New platform for simple and rapid protein-based affinity reactions

We developed a spongy-like porous polymer (spongy monolith) consisting of poly(ethylene-co-glycidyl methacrylate) with continuous macropores that allowed efficient in situ reaction between the epoxy groups and proteins of interest. Immobilization of protein A on the spongy monolith enabled high-yield collection of immunoglobulin G (IgG) from cell culture supernatant even at a high flow rate. In addition, immobilization of pepsin on the spongy monolith enabled efficient online digestion at a high flow rate.

porosimeter ( Supplementary Fig. 1), whereas no meso-pores were detected by nitrogen-gas adsorption analysis. The PEGM-SpM was packed into a stainless-steel column by a simple method ( Supplementary Fig. 2) similar to that used in our previous study. After the column was conditioned with methanol and water, a protein A solution (1.0 mg mL −1 in PBS) was passed through the column, and then incubated at 37 °C for 16 h after both ends of the column were sealed. No morphological alteration was observed following the protein A modification ( Supplementary Fig. 3). To confirm the effect of the modification, we then analyzed the column (ProA-SpM) by liquid chromatography (LC). As shown in Fig. 2.(a), IgG-1 was strongly retained on the original PEGM-SpM via hydrophobic interaction in a typical reversed-phase LC (RPLC) mode. On the other hand, the ProA-SpM exhibited significantly less hydrophobic interaction, resulting in faster elution of IgG-1. A similar phenomenon was observed when BSA was used as a solute ( Supplementary Fig. 4). These results indicated that protein A effectively covered the skeleton surface of the monolith, dramatically suppressing non-selective hydrophobicity. Next, we confirmed the affinity of the ProA-SpM via simple pH-gradient LC, which is commonly employed to evaluate protein A-immobilized columns because the interaction between protein A and IgG occurs only at pH >7. The resultant chromatograms are summarized in Fig. 2.(b). As expected, IgG-1 was selectively retained on the ProA-SpM and released by a one-step pH gradient. By contrast, BSA was quickly eluted without any retention,  and PEGM-SpM adsorbed IgG-1 due to its high hydrophobicity. Additionally, another IgG family member, IgG-2, was also effectively separated on the ProA-SpM ( Supplementary Fig. 5). In a simplified validation, we injected various amounts of IgG-1 into the ProA-SpM, and found that the linear range of peak area was at least 1.0-250 µg ( Fig. 2.(c)). In addition, we evaluated the accuracy by continuous analyses (n = 6), and estimated the relative standard deviation (RSD) as 0.7%. According to these results, this simply prepared ProA-SpM column had affinity similar to that of commercially available protein A-immobilized columns.
To be most useful, an affinity column must have abundant adsorption capacity for the ligand(s) of interest. To evaluate the maximum adsorption capacity due to immobilized protein A, we performed a frontal analysis, a method commonly utilized to evaluate capacity by LC 34,35 , of both our column and a commercially available protein A-immobilized column (ProA-Column). This analysis revealed that the densities of immobilized protein A in the ProA-SpM and ProA-Column were 1.0-4.2 nmol g −1 and 5-21 nmol g −1 , respectively ( Supplementary Fig. 6). Although the density of immobilized protein A was slightly lower in the ProA-SpM, the adsorption capacity for IgG-1 was comparable between the two columns (0.31 mg for the ProA-SpM and 0.32 mg for the ProA-Column). Therefore, our ProA-SpM had sufficient capacity to serve as an affinity column for the effective separation of IgG.
The most important advantage of the spongy monolith is its potential for high-throughput elution. To evaluate this feature, we carried out a similar affinity separation using IgG-1 as the solute under various flow rate conditions; the results are summarized in Fig. 3. When the ProA-Column was utilized at a higher flow rate, the flow-through fraction was presented in front of the solvent peak, as shown in Fig. 3(a). By contrast, the ProA-SpM allowed higher recovery, even at a high flow rate. Figure 3(c) shows the chromatograms for both the ProA-Column and ProA-SpM at a flow rate of 9.0 mL min −1 . Obviously, the collected and flow-through peaks were completely different from each other. The backpressure and recovery of IgG on the columns at each flow rate are summarized in Fig. 4(a) and (b), respectively. For the ProA-SpM, both backpressure and recovery were superior to those of the ProA-Column. A potential reason for these significant differences, especially in recovery at a high flow rate, is that the affinity interaction between a protein-based ligand and an antibody under a higher flow rate is generally not effective in a column in which spherical and porous beads are packed, due to the lower accessibility caused by slower mass transfer. Usually, in an LC analysis, the van Deemter equation (1) 36 is employed  to determine the plate height, H, which is defined as diffusion per column length and directly contributes to the separation efficiency: Here, d P , D m , and u are the diameter of the packed particle, diffusion coefficient of the solute, and linear velocity, respectively, and A, B, and C are constants corresponding to eddy diffusion, longitudinal diffusion, and mass transfer, respectively. Under higher linear velocity, mass transfer is usually predominant, resulting in low separation efficiency. For interactions among macromolecules (e.g., protein-protein interactions), slow mass transfer may provide fewer chances for encounter; thus, most of the IgG was eluted without interaction at high flow rates on the ProA-Column. On the other hand, the spongy monolith contains only macro-size flow-through pores, and protein A should be immobilized only on the surface of the monolithic skeleton. Therefore, we anticipated that the ProA-SpM would allow effective interaction between protein A and IgG-1. Additionally, the linearity of recovery at a higher flow rate (9.0 mL min −1 ) is similar to that at a lower flow rate ( Supplementary Fig. 7). Furthermore, we investigated the ruggedness of the ProA-SpM. As a result of 100 times repeated analyses with IgG under 9.0 mL min −1 , the RSDs of the retention time and recovery of IgG were estimated as 0.45% and 0.41%, respectively. Also, the recovery of IgG was kept over 99% even after washing with 0.1 M aqueous NaOH (5 times), which is commonly used as an evaluation for the ruggedness of affinity columns. These results suggested that the ProA-SpM had enough ruggedness as an affinity column. According to these results, we anticipate that the ProA-SpM could be used as a novel affinity separation medium for high-throughput purifications.
To demonstrate purification of IgG from cell culture samples, we used the ProA-SpM. Cell culture supernatant treated using typical procedures was separated with a simple pH gradient using a variety of flow rates. Then, the peak likely to contain IgG was manually fractionated, and the chromatograms of free supernatant and a standard IgG are shown in Fig. 5(a). To confirm the presence of IgG in the fraction, the collected sample was analyzed by authentic RPLC with time-of-flight (TOF) mass spectrometry (MS) (TOF-MS), which is usually employed for the proteins separation using a reversed-phase column with MS detection. As shown in the obtained chromatograms ( Supplementary Fig. 8) generated by UV detection, both the supernatant and the first fraction contained a major peak and a few minor peaks, whereas the collected fraction clearly contained only one peak, which corresponded to the standard IgG peak. A comparison of total ion chromatograms is provided in Fig. 5(b). Similar to the UV chromatograms, the collected sample exhibited a clear peak without any other minor peaks. After deconvolution of the original MS results, all the peaks from the original supernatant could be assigned, and the observed MS numbers are summarized in Table 1. In addition, the MS spectra of each peak are described in Supplementary  Fig. 9. These results indicate that the collected fraction contained the selectively separated IgG moiety from the natural cell culture sample. Finally, the concentration of IgG in the supernatant of the cell culture was estimated as 0.42 mg mL −1 . Thus, we successfully demonstrated affinity separation based on the protein A-IgG interaction at high throughput using the newly developed spongy monolith. The method exhibited a good reproducibility and efficient recovery over a wide range of concentrations.
As mentioned above, the newly developed spongy monolith was suitable for a high-throughput affinity reaction. Because the immobilization of the proteins was based on a simple reaction with epoxy groups in the monolith, we believe that this material could be used for a variety of protein-based reactions. To explore this idea further, we carried out high-throughput online digestion using a digestive enzyme, pepsin. The immobilization of pepsin was performed successfully by a method similar to the one used for protein A. Pepsin is an aspartic protease that cleaves peptide bonds between hydrophobic and preferably aromatic amino acids, such as phenylalanine, tryptophan, and tyrosine. In this evaluation, an antibody solution was introduced into the pepsin-immobilized spongy monolith (Pep-SpM), and the eluted fraction was analyzed by LC-MS. For comparison, samples in a simple solution containing pepsin and the antibody were also analyzed to confirm the cleaved peptides. The UV chromatograms are summarized in Fig. 6. As expected, longer reaction in solution yielded larger peptide fragments. On the contrary, in online digestion with the Pep-SpM, the peptide fragments were much larger, even though a faster flow rate (100 mL h −1 ) was employed. When a slower flow rate (10 mL h −1 ) was used, the detected peaks were almost the same as those in solution samples treated for 150 min. The numbers of peptides detected in LC-MS, as a function of the number of amino acids and elution time, are summarized in Fig. 7. Both figures demonstrate that a slower flow allowed for more extensive digestion. These results clearly showed that effective cleavage occurred in the Pep-SpM. To confirm the sequence of each peptide, a quantitative analysis was also carried out. The theoretical alignment of amino acids in the antibody and the theoretical pepsin digestion fragments (digestion sites: N terminal, F, I, M, Y, W, V; C terminal, C, D, E, F, L, M, T, W, Y) were compared against results generated by the PepFinder 2.0 based on the LC-MS data. All assignment data are summarized in Supplementary Table 1. Coverage for the primary amino acid alignment, as determined from those results, is shown in Table 2. In both elution flows, all 213 residues in the light chain were detected. Additionally, coverage of the heavy chain corresponded to the flow speed; i.e., a slower flow provided higher coverage. These results also supported the idea that efficient online digestion occurred in the Pep-SpM. Finally, we showed that the reproducibility of the online digestion was satisfactory at both flow rates, as shown in Fig. 8 and Supplementary  Fig. 10. All these results indicate that the spongy monolith can be used for the effective online digestion. 6.14 - Table 1. Peak identifications from a protein A load sample. Fractions 1 and 2 were collected from the separation ( Fig. 1(j)) from the front and back peaks, respectively. The peaks are corresponding to Fig. 1(k). The observed mass was estimated by a deconvolution of multi ions with MaxEnt1.

Conclusion
In summary, we proposed a new platform for protein-based affinity reaction. A spongy monolith containing epoxy groups was effectively used in affinity separation with protein A and digestion with pepsin. Both results demonstrated the utility of the new platform for rapid-flow affinity reactions. We believe that this new platform will be useful for variety of protein-based reactions with rapid flow rates and low costs. Additionally, the platform can be easily scaled up, and we anticipate that future efforts will contribute to purification of antibody-based medicines at the plant level.

Methods
Preparation of a spongy monolith. 35 w% of poly(ethylene-co-glycidyl methacrylate) (PEGM), in which glycidyl methacrylate of 8% is contained, 52 w% of pore templates (pentaerythritol), whose particle size in diameter was classified around 10 μm, and 7 w% of auxiliary of pore templates (poly(oxyethylene, oxypropylene))    triol were melted at 130 °C and homogeneously kneading. The resulting material was extruded as a columnar shape at 130 °C. The columnar material was immediately cooled in water to obtain the stick like material. After cooling, the material was washed in water under an ultrasonication to remove water-soluble compounds. At this stage, water-soluble compounds functioned as the pore templates. The porosity of the spongy monolith calculated by a void volume on LC was 65% and the diameter of its cross section across its entire length was 4.8 mm. (PEGM-SpM).
Packing of a spongy monolith. For packing spongy monoliths in a stainless steel column, we utilized an empty column with an internal diameter of 4.6 mm ( Supplementary Fig. 2). The diameter of the spongy monolithic column was greater than the internal diameter of the empty column (4.6 mm). Nevertheless, the elasticity of the spongy monolith material facilitated the packing. The procedure for packing was as follows: One end of the spongy monolith was compressed with a thermal shrinkage tube at 120 °C. After cooling, the shrinkage tube was removed; and the diameter of the compressed end of the spongy monolith was reduced less than 4.6 mm. After macerating the spongy monolith into ethylene glycol as a lubricity agent, the shrunk portion of the spongy monolith was inserted into the empty column and pulled from the other end, until the non-shrunk portion completely filled the column. Finally, the excess portion of the spongy monolith was cut and the column end module was connected. At this point, the shrunken end of the spongy monolith was completely cut and only the portion of the material with the initial diameter was packed into the column. Then, the prepared column was connected to a pump of LC for continuous elution. The mixture of methanol/water was eluted to the column for further washing to remove the pore templates and the homogenization of the packing 37, 38 condition. Fractionation and determination of IgG from cell culture. To know the possibility for the affinity separation with the ProA-SpM, the real sample was utilized for the separation. Protein A load sample, which was obtained just by simple filtration with membrane filter (0.2 μm) to remove the cells, was directly injected into the ProA-SpM with the same conditions as above. The peak of the seemed to IgG was manually collected (Fraction 2) and the flow through fraction was also collected (Fraction 1). Both fractions, the original supernatant, and a standard IgG were analyzed by LC with a TOF-mass spectrometer. For the intact-MS analysis, the samples were separated with RPLC using an LC-20 Prominence XR (Shimadzu) employing an Aeris Widepore XB-C8 300 Å 2.1 × 100 mm, 3.6 μm column (Phenomenex). The mobile phase A was water/TFA (1000/1) and  Fig. 2(a) using UV detection. For LC-MS analyses, instead mobile phase condition was follows; 0.1 vol% TFA in water as mobile phase A and 0.1 vol% TFA in 90% MeCN aqueous, 0 to 43% B for 5 to 120 min, 100% B for 120.1 to 135 min, which is corresponding to Fig. 2(b,c)  equipped with an electrospray ion source set in the positive ion mode for the m/z of 300 to 2000. MS/MS fragmentation analysis was conducted by using following conditions: the parent ions were fragmented with HCD at the isolation width of 6.0 Da and the collision energy of 35 V. Supplementary Table 1 indicates all the peptides detected by LC-MS containing the origin (light or heavy chain), the number of amino acid of each terminal, and the length. Here, the number of amino acid was assigned that the number 1 is first amino acid from the N-terminal, and the total number of amino acids are 213 and 449 in light chain and heavy chain 41,42 , respectively.