Glu1 fibrinopeptide b lock mass standard

HA-SS-np materials were produced at the Vaccine Production Program Laboratory (Gaithersburg, MD). HA-SS-np formulated in one of the five formulation buffers (containing sodium phosphate, sodium chloride, and sucrose, pH 7.2) was used for investigational purposes, and a HA-SS-np interim reference material (in 1x PBS) was used as a control. Reagents included LC-MS grade 0.1% formic acid in water (mobile phase A), 0.1% formic acid acetonitrile (mobile phase B), and ammonium bicarbonate that were purchased from JT Baker (Phillipsburg, NJ). RapiGest^™ surfactant and [Glu1]-Fibrinopeptide B lock mass standard were purchased from Waters (Milford, MA). Formic acid, urea, and Zeba^™ desalting columns (7K MWCO) were purchased from Pierce (Rockford, IL). Dithiothreitol (DTT) and iodoacetamide (IAM) were purchased from ThermoFisher Life Technologies (Grand Island, NY) and Sigma Aldrich (St. Louis, MO), respectively. Chymotrypsin and PNGase F glycosidase were purchased from Promega (Madison, WI); Lys-C was purchased from New England Biolabs (Ipswich, MA). Method details of the stability-indicating analyses (rCGE and SEC) are described in the online supporting information.

Env trimer materials were produced in-house at the Vaccine Production Lab in NIH (Bethesda, MD) and based on the BG505.DS.SOSIP.664 construct. Purified trimeric Env material was produced in the CHO cell line and formulated in either 1×PBS or final formulation buffer. For the LC-MS analysis, reagents included high-purity LC-MS grade water and acetonitrile containing formic acid (mobile phases) and ammonium bicarbonate, which were purchased from J.T. Baker (Phillipsburg, NJ). [Glu1]-Fibrinopeptide B lock mass standard was purchased from Waters (Milford, MA). Formic acid, urea and Zeba^™ spin desalting columns (7 K MWCO) were purchased from Pierce (Rockford, IL). Dithiothreitol (DTT) and iodoacetamide (IAM) were purchased from ThermoFisher Life Technologies (Grand Island, NY) and Sigma Aldrich (St Louis, MO), respectively. Rapid PNGase F glycosidase (reducing and non-reducing formats) was purchased from New England Biolabs (Ipswich, MA).