We would like to acknowledge Ibrahim Al Amedi for cultivating the transgenic tobacco plants and Dr. Thomas Rademacher for providing the tobacco expression vector. The authors wish to thank Dr. Richard M. Twyman for editorial assistance and Markus Sack for fruitful discussions about the DFE affinity ligand structure. This work was funded in part by the Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164 and the state of North-Rhine-Westphalia under the Leistungszentrum grant no. 423 &#8220;Networked, adaptive production&#8221;. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) in the framework of the Research Training Group &#8220;Tumor-targeted Drug Delivery&#8221; grant 331065168. GE healthcare supported the open-access publication of this article.

1. Cultivate the Transgenic Tobacco Plants
NOTE: The design of the DFE fusion protein and the generation of transgenic plants are described elsewhere5,17.
<ol>
	<li>Germinate the transgenic tobacco seedlings in soil. Irrigate the plants with a 1.0 g&#183;L-1 fertilizer solution. Transfer the plants to single, 100 mm x 100 mm x 60 mm (length, width, height) pots when they grow to a diameter of ~0.04 m.</li>
	<li>Cultivate the transgenic tobacco plants in a greenhouse with a 16-h photoperiod (25/22 &#176;C light/dark temperature regime), with 70% relative humidity and automated fertilization with a 1.0 g&#183;L-1 fertilizer solution for 15 min every hour.</li>
	<li>After 7 weeks, harvest all leaves except the four cotyledon leaves at the base of the plant stem. Immediately process the harvested leaves as described below.</li>
</ol>
2. Heat Precipitation of Host Cell Proteins
<ol>
	<li>Set up a water bath with a 20 L working volume in a stainless-steel heated vessel (0.3 m x 0.3 m x 0.3 m). At the start, place a magnetic pump in the water bath to agitate the liquid permanently with a flow of 5 L min-1. Mount an adjustable thermostat on the water bath for temperature control (steps 2.4 and 2.8). 
	CAUTION: All the following steps up to 2.10 involve the handling of hot liquid. Wear appropriate personal protective equipment including thermally insulated gloves and goggles.</li>
	<li>Add 15 L of deionized water to the water bath and heat the liquid to 70 &#176;C.</li>
	<li>Place a 10 L bucket filled with 5 L of deionized water on a magnetic stirrer. Add sodium phosphate to a final concentration of 120 mM. Adjust the pH to 8.14 using 10 M hydrochloric acid. When all the components have dissolved, add the resulting concentrated blanching buffer (5 L) to the water bath from step 2.2.</li>
	<li>Agitate the water bath until the temperature reaches 70 &#176;C. Use 10 M hydrochloric acid to adjust the pH to 8.14 if necessary. Continue agitating for at least 15 min after the required temperature and pH are reached to ensure that the entire assembly is in thermal equilibrium.</li>
	<li>Prepare a 20 L bucket (0.3 m x 0.3 m x 0.3 m) filled with deionized water at a temperature of ~17 &#176;C (required for step 2.9).</li>
	<li>Prepare 400 g aliquots of the harvested tobacco leaves from step 1.3. Place one aliquot in a perforated basket (0.2 m x 0.2 m x 0.2 m; pore width at least 0.02 m x 0.02 m). Avoid overfilling the basket with plant material or packing the leaves too tightly to avoid damaging the tissue.</li>
	<li>Fully submerge the basket in the hot blanching buffer from step 2.4 and make sure all leaves remain under the liquid surface. Use a thermostable silicone spoon to hold the leaves under the surface if necessary.</li>
	<li>Incubate the tobacco leaves for 1.5 min in the blanching fluid while the pump is still agitating the liquid. Monitor the liquid temperature during the entire incubation period. Avoid blocking the pump inlet with tobacco leaves.</li>
	<li>Remove the basket of tobacco leaves from the blanching buffer and drain residual buffer from the leaves for 30 s. Transfer the basket to the bucket filled with cold water (from step 2.5) and immerse the leaves for 30 s. Remove the basket and drain residual water from the leaves for 30 s before homogenization (step 3).</li>
	<li>Repeat steps 2.6 to 2.9 with fresh aliquots from 2.6 until the entire harvested biomass is processed. Constantly monitor and adjust the pH and the temperature of the blanching buffer in the water bath. 
	NOTE: Blanched plant material can be stored on ice for up to 30 min when several blanching cycles are required to process the entire biomass. Blanched leaves can also be stored in vacuum-sealed bags at &#8211;80 &#176;C for at least 3 weeks. However, immediate processing is recommended because prolonged storage can reduce the DFE yield.</li>
</ol>
3. Protein Extraction and Clarification
CAUTION: The following steps involve a blender with rotating blades. Do not work in the blender vessel when powered or mounted on the motor unit.
<ol>
	<li>Place 150 g (wet mass) of blanched tobacco leaves (step 2.9) in the blender vessel and add 450 mL of extraction buffer (50 mM sodium phosphate, 500 mM sodium chloride, 10 mM sodium disulfite, pH 7.5). Close the blender cap tightly to prevent the spilling of plant material or buffer.</li>
	<li>Homogenize the leaves for 3x for 30 s, with 30 s breaks between each pulse. Ensure that all leaves are homogenized and that none stick to the top of the blender vessel. If necessary, open the blender during the breaks and push down leaves that stick at the upper part of the vessel, using a clean silicone spoon.</li>
	<li>After homogenization, take a 1.0 mL sample of the homogenate for subsequent analysis (step 7).</li>
	<li>Collect the homogenate in a vessel of adequate size (e.g. if working with a total biomass of 1.0 kg, use a vessel with a capacity of at least a 5 L). Repeat steps 3.1 to 3.3 until all of the blanched biomass is homogenized.</li>
	<li>Mount a bag filter into a filter mount and place another adequately sized vessel (see 3.4) beneath the assembly. Apply the homogenate to the bag filter at a rate of ~0.15 L min-1. Take samples of the bag filtrate after each liter for subsequent analysis (step 7) and measure the turbidity of the bag filtrate pool as a 1:10 dilution in extraction buffer using a turbidimeter or similar device.</li>
	<li>Further clarify the bag filtrate pool using 0.02 m2 of a K700 (top) and KS50 (bottom) depth filter layer combination per liter of bag filtrate pool. Apply a flow rate of 3.0 L min-1&#183;m-2 up to a maximum pressure of 0.2 MPa. Then remove residual particles by passing the clarified filtrate through a 0.2 &#181;m sterile filter as previously described5.</li>
</ol>
4. Purification of the DFE Affinity Ligand
<ol>
	<li>Prepare a fast protein liquid chromatography system by flushing with elution buffer (10 mM sodium phosphate, 300 mM imidazole, pH 7.4), wash buffer (10 mM sodium phosphate, pH 7.4) and equilibration buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4). Mount a column containing ~50 mL of chelating cross-linked agarose resin per kilogram of leaf biomass (~2.5 L depth filtrate).</li>
	<li>Charge the column with nickel ions by applying 5 column volumes (CV) of 200 mM nickel sulfate solution and wash it with 5 CV of elution buffer. Use a flow rate of 50 cm h-1 and monitor the UV signals at 260, 280 and 558 nm for all subsequent chromatography steps.</li>
	<li>Equilibrate the column with 10 CV of equilibration buffer. Then load 50 CV of the clarified plant extract (from step 3.6) onto the conditioned column.</li>
	<li>Wash the column with 10 CV of wash buffer. Elute the DFE fusion protein with 5 CV elution buffer and collect the product-containing fraction once the UV signals at 280 nm and 558 nm have increased to more than 5 mAU above the baseline.</li>
	<li>Take a 0.2 mL sample from the elution fraction in order to measure the total soluble protein (TSP) concentration (step 7.1), DFE concentration (steps 7.2 and 7.3), and DFE purity (step 7.4).</li>
	<li>Mount a column containing ~50 mL of cross-linked dextran resin on a chromatography system. Equilibrate the column with 5 CV of coupling buffer (200 mM HEPES, 500 mM sodium chloride, pH 8.5). Inject 10 mL of the DFE elution fraction (step 4.4) for buffer exchange and monitor the UV absorbance at 280 and 558 nm.</li>
	<li>Collect the DFE-containing fraction once the UV signals at 280 nm and 558 nm have increased to more than 5 mAU above the baseline. Take a 0.2 mL sample and determine the TSP concentration, DFE concentration, and DFE purity (step 7).</li>
	<li>Concentrate the purified DFE sample (from step 4.7) to 15 g L-1 using a centrifugal concentrator tube at 3,000 x g at 4 &#176;C for 30 min in a centrifuge. Continue with the coupling reaction (step 5). 
	NOTE: Store the DFE solution at 4 &#176;C if the concentration or coupling steps cannot be carried out immediately.</li>
</ol>
5. Coupling DFE to the Activated Cross-linked Agarose Resin
NOTE: Do not replace the isopropanol used for the storage of NHS-activated columns until all equipment and solutions for coupling are ready. Never let the columns run dry.
<ol>
	<li>Setup a design of experiments (DoE) model to optimize the coupling of DFE to the activated resin, with pH, buffer composition and DFE concentration as factors. The details of the DoE method are described elsewhere15.</li>
	<li>Prepare the affinity ligand solution (from step 4.8) in a concentration range from 0.5&#8211;15 g&#183;L-1 or as defined in the DoE and store it on ice until the coupling reaction is ready (step 5.5). Fill at least ten 2 mL syringes with the DFE solution and prepare an adapter to mount the syringes on NHS-activated cross-linked agarose columns with a bed volume of 1.0 mL.</li>
	<li>For every 10 columns used for coupling, prepare 30 mL of deactivation solution (0.5 M ethanolamine, 0.5 M sodium chloride, pH 8.3), 30 mL of low-pH solution (0.1 M sodium acetate, 0.5 M sodium chloride, pH 4.0) and 10 mL of storage solution (0.05 M disodium phosphate, 0.1% (m/v) sodium azide, pH 7.0).</li>
	<li>Prepare 20 mL of 1 mM hydrochloric acid in a tube and incubate it on ice for at least 20 min. Prepare a precision scale to monitor the flow-through fractions for all steps during the coupling reaction (step 5.7).</li>
	<li>Open a sealed NHS-activated cross-linked agarose column and mount the syringe adapter at the column inlet. Prevent any air entering the column by applying a drop of buffer onto the adapter inlet before connecting it to the syringe.</li>
	<li>Wash the column with 6 mL of ice-cold 1 mM hydrochloric acid (from step 5.4) at a flow rate of &#60;1 mL min-1 and immediately proceed to step 5.7.</li>
	<li>Inject 1.5 mL of DFE solution (step 5.2) using a 2 mL syringe at a flow rate of &#60;1 mL min-1 and collect the flow-through fraction on a precision scale (step 5.4) for subsequent analysis (step 7). Seal the column at both ends and incubate for 15&#8211;45 min at 22 &#176;C, depending on the DoE setup.</li>
	<li>Inject 6 mL of deactivation solution followed by 6 mL of low-pH solution at a flow rate of &#60;1 mL min-1 to remove non-covalently bound ligands from the resin. Then inject 6 mL of deactivation solution and incubate the column for 15 min.</li>
	<li>Inject 6 mL of low-pH solution into the column, followed by 6 mL of deactivation solution. Then inject another 6 mL of low-pH solution into the column.</li>
	<li>Inject 2 mL of storage solution into the column and store at 4 &#176;C.</li>
</ol>
6. Testing the Purification of mAbs from Clarified Plants Extracts
<ol>
	<li>Prepare 100 mL of clarified plant extract containing 2F55 or the supernatant from the preferred cell-based expression system, also containing 2F5.</li>
	<li>Prepare equilibration buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4), low-pH elution buffer (0.05 M citrate, 0.05 M sodium chloride, pH 4.0&#8211;3.25), and high-ionic-strength elution buffer (1.0&#8211;4.0 M magnesium chloride, 0.1 M HEPES, pH 8.0)</li>
	<li>Flush the chromatography system with the buffers. Mount a DFE affinity column (from step 5.10) on the chromatography system and equilibrate with 5 CV of equilibration buffer at a flow rate of 1.0 mL min-1. Monitor the UV absorbance at 280 nm. 
	NOTE: Loading plant extract or cell culture supernatant onto the column can cause an increase in backpressure. Set a high-pressure alert at 0.2 MPa to avoid damage to the chromatography system or DFE column.</li>
	<li>Load 80 mL of the clarified plant extract or supernatant (step 6.1) onto the column at a flow rate of 0.5 mL min-1 to guarantee a contact time of 2 min. Collect the flow-through samples in 2 mL fractions for breakthrough curve reconstruction (step 7.3). Store the flow-through samples at 4 &#176;C if immediate sample analysis is not possible.</li>
	<li>Wash the column with 6 CV of equilibration buffer. Collect a sample of the wash at the beginning, middle and end of this step.</li>
	<li>Elute mAb 2F5 with 5 CV of low-pH elution buffer or high-ionic-strength elution buffer (0.1 M HEPES, 1.25 M magnesium chloride, pH 8.0). Collect the DFE fraction when the UV 280 nm signal has increased to 5 mAU above the baseline.
	<ol>
		<li>Optimize the elution buffer for each epitope&#8211;antibody pair. For 2F5, 1.25 M magnesium chloride achieved an optimal balance between product recovery and ligand stability. 
		NOTE: The magnesium chloride solution is prone to precipitation. Therefore, dissolve the magnesium chloride in ~700 mL of water. Separately dissolve the HEPES in 100 mL of water and adjust the pH to 8.0. Add the dissolved magnesium chloride solution to the HEPES solution and add water to a final volume of 1.0 L. Do not adjust the pH after dissolving the magnesium chloride because this will cause precipitation.</li>
	</ol>
	</li>
	<li>Analyze all samples taken during steps 6.4&#8211;6.6 using the Bradford method, lithium dodecylsulfate polyacrylamide gel electrophoresis (LDS-PAGE) and surface plasmon resonance (SPR) spectroscopy (step 7).</li>
</ol>
7. Sample Analysis
<ol>
	<li>Measure the TSP concentration using the Bradford method18,19.
	<ol>
		<li>In triplicate, pipette 2.5 &#181;L of each sample into a single well of a 96-well plate. Use eight bovine serum albumin (BSA) standards in triplicate covering the range 0&#8211;2,000 mg&#183;L-1.</li>
		<li>Add 200 &#181;L of Bradford reagent to each well and mix by gently pipetting up and down. Keep the pipette level to avoid the formation of bubbles that distort the subsequent readout.</li>
		<li>Incubate the plate for 10 min at 22 &#176;C and measure the absorbance at 595 nm in a spectrophotometer. Calculate the TSP concentration in the samples based on a standard curve through the BSA reference points.</li>
	</ol>
	</li>
	<li>Quantify DFE by fluorimetry
	<ol>
		<li>In triplicate, pipette 50 &#181;L of each sample into single wells of a black 96-well half-area plate. Include six DsRed standards covering the range 0&#8211;225 mg&#183;L-1.</li>
		<li>Measure the fluorescence twice using a 530 &#177; 30 nm excitation filter and a 590 &#177; 35 nm emission filter in a spectrophotometer. Calculate the DFE concentration in the samples based on a standard curve through the DsRed reference points.</li>
	</ol>
	</li>
	<li>Measure the 2F5 concentration by SPR spectroscopy20.
	<ol>
		<li>Prepare the HBS-EP running buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3 mM ethylenediaminetetraacetic acid (EDTA), 150 mM sodium chloride, 0.005% v/v Tween-20, pH 7.4) and filter sterilize it by passing it through a 0.2 &#181;m vacuum bottle-top filter. Degas the buffer for 15 min.</li>
		<li>Connect the HBS-EP running buffer to the SPR instrument before docking a carboxymethylated dextran surface chip and equilibrate the system using the prime function. Start a manual run with a flow rate of 30 &#956;L min-1 and condition the chip surface by alternately injecting 30 mM hydrochloric acid and 25 mM sodium hydroxide (two injections each) over flow cells 1 and 2 at a flow rate of 30 &#956;L min-1 for 1 min.</li>
		<li>Prepare 300 &#181;L of a 500 mg&#183;L-1 Protein A solution in 10 mM sodium acetate (pH 4.0). Thaw vials containing 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.1 M NHS and centrifuge at 16,000 x g for 1 min before mixing 70 &#956;L of EDC and 70 &#956;L of NHS.</li>
		<li>Transfer the EDC/NHS mixture and the Protein A solution to 7 mm plastic sample vials and place in the sample rack. Activate the carboxymethylated dextran chip surface by injecting the EDC/NHS mixture over flow cells 1 and 2 at a flow rate of 10 &#956;L min-1 for 10 min.</li>
		<li>Couple Protein A to the activated carboxymethylated dextran surface by injecting the Protein A solution over flow cell 2 at a flow rate of 15 &#956;L min-1 for 15 min.</li>
		<li>While coupling the Protein A, thaw a vial of 1.0 M ethanolamine hydrochloride (pH 8.5) and centrifuge at 16,000 x g for 1 min. Transfer 150 &#181;L to a 7-mm plastic vial and place into the instrument. When step 7.3.5 is complete, deactivate the chip surface by injecting the ethanolamine solution over flow cells 1 and 2 at a flow rate of 10 &#956;L min-1 for 7 min.</li>
		<li>Condition the chip surface by alternately injecting 30 mM hydrochloric acid and 25 mM sodium hydroxide (two injections each) over flow cells 1 and 2 at a flow rate of 30 &#956;L min-1 for 0.5 min.</li>
		<li>Prepare standards of antibody 2F5 in HBS-EP running buffer at a concentration of 500 &#181;g&#183;L-1 and dilute samples containing 2F5 in HBS-EP to a final concentration in the range 20&#8211;1,000 &#181;g&#183;L-1. Inject samples and standards over flow cells 1 and 2 at a flow rate of 30 &#956;L min-1 for 3 min. Inject a 2F5 standard after every 15 samples.</li>
		<li>Subtract the relative units (RU) signal of flow cell 1 (reference cell) from the signal of flow cell 2 (measurement cell) for each sample and calculate the antibody concentration based on the RU signal of 2F5 standard injections.</li>
		<li>After every sample or standard injection, regenerate the chip surface by injecting 30 mM hydrochloric acid at a flow rate of 30 &#956;L min-1 for 0.5 min over flow cells 1 and 2.</li>
		<li>Plot the 2F5 concentration for each flow-through sample from step 6.4 against the flow through volume to obtain the 2F5 breakthrough curve. Calculate the amount of injected 2F5 when 10% of the loading 2F5 concentration was reached to obtain the 10% dynamic binding capacity (DBC).</li>
	</ol>
	</li>
	<li>Analyze protein samples by LDS-PAGE.
	<ol>
		<li>Open a ready-to-use 4&#8211;12% BisTris LDS polyacrylamide gel and place it in an electrophoresis module. Transfer 800 mL of running buffer (50 mM MES, 50 mM Tris base, 0.1% (m/v) SDS, 1 mM EDTA, pH 7.3) into the module and add 0.5 mL of antioxidant solution.</li>
		<li>In a 1.5 mL reaction tube, mix 10 &#181;L of loading buffer with 4 &#181;L of reducing agent and 26 &#181;L of the protein sample. Incubate the tube in a heat block for 10 min at 80 &#176;C. Centrifuge the tube at 500 x g for 30 s.</li>
		<li>Load the LDS polyacrylamide gel (10 &#181;L of sample per lane) and load 5 &#181;L of pre-stained protein standard (10&#8211;180 kDa) in a separate lane.</li>
		<li>Close the electrophoresis chamber and connect the power supply. The electrophoresis should run for 40 min and at constant 200 V.</li>
		<li>Remove the gel from the electrophoresis chamber. Open the gel sheathing and wash the gel for 15 min in water on a shaker at 19 rpm. Stain the gel for 1 h in staining solution on a shaker at 19 rpm.</li>
		<li>Destain the gel for 1 h in water on a shaker at 19 rpm. Scan the gel using a film scanner.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to disclose.

Applications of the novel affinity resin
We have shown that custom affinity chromatography resins for the capture of mAbs can be manufactured by immobilizing a ligand containing a mAb-specific epitope to NHS-activated cross-linked agarose. To design such a resin, it was necessary to know the epitope sequence and to use a linear epitope. The resulting resins are advantageous for the capture of mAbs because they could potentially replace expensive Protein A affinity chromatography steps. The interaction between 2F5 and DFE in our case study was mediated by epitope&#8211;paratope binding, so our ligand should be more selective than Protein A, which binds to the Fc region of most murine and human IgGs. Because individual ligands are needed for each mAb, our method may initially seem suitable mainly for antibodies that are produced on a very large scale. However, by combining our approach with rapid plant-based transient protein expression, new affinity ligands can be prepared in less than 2 weeks27 with minimal effort28. Hence, the method is also suitable for small-scale mAb purification.
Production and potential improvements of the affinity ligand
Plants offer a fast and safe production platform for affinity ligands5,29,30, such as the DFE fusion protein featured in our case study. Blanching the plant material greatly reduced the quantity of host cell proteins in a single step and was easily integrated into a standard clarification routine. However, the recovery of the ligand was low in the current setup, probably due to its moderate thermal stability and some non-specific binding to the filter layers, as reported for other products31,32,33. Engineering the carrier to increase its thermal stability may therefore help to improve the ligand yield in the future, as described for the malaria vaccine candidate CCT, the antitumor enzyme PpADI or a mesophilic &#946;-glucosidase34,35,36. The same holds true for the depth filtration step, where protein engineering may help to reduce non-specific binding to the filter material37. The production costs for DFE and similar ligands could also be reduced by improving the overall efficiency of clarification using flocculants or filter additives38,39.
When DsRed is used as a carrier, it forms a tetrameric complex. This is advantageous because it increases the number of epitopes per ligand, but it may also render the ligand more susceptible to disassembly or denaturation during affinity chromatography. A monomeric carrier protein such as mCherry may therefore be preferable, because it is stable at low pH40, and the inclusion of tandem repeats of the epitope would increase the avidity of the ligand and thus increase resin capacity5,26,41. For simple carrier-epitope proteins (i.e., those with no disulfide bonds or post-translational modifications) microbial production systems may reduce the manufacturing costs and make the ligands more competitive with Protein A. For example, green fluorescent protein has been expressed in bacterial cells with a yield of ~1 g&#183;kg-1 biomass, which would significantly reduce ligand production costs42.
Regardless of the expression host, a purified affinity ligand was required during coupling to minimize the immobilization of host cell proteins or media components that can otherwise reduce resin selectivity and capacity. The inclusion of a poly-histidine tag for IMAC purification increased the purity to ~90% in a single step, facilitating rapid and inexpensive ligand production5,43,44. However, the position of the fusion tag is important because it has the potential to sterically hinder epitope binding or to induce the cleavage of either the tag or the epitope from the carrier45,46.
Immobilization of the affinity ligand on NHS-activated chromatography columns
Immobilization was carried out manually or using a chromatography system. The small buffer volumes per column seemed to favor manual handling (e.g., due to the minimal waste volumes). However, if multiple/larger columns are needed, the chromatography system makes the coupling conditions easier to control (e.g., regulated flow rates) and is therefore more likely to achieve reproducible results in terms of DBC. Our data suggest that the coupling buffer and pH have an important effect on the coupling efficiency and overall column costs. Screening factors that influence the coupling reaction and adjusting them for each carrier protein (or even for each carrier&#8211;ligand fusion) could therefore improve coupling efficiency and resin performance, and we recommend this approach.
Testing 2F5 isolation using the DFE affinity resin
Product yield and purity are important aspects of resin performance, and in the case of DFE we achieved a yield of 105 &#177; 11% (&#177;SD, n=3) and a purity of 97 &#177; 3% (&#177;SD, n=3), which is comparable with the performance of benchmark Protein A resins25,26. Another key performance indicator for resins in general (and particularly for those based on affinity ligands) is the DBC at 10% product breakthrough, because this parameter affects the amount of resin required for a specific process and thus the costs. For the DFE ligand, the initial DBC was ~4 g&#183;L-1 resin, which is ~13% of the corresponding value for Protein A under similar conditions (only 2 min contact time)25,47 but about 15-fold higher compared to other custom affinity resins such as the anti-FSH-immunoaffinity ligand using the same residence time of 2 min48. The DBC of DFE declined to 15% of the starting value after 25 bind-and-elute cycles, whereas more than 50 cycles are required for the same loss of DBC in commercial Protein A resins49. However, it is important to note that our carrier has not yet been optimized to the same extent as Protein A, which has been comprehensively investigated and improved over the last four decades8.
Thus far we have improved the resin stability and product recovery by switching from a low-pH elution buffer to a high concentration of magnesium chloride (Figure 3), as recommended in earlier studies13. The characteristic red color of the affinity ligand did not fade substantially during the 25 bind-and-elute cycles, so we speculate that endogenous plant proteases in the clarified plant extracts31 may have truncated and thus inactivated the epitope of the ligand. Therefore, designing protease-resistant linkers to connect the carrier and epitope may help to maintain the initial DBC over an extended number of cycles. Given the rapid and simple expression and purification of the DFE ligand, its straightforward coupling to commercial chromatography resins, and its excellent product yield and purity, we believe that our method offers a suitable alternative to Protein A for the purification of mAbs and antibody derivatives which do not bind to Protein A, especially if improvements to the carrier and linker can improve the DBC and ligand stability. This assumption was supported by the small difference in the dissociation constant of DFE-purified and Protein A-purified 2F5 antibody5, indicating that our new affinity ligand allows the recovery of high-quality mAbs.
Benefits and current limitations of the method
Producing the affinity ligand as a genetic fusion with a carrier protein increases solubility in aqueous buffers and thus compatibility with typical ligand coupling conditions. In contrast, blank peptides derived from solid phase peptide synthesis may have limited solubility under these conditions due to their sequence50, which cannot be changed because it is dictated by the amino acid sequence of epitope recognized by the mAb to be purified. Others have therefore used an on-resin synthesis of peptide ligands51. The static binding capacity of the resulting resin was high (~80 g&#183;L-1), but the process of resin preparation is lengthy, a dynamic binding capacity was not reported and the obtained purity and recovery were lower than in our approach. An additional advantage of a fusion protein ligand in laboratory scale is that the ligand and variants thereof can be rapidly produced, purified and tested with minimal effort in an easy-to-use high-through expression system52.
The two current limitations of the method presented here are the low dynamic binding capacity of 3 g&#183;L-1 and its 90% reduction over the course of 25 bind-and-elute cycles5. These limitations can be addressed in the future by applying less stringent loading conditions and replacing the current DsRed carrier with an engineered, more stable variant respectively. For example, doubling the current contact time from 2 to 4 minutes has the potential to double the dynamic binding capacity as was shown for some Protein A resins26.
Troubleshooting
The following table highlights potential problems that can be encountered during this protocol and provides hints on how to solve them (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Table 1: Potential problems that can be encountered and possible fixes.</td>
		</tr>
		<tr>
			<td>Protocol step</td>
			<td>Problem</td>
			<td>Couse</td>
			<td>Fix</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Plants do not grow</td>
			<td>Compromised growth conditions</td>
			<td>Check the pH and conductivity of the fertilizer</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Check the temperature and light conditions</td>
		</tr>
		<tr>
			<td>2 and 3</td>
			<td>Large quantities of host cell proteins are present after extraction</td>
			<td>Incomplete precipitation</td>
			<td>Check the temperature during blanching</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Check the agitation in the blanching bath</td>
		</tr>
		<tr>
			<td>2 and 3</td>
			<td>No product found in the plant extract</td>
			<td>Blanching temperature too high</td>
			<td>Check temperature and pH during blanching</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>pH in blanching buffer too low</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>Large stem or leaf parts remain after extraction</td>
			<td>Incomplete mixing in blender</td>
			<td>Make sure the plant material does not form a plug in the blender</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Rapid pressure increase during depth filtration</td>
			<td>Incorrect filter selection and/or orientation</td>
			<td>Check the filter type and orientation</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Little fusion protein during elution / a lot of fusion protein in flow-through</td>
			<td>IMAC resin was not charged with metal ions</td>
			<td>Check if the IMAC resin was correctly charged with ions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Fusion protein lost the affinity tag</td>
			<td>Avoid intense sunlight and high temperatures during plant cultivation</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Fusion protein lost during concentration</td>
			<td>Fusion protein bound to the membrane</td>
			<td>Check the membrane type</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Make sure the concentration factor was not too high</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Low coupling yield</td>
			<td>Incorrect sequence of coupling reagent addition</td>
			<td>Check the reagents labels and sequence of addition</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Incorrect preparation of the columns before coupling</td>
			<td>Check the conditions of column preparaiton</td>
		</tr>
		<tr>
			<td>5 and 6</td>
			<td>Low mAb yield</td>
			<td>Low mAb expression in the plant biomass</td>
			<td>Test mAb expression in biomass</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Low ligand density</td>
			<td>Check the purity of the fusion protein preparation</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Very low/high protein concentrations in Bradford assay</td>
			<td>Bubble formation during pipetting</td>
			<td>Check for bubbles in the 96-well palte</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Low mAb concentration during SPR measurement</td>
			<td>Compromised Protein A chip</td>
			<td>Compare with results of standard mAb with known concentration</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Incorrect sample dilution</td>
			<td>Check the dilution rate and buffer</td>
		</tr>
	</tbody>
</table>
Table 1: Trouble-shooting.

Biopharmaceutical products are important for the treatment of a broad spectrum of diseases in nearly every branch of medicine1. Monoclonal antibodies (mAbs) dominate the biopharmaceutical market, with worldwide sales expected to reach almost &#8364;110 billion in 20202. The favored expression platform for mAbs are Chinese hamster ovary cell lines, which typically produce high mAb titers of up to 10 g&#8729;L-1 in the culture supernatant3,4. However, the production of mAbs in mammalian cell cultures is expensive due to the high cost of the medium and the need for sterile fermentation5. Alternative expression platforms such as plants potentially offer a faster, simpler, less expensive and more scalable approach for industrial manufacturing6,7.
In addition to the costs associated with mammalian cell cultures, the widespread use of Protein A affinity chromatography for product capture is a major cost driver for the industrial production of mAbs. Protein A is naturally found on the surface of Staphylococcus aureus cells and it binds to the fragment crystallizable (Fc) region of certain murine and human antibodies, thereby acting as a defense mechanism to evade the humoral immune system8. Protein A has become the industry gold standard for the capture of mAbs from cell culture supernatants and is also widely used by the research community because it is highly selective, typically achieving mAb purities of ~95% in a single step8. Unsurprisingly, sales of Protein A over the last two decades have closely mirrored the sales of mAbs8. Depending on the production scale, the costs of Protein A can correspond to more than 25% of the total process costs and thereby affect the market price of therapeutic mAbs, which can be up to &#8364;2,000 g-1 5,9. Therefore, alternative chromatography resins with a similar purification performance have the potential to substantially reduce production costs, making antibody-based therapies accessible for a larger number of patients10,11,12. Such alternatives may also circumvent the disadvantages of Protein A chromatography, including the harsh elution conditions at low pH (typically &#60;3.5) that can potentially cause mAbs to undergo conformational changes that promote aggregation13. Importantly, Protein A is selective only for the Fc region of certain IgG subclasses, so non-functional molecules with truncated binding domains may co-purify with the intact product5, whereas mAb derivaties such as single-chain variable fragments do not bind to Protein A at all.
Here, we describe an alternative affinity chromatography resin for the capture of the HIV-neutralizing mAb 2F5 using its linear epitope ELDKWA (one letter amino acid code)5,14. We genetically fused the 2F5 epitope to the C-terminus of the fluorescent protein DsRed, which functioned as a carrier and reporter molecule, and produced the resulting protein DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants. DFE was purified by single-step immobilized metal-ion affinity chromatography (IMAC). The immobilization of the purified DFE affinity ligand onto a cross-linked agarose resin was achieved by chemical coupling using N-hydroxysuccinimide (NHS)-activated cross-linked agarose columns. Statistical experimental designs were then used to optimize the immobilization procedure and coupling efficiency15. The purification strategy for mAb 2F5 was evaluated in terms of antibody purity, yield and ligand stability. In contrast to Protein A, which binds the Fc region, DFE bound to the complementarity-determining region of 2F5, ensuring the purification of molecules with an intact paratope. Our concept can easily be adapted to any mAb with a linear epitope or to other peptide-based affinity ligands which can be easily identified by microarray studies16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59933/59933fig01.jpg" /> 
Figure 1: Process flow scheme for the preparation of epitope affinity resins that can be used for the capture of mAbs from crude plant extracts or cell culture supernatants.&#160;(A) The affinity ligand DFE was expressed in transgenic tobacco plants. A heat precipitation step (B) was included before harvested leaves were homogenized (C). (D) The crude plant extract was clarified by bag filtration, depth filtration and 0.2 &#181;m sterile filtration. (E) DFE was then purified by IMAC. (F, G) The purified DFE affinity ligand was immobilized on EDC/NHS-activated cross-linked agarose columns. (H) Bacterial cultures carrying T-DNA encoding antibody 2F5 were used for transient expression in N. benthamiana plants (I) grown in a phytotron. (J) N. benthamiana leaves were harvested and processed as described in D. (K) mAb 2F5 was purified from the clarified extract using the DFE affinity columns (L). <a href="https://www.jove.com/files/ftp_upload/59933/59933fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>10L/20L Bucket</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Blanching equipment</td>
		</tr>
		<tr>
			<td>2100P Portable Turbidimeter</td>
			<td>Hach</td>
			<td>4650000</td>
			<td>Turbidimeter</td>
		</tr>
		<tr>
			<td>&#196;KTApure</td>
			<td>GE Helthcare</td>
			<td>29018226</td>
			<td>Chromatography system</td>
		</tr>
		<tr>
			<td>Allegra 25R</td>
			<td>Beckman Coulter</td>
			<td>369434</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Amine Coupling Kit</td>
			<td>GE Healthcare</td>
			<td>BR100050</td>
			<td>SPR chip coupling kit</td>
		</tr>
		<tr>
			<td>Amine Coupling Kit</td>
			<td>GE Healthcare</td>
			<td>BR100050</td>
			<td>SPR chip coupling kit</td>
		</tr>
		<tr>
			<td>Antibody 2G12</td>
			<td>Fraunhofer IME</td>
			<td>n/a</td>
			<td>Standard for SPR quantification</td>
		</tr>
		<tr>
			<td>Blender</td>
			<td>Waring</td>
			<td>800EG</td>
			<td>Blender</td>
		</tr>
		<tr>
			<td>BP-410</td>
			<td>Fuhr</td>
			<td>2632410001</td>
			<td>Bag filter</td>
		</tr>
		<tr>
			<td>CanoScan 5600F</td>
			<td>Canon</td>
			<td>2925B009</td>
			<td>Scanner</td>
		</tr>
		<tr>
			<td>Centrifuge tube 50 mL self-standing</td>
			<td>Labomedic</td>
			<td>1110504</td>
			<td>Reaction tube</td>
		</tr>
		<tr>
			<td>Chelating Sepharose FF</td>
			<td>GE Helthcare</td>
			<td>17-0575-01</td>
			<td>Chromatography resin</td>
		</tr>
		<tr>
			<td>Cond 3320</td>
			<td>WTW</td>
			<td>EKA 3338</td>
			<td>Conductometer</td>
		</tr>
		<tr>
			<td>Design-Expert(R) 8</td>
			<td>Stat-Ease, Inc.</td>
			<td>n/a</td>
			<td>DoE software</td>
		</tr>
		<tr>
			<td>Discovery Compfort</td>
			<td>Gilson</td>
			<td>F81029</td>
			<td>Multichannel pipette</td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>Carl Roth GmbH</td>
			<td>4984.3</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Diverse bottles</td>
			<td>Schott Duran</td>
			<td>n/a</td>
			<td>Glas bottles</td>
		</tr>
		<tr>
			<td>Dri Block DB8</td>
			<td>Techne</td>
			<td>Z381373</td>
			<td>Heat block</td>
		</tr>
		<tr>
			<td>DsRed</td>
			<td>Fraunhofe IME</td>
			<td>n/a</td>
			<td>Standart</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth GmbH</td>
			<td>8043.2</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>EnSpire</td>
			<td>Perkin Elmer</td>
			<td>2390-0000</td>
			<td>Plate reader</td>
		</tr>
		<tr>
			<td>ETHG-912</td>
			<td>Oregon Scientific</td>
			<td>086L001499-230</td>
			<td>Thermometer</td>
		</tr>
		<tr>
			<td>F9-C</td>
			<td>GE Helthcare</td>
			<td>29027743</td>
			<td>Fraction collector</td>
		</tr>
		<tr>
			<td>Ferty 2 Mega</td>
			<td>Kammlott</td>
			<td>5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr>
			<td>Forma -86C ULT freezer</td>
			<td>ThermoFisher</td>
			<td>88400</td>
			<td>Freezer</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth GmbH</td>
			<td>9105.3</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Hettich Centrifuge Mikro 200</td>
			<td>Hettich</td>
			<td>2400</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>HiPrep 26/10</td>
			<td>GE Helthcare</td>
			<td>GE17-5087-01</td>
			<td>Chromtography column</td>
		</tr>
		<tr>
			<td>HiTrap NHS-activated Sepharose HP, 1 mL</td>
			<td>GE Helthcare</td>
			<td>17-0716-01</td>
			<td>Chromatography columns</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Carl Roth GmbH</td>
			<td>4625.1</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Carl Roth GmbH</td>
			<td>3899.2</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>K700</td>
			<td>Pall</td>
			<td>5302305</td>
			<td>Depth filter layer</td>
		</tr>
		<tr>
			<td>KM02 basic</td>
			<td>IKA</td>
			<td>n/a</td>
			<td>Magnetic stirrer</td>
		</tr>
		<tr>
			<td>KS50P 60D</td>
			<td>Pall</td>
			<td>B12486</td>
			<td>Depth filter layer</td>
		</tr>
		<tr>
			<td>L/S 24</td>
			<td>Masterflex</td>
			<td>SN-06508-24</td>
			<td>Tubing</td>
		</tr>
		<tr>
			<td>Lauda E300</td>
			<td>Lauda Dr Wobser GmbH</td>
			<td>Z90010</td>
			<td>Immersion circulator</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Carl Roth GmbH</td>
			<td>KK36.2</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Masterflex L/S</td>
			<td>Masterflex</td>
			<td>HV-77921-75</td>
			<td>Peristaltic pump</td>
		</tr>
		<tr>
			<td>Minisart 0.2 &#181;m</td>
			<td>Sartorius</td>
			<td>16534K</td>
			<td>Filter unit</td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow PES bottle top filter</td>
			<td>Thermo Fischer Scientific</td>
			<td>595-4520</td>
			<td>Vacuum filtration of SPR buffers</td>
		</tr>
		<tr>
			<td>Nickel sulphate</td>
			<td>Carl Roth GmbH</td>
			<td>T111.1</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Novex NuPAGE 4-12% BisTris LDS gels</td>
			<td>Invitrogen</td>
			<td>NP0336BOX</td>
			<td>LDS-PAA gels</td>
		</tr>
		<tr>
			<td>Novex X-cell Mini Cell</td>
			<td>Invitrogen</td>
			<td>EI0001</td>
			<td>PAGE chamber</td>
		</tr>
		<tr>
			<td>NuPAGE 20x running buffer</td>
			<td>Invitrogen</td>
			<td>NP0002</td>
			<td>Buffer concentrate</td>
		</tr>
		<tr>
			<td>NuPAGE antioxidant</td>
			<td>Invitrogen</td>
			<td>NP0005</td>
			<td>Antioxidant</td>
		</tr>
		<tr>
			<td>PageRuler protein ladder (10-180 kDa)</td>
			<td>Invitrogen</td>
			<td>26616</td>
			<td>Protein standart</td>
		</tr>
		<tr>
			<td>Perforated bucked</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Blanching</td>
		</tr>
		<tr>
			<td>PH 3110</td>
			<td>WTW</td>
			<td>2AA110</td>
			<td>PH meter</td>
		</tr>
		<tr>
			<td>PowerPac HC</td>
			<td>Biorad</td>
			<td>1645052</td>
			<td>Electrophoresis module</td>
		</tr>
		<tr>
			<td>Protein A from Staphylococcus aureus</td>
			<td>Sigma-Aldrich</td>
			<td>P7837-5MG</td>
			<td>Coating of SPR chips</td>
		</tr>
		<tr>
			<td>Sephadex G-25 fine, cross linked dextran</td>
			<td>GE Helthcare</td>
			<td>17003301</td>
			<td>Chromatography resin</td>
		</tr>
		<tr>
			<td>Silicone spoon</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Spoon</td>
		</tr>
		<tr>
			<td>Simply Blue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>Gel staining solution</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Carl Roth GmbH</td>
			<td>6773.1</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Carl Roth GmbH</td>
			<td>X891.1</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>S2002-100G</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth GmbH</td>
			<td>P029.2</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Carl Roth GmbH</td>
			<td>HN13.2</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Sodium bisulfite</td>
			<td>Carl Roth GmbH</td>
			<td>243973-100G</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Sodium phosophate</td>
			<td>Carl Roth GmbH</td>
			<td>T877.2</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>SPR Affinity Sensor - High Capacity Amine</td>
			<td>Sierra Sensors GmbH/Bruker Daltonics</td>
			<td>SPR-AS-HCA</td>
			<td>SPR chip</td>
		</tr>
		<tr>
			<td>SPR-2/4 Surface Plasmon Resonance Analyzer</td>
			<td>Sierra Sensors GmbH/Bruker Daltonics</td>
			<td>n/a</td>
			<td>SPR device</td>
		</tr>
		<tr>
			<td>SSM3</td>
			<td>Stuart</td>
			<td>10034264</td>
			<td>Mini Gyro-rocker</td>
		</tr>
		<tr>
			<td>Heated vessel, 20 L</td>
			<td>Clatronic</td>
			<td>n/a</td>
			<td>Blanching chamber</td>
		</tr>
		<tr>
			<td>Sterile syringes, 2 mL</td>
			<td>B. Braun</td>
			<td>4606027V</td>
			<td>Syringes</td>
		</tr>
		<tr>
			<td>Syringe adpter (Union Luer F)</td>
			<td>GE Helthcare</td>
			<td>181112-51</td>
			<td>Syringe adapter</td>
		</tr>
		<tr>
			<td>TE6101</td>
			<td>Sartorius</td>
			<td>TE6101</td>
			<td>Precision scale</td>
		</tr>
		<tr>
			<td>Tween-20 (Polysorbate)</td>
			<td>Merck</td>
			<td>8170721000</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Unicorn 6.4</td>
			<td>GE Helthcare</td>
			<td>29056102</td>
			<td>Chromatography software</td>
		</tr>
		<tr>
			<td>Vacuum bags</td>
			<td>Ikea</td>
			<td>203.392.84</td>
			<td>Plant storge</td>
		</tr>
		<tr>
			<td>VelaPad 60</td>
			<td>Pall</td>
			<td>VP60G03KNH4</td>
			<td>Filter housing</td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific industries</td>
			<td>SI-0236</td>
			<td>Vortex</td>
		</tr>
		<tr>
			<td>XK-26/20 column housing</td>
			<td>GE Helthcare</td>
			<td>28-9889-48</td>
			<td>Chromtography column</td>
		</tr>
	</tbody>
</table>

activated cross-linked agarose for the rapid development of affinity chromatography resins - antibody capture as a case study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

Expression and purification of the affinity ligand
The fusion protein DFE was expressed in transgenic tobacco plants grown in a greenhouse. The yield was ~120 mg&#183;kg-1 leaf mass with an average biomass of ~130 g per plant. The DFE purity was &#60;5% of TSP in crude plant extracts before blanching but increased to ~40% after heat treatment at 70 &#176;C for 1.5 min, which precipitated &#62;97% of the host cell proteins. The blanching step was easily integrated into the harvesting and extraction routine (Figure 1) and took less than 2 h of extra time, including setting up the water bath. The overall recovery of DFE was 23.5 mg kg1 with a purity of &#62;90%. The steps responsible for product loss were blanching, depth filtration and IMAC, with specific losses of 40%, 27% and 45%, respectively. The depth filter capacity was on average 135 &#177; 36 L m-2 (&#177;SD, n=3) and thus in the upper range of values reported in the literature21. The DFE yield increased with plant age (Figure 2).
Immobilization of the affinity ligand on NHS-activated chromatography columns
During initial coupling tests, we found that HEPES buffer (pH 8.3) increased the coupling efficiency to 89 &#177; 6% (&#177;SD, n=3) compared to 78 &#177; 9% (&#177;SD, n=3) for the bicarbonate buffer recommended by the manufacturer. Therefore, HEPES was used for all subsequent coupling experiments. A DoE approach was selected to optimize the coupling efficiency of DFE to NHS-activated cross-linked agarose resin. The absolute amount of DFE immobilized on the resin increased with the mass of DFE injected into the column and plateaued at ~15 g&#183;L-1 whereas the coupling yield declined continuously as more DFE was injected (Figure 2). The coupling yield was also &#62;50% lower in an acidic buffer, indicating the need to screen for suitable coupling conditions for each ligand on a case-by-case basis. Ideal conditions in terms of coupling yield, absolute quantity of immobilized DFE and column costs were identified using the numerical optimization tool of the DoE software. The most desirable conditions (pH 9.0 and 7.0 mg of DFE per 1 mL of cross-linked agarose resin) were located on a large plateau and were therefore robust. The DFE molecules retained their red fluorescence even after coupling, and the color intensity corresponded to the total amount of immobilized DFE (Figure 2). Therefore, column color can be used as a simple quality control parameter to estimate the coupling efficiency and column quality. The fluorescence also confirmed that DFE fusion protein assembled in the tetrameric state of native DsRed.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59933/59933fig02.jpg" /> 
Figure 2: Optimization of DFE immobilization to NHS-activated cross-linked agarose resin.&#160;(A) LDS-PAA gel with western blot overlay of homogenate and elution samples from unblanched and blanched transgenic DFE plant extracts. Harvest of plants was performed 38, 45 or 52 days after seeding. Western blots were performed using an anti-His6-antibodies5. (B) Total amount of coupled DFE affinity ligand in dependence if coupling pH and overall mass of purified DFE injected onto NHS-activated cross-linked agarose columns. Red dots indicate the actual experiments performed to build the response surface model. (C) DFE affinity columns after the coupling procedure. The numbers correspond to the coupling conditions highlighted in panel B. dps = days post seeding. <a href="https://www.jove.com/files/ftp_upload/59933/59933fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Testing 2F5 isolation using the DFE affinity resin
The recombinant 2F5 antibody was transiently produced in Nicotiana benthamiana plants grown in a phytotron5. The capture of 2F5 from the crude plant extract was tested using affinity columns coupled with ~7.0 mg purified DFE (step 6). Elution from Protein A resins usually involves an acidic buffer (pH ~3.3)13. Therefore, we initially evaluated different low-pH elution buffers (pH 6.0&#8211;3.25) for the DFE columns. The elution of 2F5 was successful at pH values below 4.5 with the highest recovery of ~35% at pH 3.25. However, low-pH elution inactivated both the antibody (as confirmed by SPR spectrometry) and the DFE ligand (as indicated by the loss of color and the lower DBC, Figure 3). The latter was anticipated given that native DsRed denatures at pH &#60;4.022,23. To avoid product and ligand denaturation, we tested magnesium chloride as an alternative elution agent because it has previously been used to elute mAbs from other affinity resins24. A magnesium chloride concentration of 1.25 M was sufficient to elute 2F5 from the DFE affinity resin with a recovery of 105 &#177; 11% (&#177;SD, n=3) and a purity of 97 &#177; 3% (&#177;SD, n=3). This performance was comparable to Protein A resins25,26. The equilibrium dissociation constant (KD) of DFE-purified 2F5 antibody and the synthetic ligand Fuzeon was 791 pM whereas that of a Protein A-purified counterpart was 763 pM5. Furthermore, no substantial color loss was observed in the resin over a total of 25 bind-and-elute cycles. The DBC of the DFE affinity resin at 10% 2F5 breakthrough declined linearly over the course of 25 cycles to ~15% of the initial value (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59933/59933fig03.jpg" /> 
Figure 3: Testing the isolation of 2F5 from clarified plant extracts using the DFE affinity resins.&#160;(A) DFE affinity resins after one elution cycle using buffers with different pH values in the range 4.0&#8211;3.0 and the same resins after a neutralization step at pH 6.0. (B) DFE affinity resins after one and six cycles of 2F5 purification using 1.25 M or 4.00 M magnesium chloride as eluent. (C) Chromatograms of frontal loading experiments (break-through curves) to determine the cycle-dependent dynamic binding capacity of DFE resin using magnesium chloride as eluent. The break-through curves were measured for 4.0 M and 1.25 M magnesium chloride elution buffers and various cycle numbers. (D) Cycle-dependent dynamic binding capacity of DFE using 1.25 M magnesium chloride as eluent. <a href="https://www.jove.com/files/ftp_upload/59933/59933fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study at JoVE.com

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

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Research

JoVE Journal

Biochemistry

9.1K Views.  RWTH Aachen University. In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

Video: Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...