The Growing Importance of Downstream Processing in Plant-Based Biologics

Plant-based biologics, also known as molecular farming, have emerged as a transformative platform for producing therapeutic proteins, vaccines, antibodies, and other high-value biomolecules. By leveraging the natural protein synthesis machinery of plants such as tobacco (Nicotiana benthamiana), rice, and duckweed, manufacturers can avoid the high capital costs, complex culture media, and contamination risks associated with mammalian cell culture. However, the economic and regulatory viability of plant-made pharmaceuticals hinges on efficient downstream processing (DSP). Recovery and purification steps can account for 50% to 80% of total production costs. As the field matures, several emerging trends in DSP are addressing these cost barriers while improving product quality, scalability, and environmental sustainability.

This article explores the key innovations reshaping downstream processing for plant-based biologics, from novel extraction techniques to automation, advanced chromatography, process integration, and green engineering. Each trend is supported by recent research and industrial case studies, providing a practical roadmap for process developers, biomanufacturers, and regulatory strategists.

Advancements in Extraction Techniques

The first step in plant-based biologics DSP — extraction of the target protein from plant biomass — presents unique challenges. Plant cells contain rigid cell walls, high levels of proteases, phenolic compounds, and other secondary metabolites that can degrade or modify the product. Traditional extraction methods such as mechanical pressing, homogenization, and buffer-based leaching often suffer from low yields, excessive shear stress, and co-extraction of impurities. Newer techniques address these limitations with greater selectivity and gentler conditions.

Aqueous Two-Phase Extraction (ATPE)

Aqueous two-phase extraction has gained traction for plant-based systems. By using two immiscible water-based phases (e.g., polyethylene glycol and salt or polymer/polymer systems), ATPE partitions the target protein into one phase while most contaminants remain in the other. This mild, non-denaturing method can be applied directly to homogenized plant extracts, combining clarification and early purification in a single step. Recent studies have demonstrated ATPE for monoclonal antibodies expressed in N. benthamiana, achieving recovery yields above 90% with significant reduction of host cell proteins and pigments.

Supercritical Fluid Extraction

Supercritical carbon dioxide (scCO2) extraction offers an environmentally friendly alternative to organic solvents for recovering lipophilic biologics or for defatting plant material prior to aqueous extraction. By adjusting pressure and temperature, scCO2 can be tuned to selectively extract lipids, chlorophylls, and other non-polar impurities, leaving the target protein intact. Although more common for small molecules, scCO2 pre-treatment is being investigated for plant-made enzymes and vaccine antigens. The technique is valuable for reducing the burden on downstream chromatography and for improving the shelf life of clarified extracts.

Microwave-Assisted and Pressurized Liquid Extraction

Microwave-assisted extraction (MAE) and pressurized liquid extraction (PLE) use controlled heating and pressure to disrupt plant cell walls rapidly. These methods reduce extraction time from hours to minutes and can improve protein recovery from recalcitrant tissues such as seeds or roots. When combined with aqueous buffers containing reducing agents and protease inhibitors, MAE and PLE have shown promise for extracting recombinant human growth hormone and therapeutic enzymes from transgenic rice and maize. However, thermal degradation remains a concern for thermolabile biologics, so careful optimization of temperature and residence time is critical.

Automation and Process Optimization

Downstream processing in biomanufacturing has historically been batch-oriented, with manual intervention for column packing, buffer preparation, and sample handling. The plant-based biologics industry is rapidly adopting automation to increase throughput, reproducibility, and compliance with current Good Manufacturing Practice (cGMP).

Robotic Liquid Handling and Integrated Work Cells

Robotic platforms for high-throughput process development are now standard in many labs. Automated liquid handlers can execute design-of-experiments (DoE) protocols across multiple resin types, buffer conditions, and flow rates simultaneously, generating robust operating windows in days rather than weeks. At manufacturing scale, integrated work cells combine centrifugation, filtration, and chromatography modules under a single control system. These systems reduce manual errors, enable real-time data logging, and facilitate unattended operation during long purification runs.

Process Analytical Technology (PAT) and Real-Time Monitoring

The adoption of Process Analytical Technology (PAT) is transforming DSP from a black-box approach to a transparent, data-rich operation. Near-infrared (NIR) and Raman spectroscopy probes inserted inline can measure protein concentration, aggregate levels, and impurity profiles in real time. For plant-based biologics, PAT is particularly useful for detecting fluctuations in feed quality caused by batch-to-batch plant growth variability. Combined with multivariate data analysis, PAT enables dynamic adjustment of chromatography gradients or filtration flow rates, ensuring consistent product quality even when the starting material varies.

Continuous Processing

Moving from batch to continuous downstream processing is a megatrend across the biopharmaceutical industry, and plant-based biologics are no exception. Continuous chromatography systems such as periodic counter-current chromatography (PCC) and simulated moving bed (SMB) systems increase resin utilization, reduce buffer consumption, and enable steady-state operation. In continuous mode, the capture step (typically Protein A affinity or ion exchange) is run in sequence with two or more columns, allowing one column to be loaded while another is eluted and regenerated. The result is a 2- to 3-fold increase in productivity per unit of resin. For plant-based biologics, continuous processing also helps manage the rapid degradation of proteins in crude extracts by minimizing hold times.

Use of Affinity and Chromatography Techniques

Chromatography remains the workhorse of biologic purification. For plant-based products, the selection of adsorbents must account for the presence of plant-specific contaminants such as chlorophyll, polysaccharides, and alkaloids. Emerging trends include the development of plant-tailored affinity ligands, membrane-based chromatography, and multifunctional mixed-mode resins.

Plant-Specific Affinity Ligands

Traditional affinity ligands such as Protein A for antibody capture are expensive and may leach into the product. For plant-made antibodies, researchers have engineered custom ligands that recognize the glycan structures unique to plant expression systems (e.g., beta-1,2-xylose and alpha-1,3-fucose). These ligands offer high selectivity while avoiding cross-reactivity with mammalian host cell proteins. Similarly, peptide-based and aptamer-based affinity ligands are being designed for other classes of plant-made biologics, including cytokines and growth factors. The trend toward fully synthetic, plant-specific affinity resins reduces cost and improves purity in a single capture step.

Membrane Chromatography and Monolithic Columns

Traditional packed-bed chromatography columns suffer from high pressure drops and limited flow rates when processing viscous or particulate-laden plant extracts. Membrane adsorbers — porous sheets of regenerated cellulose or polyethersulfone functionalized with ion-exchange or affinity groups — offer a convective mass transport mechanism that allows processing at much higher flow rates with lower backpressure. Membrane chromatography is especially effective for the flow-through polishing step, where high capacity for contaminants is not required. Monolithic columns, which consist of a single piece of porous polymer, provide similar advantages for rapid purification of large volumes and are increasingly used for plant-derived viral nanoparticles and virus-like particles.

Mixed-Mode and Multimodal Chromatography

Mixed-mode resins combine two or more types of interaction (e.g., ion exchange and hydrophobic interaction) on the same ligand. This orthogonal selectivity can resolve species that are difficult to separate using single-mode chromatography. For plant extracts, mixed-mode resins like Capto MMC or Nuvia cPrime show excellent ability to remove nucleic acids, endotoxins, and colored pigments while maintaining high recovery of the target protein. The trend toward multimodal screening libraries, where dozens of resin chemistries are tested in parallel, allows rapid identification of optimal conditions for each plant-based biologic candidate.

Integration of Downstream Steps

Traditional DSP involves a linear sequence of discrete unit operations: clarification, capture, intermediate purification, polishing, and formulation. Each step introduces product loss, dilution, and time delays. Process intensification through step integration is a key emerging trend that reduces these inefficiencies.

Continuous Capture with Direct Feed

Systems such as the BioSC® from Novasep or the ÄKTA™ pcc from Cytiva allow the capture column to be fed directly with a pre-filtered but not fully clarified plant extract. By using a guard column or a depth filter in front of the capture column, these systems can tolerate some particulate matter, eliminating a separate centrifugation or microfiltration step. The direct coupling of extraction and capture reduces total processing time and minimizes exposure to plant proteases.

Inline Filtration and Conditioning

Instead of collecting eluates in tanks and then adjusting pH and conductivity before the next chromatography step, modern DSP systems incorporate inline dilution and pH adjustment using dynamic mixers. Inline diafiltration using tangential flow filtration modules can concentrate and buffer-exchange the product continuously, feeding directly into a downstream polishing column. These closed-loop configurations reduce the number of hold vessels, lower the risk of contamination, and improve overall yield by avoiding repeated pump transfers and dilution steps.

Integrated Disposable Solutions

Single-use technologies have become standard in early-phase manufacturing and are now migrating to commercial-scale production of plant-based biologics. Disposable depth filters, membrane chromatography capsules, and bioreactor bags for intermediate storage enable rapid changeover between products and reduce cleaning validation burdens. Integration of disposable modules into a skid-mounted platform (e.g., the KUBio™ concept) allows manufacturers to set up a complete downstream train in a pre-validated, plug-and-play configuration. This flexibility is particularly valuable for plant-based biologics aimed at emerging market or pandemic response scenarios where speed and scalability are critical.

Focus on Sustainability and Green Processing

The environmental profile of biopharmaceutical manufacturing is under increasing scrutiny. Plant-based biologics already offer a greener upstream by eliminating the need for animal-derived components and reducing energy consumption compared to bacterial or mammalian fermentation. However, downstream processing traditionally uses large volumes of water, organic solvents, and high-energy equipment. Emerging trends aim to minimize this footprint.

Reduction of Water and Buffer Consumption

Water usage in DSP can be substantial — several thousand liters per batch for a moderate-scale plant. Continuous chromatography systems reduce buffer consumption by 50–70% compared to batch mode because columns are loaded to near breakthrough and eluted with smaller volumes. Inline concentration and diafiltration also minimize the number of dilution and re-concentration cycles. Furthermore, the use of high-capacity resins and membrane adsorbers reduces the bed volumes required, directly lowering water demand.

Green Solvents and Disposable Modules

Efforts are underway to replace traditional solvents (e.g., acetonitrile for HPLC polishing) with greener alternatives such as ethanol, isopropanol, or supercritical carbon dioxide. For plant extracts, ethanol-based precipitation or crystallization steps are being explored as non-chromatographic alternatives for the initial capture of proteins, reducing the need for expensive resins. Disposable modules, while generating plastic waste, can be incinerated for energy recovery and avoid the water, chemicals, and energy required for cleaning reusable equipment. Lifecycle assessments suggest that single-use systems often have a lower overall environmental impact for clinical-scale production.

Waste Valorization

Another sustainability trend is the valorization of plant biomass after extraction. Rather than discarding spent plant material, companies are investigating the recovery of secondary metabolites, dietary fibers, or biofuels from the residual biomass. For example, N. benthamiana leaves after recombinant protein extraction can be processed to extract polyphenols with antioxidant properties, creating an additional revenue stream. This circular approach aligns with the principles of green chemistry and improves the overall economics of plant-based biologics manufacturing.

Challenges and Future Outlook

Despite significant progress, several hurdles remain before plant-based biologics achieve parity with established mammalian platforms at industrial scale. The most pressing challenges include the scalability of novel extraction and chromatography techniques, the cost of custom affinity ligands, and the regulatory standardization of plant-specific impurities.

Scalability of New Technologies

While ATPE, membrane chromatography, and continuous processing work well at the bench and pilot scales, scale-up to thousands-of-kilograms of biomass per batch is not always straightforward. Mixing times, mass transfer coefficients, and pressure drop constraints change with scale, and the lack of commercial-scale equipment for some novel technologies (e.g., large-format membrane stacks for direct viral vectors) slows adoption. Collaborations between equipment vendors and plant biologic developers are essential to bridge this gap.

Cost of Custom Ligands and Media

Plant-specific affinity ligands are expensive to develop and manufacture at scale. For early-phase products, the cost of a custom resin may be prohibitive. However, as the number of plant-made biologics entering phase III trials increases, economies of scale will drive down ligand costs. Meanwhile, manufacturers are exploring reusable affinity resins and low-cost alternative scaffolds such as designed ankyrin repeat proteins (DARPins) as capture agents.

Regulatory Considerations

Regulatory agencies such as the FDA and EMA have increasingly accepted plant-based systems, as demonstrated by the approval of Elelyso® (taliglucerase alfa) from carrot cells and the Emergency Use Authorization for plant-made COVID-19 vaccines (sanofi/GSK and Medicago). However, concerns about glycosylation patterns (especially the presence of plant-specific glycans) and the potential for adventitious agents require robust clearance studies during DSP. The trend toward using gene-edited plants with humanized glycosylation pathways (e.g., glyco-engineered N. benthamiana) simplifies downstream processing by reducing plant-specific impurities at source.

Future Directions

Looking ahead, the convergence of machine learning with high-throughput process development promises to accelerate the optimization of DSP for each plant-based biologic. AI algorithms trained on large datasets of resin screens, breakthrough curves, and impurity profiles can recommend optimal chromatography sequences and operating conditions in silico, reducing experimental burden. Additionally, the advent of modular, single-use manufacturing facilities designed for rapid deployment in geographically distributed locations aligns with the decentralized production model that plant-based platforms enable. These modular factories, equipped with standardised DSP skids, can be shipped and commissioned in months rather than years, making plant-based biologics a compelling option for addressing global health emergencies.

In summary, downstream processing for plant-based biologics is evolving rapidly through innovations in extraction, automation, chromatography, process integration, and sustainability. By adopting these emerging trends, developers can overcome the historical cost and scalability barriers of plant-made therapeutics, paving the way for a new generation of affordable, safe, and accessible biologics.

Further Reading