In particular, protein folding is known to occur on the order of microseconds to seconds , while atomistic protein simulation is generally limited to hundreds of nanoseconds due to limited computing resources.The length-scale of atomistic protein simulations is also computationally restricted, allowing only one rCMG2-Fc dimer to be simulated. Despite these limitations, this work shows MD simulation data is capable of providing insight into the effects of glycosylation on protein structure, and improving our understanding and interpretation of experimental observations. To the best of our knowledge, no study has been conducted on Fc-fusion protein considering that many experimental and molecular simulation factors. This study provides an integrated experimental and computational approach to evaluate Fc N-glycosylation impacts on rCMG2-Fc properties, and potentially serves as a guideline for general glyco protein based therapeutic design, especially for Fc-fusion proteins.The codon optimized CMG2-Fc sequence includes the extracellular domain of CMG2 , followed by two serine residues, the upper hinge of IgG2 , and Fc region of human IgG1 . The resulting sequence corresponds to the APO variant as described previously . A SEKDEL C-terminal motif was included to make the ER variant; a point mutation of N268Q on Fc was included to make the Agly variant. The genes encoding rCMG2-Fc variants were codon-optimized for expression in Nicotiana benthamiana. The full construct consists of the CaMV 35S promoter, Ω leader sequence, gene encoding the Ramy3D signal peptide,vertical growing systems followed by rCMG2-Fc gene and octopine synthase terminator . Agrobacterium tumefaciens EHA105 with the helper plasmid was transfected with the resulting binary expression vectors separately via electroporation.

A binary vector capable of expressing P19 to suppress RNAi-mediated gene silencing in Nicotiana benthamiana plants was co-infiltrated with the rCMG2-Fc-APO binary vector as previously described .Plant tissue was collected at day 6 after infiltration. To evaluate the average expression level, leaves from 10 plants were collected and stored at −80°C prior to extraction. Leaves were ground to fine powder using mortar and pestle with liquid nitrogen. The leaf powder was weighted and mixed with extraction buffer at a leaf mass to buffer volume ratio of 1:7. The mixture was incubated on a shaker at 4°C for 1 h and then centrifuged at 1,800g at 4°C for 1 h, followed by 0.22 μm filtration to remove insoluble particles. Filtered plant extract was loaded to protein A column and eluted with glycine-HCl buffer at pH of 3.0. Purified protein was immediately titrated to neutral pH with 1 M tris buffer, and buffer exchanged to 1X PBS through overnight dialysis at 4°C.Expression of rCMG2-Fc in crude plant extract was quantified by a sandwich ELISA. First, ELISA microplate wells were coated with Protein A at a concentration of 50 μg/ml in 1X PBS buffer for 1 h, followed with plate blocking with 5% nonfat milk in 1X PBS buffer for 20 min. Crude plant extracts and purified standards were loaded to the plate and incubated from 1 h . The bound rCMG2-Fc was detected by incubating a horseradish peroxidase -conjugated goat antihuman IgG at a concentration of 0.5 μg/ml for 1 h. Plates were washed three times with 1X PBST between each of these steps. All incubation steps were done at 37°C, with an incubation volume of 50 μl. Next, 100 μl of ELISA colorimetric TMB substrate was added to each well and incubated for 10 min, followed by the addition of 100 μl of 1 N HCl to stop the reaction. The absorbance at 450 nm was measured with a microplate reader . The absorbance of protein standard was plotted as a function of rCMG2-Fc concentration, and was fitted to the 4-parameter model in SoftMax Pro software. The concentration of rCMG2-Fc in crude plant extract was determined by interpolating from the linear region of the standard curve. SDS-PAGE and Western blot analyses were performed on purified rCMG2-Fc variants. Protein was denatured and reduced by treating samples at 95°C for 5 min with 5% of 2-mercaptoethanol . For nonreducing SDS-PAGE, samples were denatured by heat treatment at 95°C for 5 min. Samples were loaded to precast 4–20% SDS-Tris HCl polyacrylamide gels , running at 200 V for 35 min. For SDS-PAGE, the gel was washed three times with water and stained with Coomassie Brilliant Blue R-250 Staining Solution . For Western blot analysis, samples were transferred to a nitrocellulose membrane by electrophoretic transfer using the iBlot Gel Transfer Device . For Western blot detecting the CMG2 domain, the membrane was probed with a goat anti-CMG2 polyclonal antibody at a concentration of 0.3 μg/ml, followed by incubation of a polyclonal AP-conjugated rabbit anti-goat IgG antibody at 1:10,000 dilution.

For Western blot detecting the Fc domain, the membrane was incubated with a polyclonal AP-conjugated goat anti-human IgG antibody at 1:3,000 dilution. The blots were developed using SIGMAFAST BCIP/NBT according to the product instruction.CMG2-Fc standard from Planet Biotechnology and rCMG2-Fc variants were reduced, denatured, and run on a 4–12% Bis-Tris gel gel at 50 mA for 1.5 h. After staining for 1 h with Coomassie Brilliant Blue R-250 staining solution , the gel was washed with water overnight. Next morning, the gel was scanned with Gel Doc™ XR+ System , and a standard curve was established by plotting total protein mass of standards as a function of band intensity. Then, the band intensity for the ~50 kDa band of rCMG2-Fc variants was interpolated onto the standard curve to determine the mass of intact rCMG2-Fc, and calculate their concentrations.Purified rCMG2-Fc variants were subjected to protein sequence identification by mass spectrometry . First, 10 μg of purified rCMG2-Fc variants were subjected to SDS-PAGE analyses under reducing conditions as described in SDS-PAGE and Western Blotting section. After staining the gel in Coomassie Brilliant Blue R-250 and rinsing in water, the rCMG2-Fc protein band was excised from the gel and submitted to the Proteomics Core facility of University of California, Davis for LC-MS/MS-based protein identification. Briefly, the protein was digested with sequencing grade trypsin per manufacturer’s recommendations . Specific conditions can be found on UC Davis Proteomics Core Facility website1 . Peptides were dried using vacuum concentrator and resolubilized in 2% acetonitrile/ 0.1% trifluoroacetic acid. Peptides were analyzed by LC-MS/MS on a Thermo Scientific Q Exactive Orbitrap Mass Spectrometer in conjunction Proxeon Easy-nLC II HPLC and Proxeon nanospray source. The digested peptides were loaded on a Magic C18 200 Å 3 U reverse phase column and eluted using a 90-min gradient with a flow rate of 300 nl/min. An MS survey scan was obtained for the m/z range 300–1,600, spectra of MS/MS were developed using a top 15 method. An isolation mass window was used for the precursor ion selection, and normalized collision energy was used for fragmentation. Tandem MS spectra were extracted and charge state deconvoluted by Proteome Discoverer . The MS/ MS samples were analyzed using X! Tandem. X! Tandem was set up to search UniProt-Nicotiana benthamiana_database , the cRAP database of common laboratory contaminants2 plus an equal number of reverse protein sequences assuming the trypsin enzyme digestion. Scaffold Proteome Software version 4.0.6.1 was used to confirm protein identifications. X! Tandem identifications required at least –Log scores of greater than 1.2 with a mass accuracy of 5 ppm.

Protein identifications were accepted if they contained at least two identified peptides. Using the parameters above,outdoor vertical plant stands the Decoy False Discovery Rate was calculated to be 4.5% on the protein level and 1.94% on the spectrum level. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.rCMG2-Fc protein dissolved in 50 mM NH4HCO3 was denatured with 2 μl of dithiothreitol in a 65°C water bath for 50 min, followed by the alkylation with 4 μl of iodoacetamide in the dark for 20 min. The protein was then digested with 1 μg of trypsin in a 37°C water bath for 18 h. After the digestion, the mixture was frozen at −20°C for 1 h to deactivate the trypsin. For N-glycosylation analysis, 2 μl of the mixture was separated with an Agilent Eclipse plus C18 column coupled to an Agilent Eclipse plus C18 guard column , using a 10-min-gradient where solvent A with 0.1% formic acid and 3% of ACN in water, and solvent B with 0.1% of FA and 90% of ACN in water were used for separation. The analysis was conducted on an Agilent 1290 infinity ultra-high-pressure liquid chromatography system coupled to an Agilent 6495 triple quadrupole mass spectrometer, which was operated in a dynamic multiple reaction monitoring mode. The glycosylation site of the protein is on the infused IgG Fc region so that the transition list used here was adapted from the dMRM method of serum IgG, the development of which was described in great details in the study conducted by Hong et al. . To modify the method for rCMG2-Fc glycosylation quantitation, the plant N-glycan compositions containing xylose rather than sialic acid were used. In total, nearly 30 unique transitions for targeted glycopeptides and peptides of the rCMG2-Fc protein composed of the precursor ion, the product ion, the collision energy, and the retention time for each individual compound of the protein were developed for the dMRM method. The targeted glycopeptides were selected as precursor ions and several common oxonium fragments with m/z values as 204.08 and 366.14 were used as product ions. The software used for data analysis was Agilent MassHunter Quantitative Analysis B.05.02 software. To calculate the relative abundance of each glycopeptide, the abundance of individual glycopeptide was normalized to the abundance of the quantitating peptide. To validate the glycopeptides quantitated with the dMRM method, glycoproteomic analysis was conducted on rCMG2-Fc proteins. After the trypsin digestion of protein samples, glycopeptides were enriched with iSPE-HILIC cartridges. Then enriched samples were dried completely and reconstitute with 30 μl of water for LC-MS/MS analysis. One microgram of sample was separated with a Thermo Acclaim PepMap RSLC C18 column using a 180-min gradient. The analysis was conducted on a Thermo UltiMate 300 nano LC system coupledto an Orbitrap Fusion Lumos Tribrid mass spectrometer. The collected raw data were inspected with the software FreeStyle and the MS/MS spectra were search with the software Byonic.About 100 ns simulations of Agly, MAN8, and GnGnXF rCMG2-Fc glycoforms were performed in GROMACS with the AMBER ff14SB and GLYCAM06-j force fields. The AMBER topology files were exported to GROMACS format using ACPYPE with updated modifications which enable simulations with the GLYCAM forcefield in GROMACS . The 100 ns production simulations were first preceded by energy minimization in vacuum, solvation, solvated energy minimization, a 100 ps NVT equilibration, and finally a 100 ps NPT equilibration. Both energy minimizations were terminated with a maximum force tolerance of 1,000 kJ mol−1 nm−1. Each glycoform was solvated with explicit water with a minimum distance of 1.2 nm between the glycoprotein and the edge of the periodic box. The solvated systems were then neutralized with either sodium or chloride ions, and then concentrated to 0.155 M NaCl. The velocity-rescale thermostat was used with a reference temperature of 310 K and a time constant of 0.1 ps. The isotropic Parrinello-Rahman barostat was used with a reference pressure of 1 bar, a time constant of 2 ps, and an isothermal compressibility of 4.5 × 10−5 bar−1. All nonbonded interactions employed a short-range cutoff of 1 nm, with vertically shifted potentials such that the potential at the cutoff range is zero. The Particle-Mesh Ewald method with cubic interpolation was used to model long range electrostatic interactions. All non-water bonds were constrained with LINCS , while water bonds were constrained with SETTLE . A 2 fs time step was used with a sampling interval of 0.1 ns, for a total of 1,000 data points per 100 ns simulation.Protein stability is an important property of biopharmaceuticals, as it is often required that a protein remains stable and active during both storage and circulation in the target system after injection. To assess the stability of rCMG2-Fc variants and understand the EC50 differences observed in TNAs, a functional ELISA was developed to measure the amount of active rCMG2-Fc after incubation at 37°C for a range of time periods. Four time periods were tested: 1, 2, 3 h and overnight .