Table 3.

Critical Quality Attributes and methods of their assessment for mRNA-LNP Drug Product as outlined in draft USP guidance ⁴⁹ and Daniel et al.⁴² Stability-indicating attributes for DP are highlighted in bold.

Quality	Attribute	Method
Identity	mRNA Sequence confirmation	Next generation sequencing (NGS), Sanger sequencing, Reverse Transcriptase–PCR
Content	mRNA content	RT-qPCR and RT–dPCR, Ultraviolet Spectroscopy, Ribogreen assay
	mRNA Encapsulation	Ribogreen assay, IEX chromatography
Potency	In vitro expression	Bioassay
Purity and Impurities	Percentage of mRNA main peak, pre- and post -main peaks	IP-RP-HPLC, Capillary gel electrophoresis
	5’ cap	IP–RP–HPLC
	3’ poly(A)	IP-RP–HPLC
DP specific attributes	Lipid content	LC-CAD, LC-MS, FFF-MALS-UV-dRI
	Lipid identity	LC-CAD, LC-MS, FFF-MALS-UV-dRI
	LNP size	Dynamic light scattering
	LNP polydispersity	Dynamic light scattering
	LNP surface charge	Electrophoretic light scattering, PALS, Capillary Electrophoresis
Purity and Impurities	Individual and total lipid impurities	LC-CAD, LC-MS, FFF-MALS-UV-dRI
	mRNA-lipid adducts	IP–RP–HPLC
	Residual solvents	Gas Chromatography
Safety	Endotoxin	USP <85>
	Bioburden	USP <61>, <62>, <1115>
	Sterility	USP <71>
Other	Appearance	USP <790>
	pH	USP <791>
	Particulate Matter	USP <787>, USP <788>
	Osmolality	USP <785>
	LNP morphology	CryoEM, SANS, SAXS, FFF-MALS-UV-dRI, DSC

Abbreviations: RT-qPCR, reverse transcriptase, quantitative polymerase chain reaction; dPCR, digital PCR; IEX, ion-exchange; IP-RP-HPLC, ion pairing reverse phase high performance liquid chromatography; LC, liquid chromatography; CAD, charged aerosol detector; MS, mass spectrometry; FFF, field-flow fractionation; MALS, multi-angle light scattering; UV, ultraviolet; dRI differential refractive index; CryoEM, cryo electron microscopy; SANS, small angle neutron scattering; SAXS small angle X-ray scattering; DSC, differential scanning calorimetry