Optilab dri detector

″LC system 1″ was used
for the SEC-UV analysis of the mRNAs and mRNA-LNPs. The setup was
composed of a biocompatible Vanquish Binary pump module, a Split Sampler
HT module, a thermostatic rapid separation (RS) column compartment,
and an UltiMate 3000 diode array detector (DAD) with a 2.5 μL
cell volume (Thermo Scientific, Waltham, Massachusetts). UV absorption
was monitored at 260 and 220 nm (10 Hz data collection rate and 0.5
s response time).
The AEC and SEC analyses of the pDNA samples
were performed using a herein described “LC system 2″.
A Vanquish quaternary pump F, Split Sampler FT, and column compartment
H were coupled to a DAD-FG module equipped with a 2.5 μL flow
cell volume and operated at 260 nm (Thermo Fisher Scientific, Waltham,
Massachusetts).
″LC system 3″ was used for SEC-MALS-dRI
analysis.
For separations, an Agilent 1260 series instrument with a quaternary
pump, thermostated column compartment, autosampler, and DAD was employed
(Agilent Technologies, Santa Clara, California). For detection, the
LC-DAD system was further coupled to a DAWN MALS Detector 8 (Wyatt
Technology, Santa Barbara, California) and an Optilab dRI Detector
(Wyatt Technology, Santa Barbara, California) for molecular weight
(MW) and radius of gyration (R_g) analysis,
respectively.

The molar mass, oligomerization state, and degree of glycosylation of S1 were determined by SEC-MALS. The S1 protein from the scaled-up production run was purified by Strep-Tactin purification followed by removal of the strep-tag by TEV protease incubation and SEC. After SEC, the second peak on the chromatogram (Fig. S2, elution 99 to 117 ml) was selected for SEC-MALS analyses to exclude large aggregates. An Infinity 1260 II high-performance liquid chromatography system (Agilent) was coupled to an Optilab dRI detector and miniDawn MALS detector (Wyatt Technologies, USA). The column thermostat and autosampler were both set to 20°C. The protein was diluted to 0.83 mg/ml in PBS (Sigma-Aldrich) and was then resolved in duplicate experiments on a Superdex 200 increase 10/30 GL column (GE Life Sciences) equilibrated in PBS. With ASTRA software (Wyatt Technologies), the absorption at 280 nm, light scattering, and refractive index properties of the eluates were collected and analyzed. The contribution of protein and glycosylation to the eluting species was determined by the conjugate analysis method in ASTRA using dn/dc values of 0.185 ml/g and 0.14 ml/g for protein and glycans, respectively, and an extinction coefficient for the protein at 280 nm of 1.2 ml/(mg·cm). The dn/dc value obtained for the conjugate was used for further molecular weight determination.