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 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.
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 €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∙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 €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 <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.
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. (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 µm 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). Please click here to view a larger version of this figure.
1. Cultivate the Transgenic Tobacco Plants
NOTE: The design of the DFE fusion protein and the generation of transgenic plants are described elsewhere5,17.
2. Heat Precipitation of Host Cell Proteins
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.
4. Purification of the DFE Affinity Ligand
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.
6. Testing the Purification of mAbs from Clarified Plants Extracts
7. Sample Analysis
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·kg-1 leaf mass with an average biomass of ~130 g per plant. The DFE purity was <5% of TSP in crude plant extracts before blanching but increased to ~40% after heat treatment at 70 °C for 1.5 min, which precipitated >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 >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 ± 36 L m-2 (±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 ± 6% (±SD, n=3) compared to 78 ± 9% (±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·L-1 whereas the coupling yield declined continuously as more DFE was injected (Figure 2). The coupling yield was also >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.
Figure 2: Optimization of DFE immobilization to NHS-activated cross-linked agarose resin. (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. Please click here to view a larger version of this figure.
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 6.0–3.25) 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 <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 ± 11% (±SD, n=3) and a purity of 97 ± 3% (±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).
Figure 3: Testing the isolation of 2F5 from clarified plant extracts using the DFE affinity resins. (A) DFE affinity resins after one elution cycle using buffers with different pH values in the range 4.0–3.0 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. Please click here to view a larger version of this figure.
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–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 β-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·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–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 ± 11% (±SD, n=3) and a purity of 97 ± 3% (±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·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·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·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 1: Potential problems that can be encountered and possible fixes. | |||
Protocol step | Problem | Couse | Fix |
1 | Plants do not grow | Compromised growth conditions | Check the pH and conductivity of the fertilizer |
Check the temperature and light conditions | |||
2 and 3 | Large quantities of host cell proteins are present after extraction | Incomplete precipitation | Check the temperature during blanching |
Check the agitation in the blanching bath | |||
2 and 3 | No product found in the plant extract | Blanching temperature too high | Check temperature and pH during blanching |
pH in blanching buffer too low | |||
3 | Large stem or leaf parts remain after extraction | Incomplete mixing in blender | Make sure the plant material does not form a plug in the blender |
3 | Rapid pressure increase during depth filtration | Incorrect filter selection and/or orientation | Check the filter type and orientation |
4 | Little fusion protein during elution / a lot of fusion protein in flow-through | IMAC resin was not charged with metal ions | Check if the IMAC resin was correctly charged with ions |
Fusion protein lost the affinity tag | Avoid intense sunlight and high temperatures during plant cultivation | ||
4 | Fusion protein lost during concentration | Fusion protein bound to the membrane | Check the membrane type |
Make sure the concentration factor was not too high | |||
5 | Low coupling yield | Incorrect sequence of coupling reagent addition | Check the reagents labels and sequence of addition |
Incorrect preparation of the columns before coupling | Check the conditions of column preparaiton | ||
5 and 6 | Low mAb yield | Low mAb expression in the plant biomass | Test mAb expression in biomass |
Low ligand density | Check the purity of the fusion protein preparation | ||
7 | Very low/high protein concentrations in Bradford assay | Bubble formation during pipetting | Check for bubbles in the 96-well palte |
7 | Low mAb concentration during SPR measurement | Compromised Protein A chip | Compare with results of standard mAb with known concentration |
Incorrect sample dilution | Check the dilution rate and buffer |
Table 1: Trouble-shooting.
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 “Networked, adaptive production”. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) in the framework of the Research Training Group “Tumor-targeted Drug Delivery” grant 331065168. GE healthcare supported the open-access publication of this article.
Name | Company | Catalog Number | Comments |
10L/20L Bucket | n/a | n/a | Blanching equipment |
2100P Portable Turbidimeter | Hach | 4650000 | Turbidimeter |
ÄKTApure | GE Helthcare | 29018226 | Chromatography system |
Allegra 25R | Beckman Coulter | 369434 | Centrifuge |
Amine Coupling Kit | GE Healthcare | BR100050 | SPR chip coupling kit |
Amine Coupling Kit | GE Healthcare | BR100050 | SPR chip coupling kit |
Antibody 2G12 | Fraunhofer IME | n/a | Standard for SPR quantification |
Blender | Waring | 800EG | Blender |
BP-410 | Fuhr | 2632410001 | Bag filter |
CanoScan 5600F | Canon | 2925B009 | Scanner |
Centrifuge tube 50 mL self-standing | Labomedic | 1110504 | Reaction tube |
Chelating Sepharose FF | GE Helthcare | 17-0575-01 | Chromatography resin |
Cond 3320 | WTW | EKA 3338 | Conductometer |
Design-Expert(R) 8 | Stat-Ease, Inc. | n/a | DoE software |
Discovery Compfort | Gilson | F81029 | Multichannel pipette |
Disodium phosphate | Carl Roth GmbH | 4984.3 | Media component |
Diverse bottles | Schott Duran | n/a | Glas bottles |
Dri Block DB8 | Techne | Z381373 | Heat block |
DsRed | Fraunhofe IME | n/a | Standart |
EDTA | Carl Roth GmbH | 8043.2 | Buffer component |
EnSpire | Perkin Elmer | 2390-0000 | Plate reader |
ETHG-912 | Oregon Scientific | 086L001499-230 | Thermometer |
F9-C | GE Helthcare | 29027743 | Fraction collector |
Ferty 2 Mega | Kammlott | 5.220072 | Fertilizer |
Forma -86C ULT freezer | ThermoFisher | 88400 | Freezer |
HEPES | Carl Roth GmbH | 9105.3 | Buffer component |
Hettich Centrifuge Mikro 200 | Hettich | 2400 | Centrifuge |
HiPrep 26/10 | GE Helthcare | GE17-5087-01 | Chromtography column |
HiTrap NHS-activated Sepharose HP, 1 mL | GE Helthcare | 17-0716-01 | Chromatography columns |
Hydrochloric acid | Carl Roth GmbH | 4625.1 | Buffer component |
Imidazole | Carl Roth GmbH | 3899.2 | Buffer component |
K700 | Pall | 5302305 | Depth filter layer |
KM02 basic | IKA | n/a | Magnetic stirrer |
KS50P 60D | Pall | B12486 | Depth filter layer |
L/S 24 | Masterflex | SN-06508-24 | Tubing |
Lauda E300 | Lauda Dr Wobser GmbH | Z90010 | Immersion circulator |
Magnesium chloride | Carl Roth GmbH | KK36.2 | Buffer component |
Masterflex L/S | Masterflex | HV-77921-75 | Peristaltic pump |
Minisart 0.2 µm | Sartorius | 16534K | Filter unit |
Nalgene Rapid-Flow PES bottle top filter | Thermo Fischer Scientific | 595-4520 | Vacuum filtration of SPR buffers |
Nickel sulphate | Carl Roth GmbH | T111.1 | Buffer component |
Novex NuPAGE 4-12% BisTris LDS gels | Invitrogen | NP0336BOX | LDS-PAA gels |
Novex X-cell Mini Cell | Invitrogen | EI0001 | PAGE chamber |
NuPAGE 20x running buffer | Invitrogen | NP0002 | Buffer concentrate |
NuPAGE antioxidant | Invitrogen | NP0005 | Antioxidant |
PageRuler protein ladder (10-180 kDa) | Invitrogen | 26616 | Protein standart |
Perforated bucked | n/a | n/a | Blanching |
PH 3110 | WTW | 2AA110 | PH meter |
PowerPac HC | Biorad | 1645052 | Electrophoresis module |
Protein A from Staphylococcus aureus | Sigma-Aldrich | P7837-5MG | Coating of SPR chips |
Sephadex G-25 fine, cross linked dextran | GE Helthcare | 17003301 | Chromatography resin |
Silicone spoon | n/a | n/a | Spoon |
Simply Blue SafeStain | Invitrogen | LC6060 | Gel staining solution |
Sodium acetate | Carl Roth GmbH | 6773.1 | Buffer component |
Sodium acetate | Carl Roth GmbH | X891.1 | Media component |
Sodium azide | Sigma Aldrich | S2002-100G | Media component |
Sodium chloride | Carl Roth GmbH | P029.2 | Buffer component |
Sodium citrate | Carl Roth GmbH | HN13.2 | Buffer component |
Sodium bisulfite | Carl Roth GmbH | 243973-100G | Media component |
Sodium phosophate | Carl Roth GmbH | T877.2 | Media component |
SPR Affinity Sensor - High Capacity Amine | Sierra Sensors GmbH/Bruker Daltonics | SPR-AS-HCA | SPR chip |
SPR-2/4 Surface Plasmon Resonance Analyzer | Sierra Sensors GmbH/Bruker Daltonics | n/a | SPR device |
SSM3 | Stuart | 10034264 | Mini Gyro-rocker |
Heated vessel, 20 L | Clatronic | n/a | Blanching chamber |
Sterile syringes, 2 mL | B. Braun | 4606027V | Syringes |
Syringe adpter (Union Luer F) | GE Helthcare | 181112-51 | Syringe adapter |
TE6101 | Sartorius | TE6101 | Precision scale |
Tween-20 (Polysorbate) | Merck | 8170721000 | Buffer component |
Unicorn 6.4 | GE Helthcare | 29056102 | Chromatography software |
Vacuum bags | Ikea | 203.392.84 | Plant storge |
VelaPad 60 | Pall | VP60G03KNH4 | Filter housing |
Vortex-Genie 2 | Scientific industries | SI-0236 | Vortex |
XK-26/20 column housing | GE Helthcare | 28-9889-48 | Chromtography column |
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