Table 1.

Host Cell Protein Impurities with N-Linked Glycans from Baculovirus-Sf9 Productions of rAAV8

Source	Sf9 HCP Impurity Peptides with N-linked Glycans	Unmodified Peptide Mass (Da)	Modified Peptide Mass (Da)	HexNAc	Hex	Me-HexA	Fuc
Cell lysate	NYTVELHELEALAK	1,629.8483	2,668.2303	2	3		1
			2,871.3117	3	3		1
			2,506.1707	2	2		1
			2,696.2244	2	2	1	1
			2,522.1711	2	3		1
			1,978.9856	1			1
			1,832.9276	1
	NYTVELHELEALAAK	1,700.8854	1,903.9647	1
	ANYTVELHELEALAK	1,700.8854	1,903.9647	1
	N(+28)YTVELHELEALAK	1,657.8796	1,860.9589	1
			2,696.2200	2	3		1
Media	NYTVELHELEALAK	1,,629.8483	2,668.2303	2	3		1
			2,871.3117	3	3		1
			2,506.1757	2	2		1
			1,978.9881	1			1
			2,522.1714	2	3
			2,696.2214	2	2	1	1
			1,832.9298	1
	KNYTVELHELEALAK	1,757.9436	2,650.2657	2	3
			2,796.3262	2	3		1
			2,999.3988	3	3		1
	AKNYTVELHELEALAK	1,828.9803	2,721.3024	2	3
			2,867.3606	2	3		1
	NYTVELHELEALANR	1,771.8973	2,810.304	2	3		1
	NYTVELHELEALNR	1,700.8602	2,739.2646	2	3		1
			2,593.2048	2	3
	NYTVELHELEALAAK	1,700.8854	1,903.9647	1
	ANYTVELHELEALAK	1,700.8854	1,903.9647	1

N-linked glycans (modified residue is shown in bold) are identified on the common Sf9 HCP impurity ferritin identified by LC-MS/MS for rAAV8 from both cell lysate and media-purified vectors. Excluded from the list are common process contaminants that occur in routine sample preparation (e.g., human keratin, trypsin), impurities with mutations/aberrations such that they did not map to known proteins by BLASTp search, and any modification that could not be site localized. Unmodified and modified peptide masses are shown, as well as the number of glycan moieties each modified mass represents. HexNAc, N-acetylhexoseamine; Hex, hexose; Me-HexA, methylated hexuronic acid; Fuc, fucose. No HCP impurity peptides in the human rAAV8 preparations were observed with N-linked glycans.