, achieving a purity of 0.31 ± 0.01% (n = 12) for Vax8 and 27.6 ± 2.0% (n = 12) for DsRed (Figure 3). The average coefficient of variation was 13% (n = 24) indicating that the repeatability of this procedure was even better than blanching. The desired extract temperature was achieved after ~ 3 min if the water bath temperature was set 4.5 °C higher. As for the heated vessel, a dedicated cooling step was required after heat treatment. The heat exchanger involved more handling effort than the other methods because the pumping apparatus and heat exchanger required intensive cleaning due to the precipitate adhering to the walls of the narrow bore stainless-steel tubing. The heat exchanger also achieved the lowest downstream depth filter capacity, clarifying only 13.5 ± 6.0 (n = 3) L m-2 before clogging.
The three methods for heat precipitation described above can effectively remove tobacco HCPs prior to any chromatographic purification step 16,17. They complement other strategies that aim to increase initial product purity, e.g., guttation 29, rhizosecretion 30 or centrifugal extraction 31,32, all of which are limited to secreted proteins. However, the heat-based methods can only be used in a meaningful way if the target protein to be purified can withstand the minimum precipitation temperature of ~ 60 °C for more than 1 min. Therefore, the first step in any of the three methods is to design a target molecule with a sufficiently high melting temperature, which has been described for several malaria vaccine candidate proteins consisting of different domains from several Plasmodium falciparum antigens 16,18,21. Once the thermal stability of the target protein has been demonstrated, one of the three methods can be selected based on the available equipment and media, anticipated final process scale and subsequent DSP operations 16.
Blanching was the fastest of the methods and additional equipment requirements were minimal, so it can easily be implemented into existing laboratory-scale purification protocols for plant-derived recombinant proteins. Thorough agitation of the blanching liquid is an important process parameter that affects the efficiency of HCP precipitation based on both empirical data and theoretical calculations 21,33. Failing to achieve good mixing can impair heat transfer and result in only partial HCP removal, which in turn can be detrimental to the product if host proteases remain active 21,34. Several other parameters can also affect HCP precipitation, e.g., the heating temperature and incubation time, and a DoE approach can therefore be useful to characterize the most relevant factors and provide predictive models to quantify their effects on responses such as product purity, recovery and the performance of subsequent DSP steps 22.
In the vessel setup, longer incubation times were required to achieve complete HCP precipitation and this may increase the likelihood of undesirable target protein denaturation reflecting the extended exposure to high temperatures. More thorough mixing in the vessel could improve the heat transfer and reduce the duration of heating. The long temperature ramp in this setup can also be challenging if proteases in the extract 21,34 become more active before final heat inactivation, causing product losses.
The increased depth filter capacity observed for the vessel setup can help to reduce consumables costs, allowing a larger number of samples to be handled in a project with a fixed budget or reducing the overall funding requirements for a given set of experiments. However, this benefit may be outweighed by the cost of the additional vessel, which is necessary in addition to the homogenizer to prevent the introduction of process hold steps if several extraction runs are required in a series of experiments, e.g., as part of a DoE approach. The positive effect on filter capacity may also diminish if a more intensive mixing regime is used to reduce heating times as suggested above.
A dedicated cooling step is necessary for both extract-based heat precipitation methods, requiring not only additional resources but also prolonging the overall processing time per sample, which can also conflict with fluent DoE procedures or experimental sequences in general. The heat exchanger setup is well characterized from an engineering perspective 35 and can easily be designed and scaled up for specific temperature differences, in contrast to the vessel, whose surface-to-volume ratio changes during scale up. However, once the heat exchanger size is defined, it can be difficult to adjust to alternative temperature differences because its length and thus the heat transfer area are fixed.
Changing other parameters, such as the residence time (or flow rate) and temperature of the heat exchanger medium, can restore flexibility to some degree, but only in small-scale experiments because these factors are typically operated in narrow windows in process scale operations due to restrictions imposed by the available equipment and media. The demand for a combination of short incubation times of 3 – 5 min and a temperature difference of 40 – 60 °C becomes increasingly difficult to solve at the device level as the process scale increases because the dimensions of the heat exchanger become larger. This is especially true for the cooling step because the temperature difference between the medium and desired extract temperature is often smaller (ΔT = 10−15 °C) than the heating step (ΔT = 20−40 °C) resulting in large equipment dimensions or longer cool-down times.
In the future, the protocol can be adapted to biopharmaceutical proteins other than vaccine candidates which in this study were specifically designed for thermal stability. Many antibodies can withstand temperatures of > 70 °C 36,37 which is already compatible with the current heat treatment protocol. This natural thermal stability can be increased further by engineering the different antibody domains 38, thereby increasing the number of proteins that can be subjected to the method (and its variations) presented here. The blanching method has already been applied to transgenic tobacco plants expressing a monoclonal antibody (2G12) 17 which has not been subject to selection for thermal stability or protein engineering. Heat treatment at 65 °C increased the purity of the antibody by a factor of two prior to chromatographic purification while the recovery was similar to that observed without blanching.
Additionally, characterizing the individual HCP melting temperatures of an expression system could facilitate the identification of a temperature with which the process could be conducted similar to pasteurization of milk: high temperature, short time 39. The heat treatment (except for blanching) may also be applied to other biological starting materials to remove HCPs if the product can withstand the necessary temperatures. The latter may deviate from the ones discussed here if other expression platforms such as mammalian cell culture supernatants are being processed. In any case, the cost-benefit-ratio should be taken into account, i.e., does the benefit of reduced HCP levels outweigh the cost for implementing a heat treatment step that causes additional investment costs, increases the process time and may reduce the product yield 16. A critical parameter in this context is the product activity. If it depends on the presence of linear epitopes as for some protein-based vaccines, then heat treatment is unlikely to have an effect 40. In contrast, if protein structure is important, e.g., for conformational epitopes, the precise orientation of amino acid side chains in an enzyme's active site or the correct folding of antibody complementarity determining regions, a heat treatment may interfere with protein activity 41,42. Therefore, suitable analysis assays should be established to monitor product performance before and after heat treatment.
The authors have nothing to disclose.
We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Beiß for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as Güven Edgü for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ”Future-Pharma”, proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.
2100P Portable Turbidimeter | Hach | 4650000 | Turbidimeter |
Amine Coupling Kit | GE Healthcare | BR100050 | SPR chip coupling kit |
Autoclaving basket | Nalgene | 6917-0230 | Basket for leaf blanching |
Biacore T200 | GE Healthcare | 28-9750-01 | SPR device |
Bio Cell Analyser BCA 003 R&D with 3D ORM | Sequip | n.a. | Particle size analyzer |
Blender | Waring | 800EG | Blender |
BP-410 | Furh | 2632410001 | Bag filter |
Centrifuge 5415D | Eppendorf | 5424 000.410 | Centrifuge |
Centrifuge tube 15 mL | Labomedic | 2017106 | Reaction tube |
Centrifuge tube 50 mL self-standing | Labomedic | 1110504 | Reaction tube |
CM5 chip | GE Healthcare | BR100012 | Chip for SPR measurements |
Cuvette 10x10x45 | Sarsted | 67.754 | Cuvette for Zetasizer Nano ZS |
Design-Expert(R) 8 | Stat-Ease, Inc. | n.a. | DoE software |
Disodium phosphate | Carl Roth GmbH | 4984.3 | Media component |
Ferty 2 Mega | Kammlott | 5.220072 | Fertilizer |
Forma -86C ULT freezer | ThermoFisher | 88400 | Freezer |
Greenhouse | n.a. | n.a. | For plant cultivation |
Grodan Rockwool Cubes 10x10cm | Grodan | 102446 | Rockwool block |
Twentey-loop heat exchanger (4.8 m length) | n.a. (custom design) | n.a. | Heat exchanger |
HEPES | Carl Roth GmbH | 9105.3 | Media component |
K200P 60D | Pall | 5302303 | Depth filter layer |
KS50P 60D | Pall | B12486 | Depth filter layer |
Lauda E300 | Lauda Dr Wobser GmbH | Z90010 | Water bath thermostat |
L/S 24 | Masterflex | SN-06508-24 | Tubing |
mAb 5.2 | American Type Culture Collection | HB-9148 | Vax8 specific antibody |
Masterflex L/S | Masterflex | HV-77921-75 | Peristaltic pump |
Miracloth | Labomedic | 475855-1R | Filter cloth |
MultiLine Multi 3410 IDS | WTW | WTW_2020 | pH meter / conductivity meter |
Osram cool white 36 W | Osram | 4930440 | Light source |
Phytotron | Ilka Zell | n.a. | For plant cultivation |
Sodium disulfit | Carl Roth GmbH | 8554.1 | Media component |
Sodium chloride | Carl Roth GmbH | P029.2 | Media component |
Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm | n.a. (custom design) | n.a. | Container for heat precipitation |
Synergy HT | BioTek | SIAFRT | Fluorescence and spectrometric plate reader |
VelaPad 60 | Pall | VP60G03KNH4 | Filter housing |
Zetasizer Nano ZS | Malvern | ZEN3600 | DLS particle size distribution measurement |
Zetasizer Software v7.11 | Malvern | n.a. | Software to operate the Zetasizer Nano ZS device |