Detailed statistics are shown in Table S23

Detailed statistics are shown in Table S23. To determine whether the L-371,257 significant functional and sexually dimorphic hepatic expression differences were restricted to rAAV1, we repeated the same experiment with rAAV8. as well as cost considerations. (when scale-up was needed. This often corresponded to an increased vector dose being administered to achieve relevant expression, and in some cases patients developed severe adverse events directly related to treatment.8,9 Thus, while poised to revolutionize treatment of rare and common diseases alike, a thorough characterization of differences in rAAV vector safety and potency produced by the two developing platforms was needed. We characterized differences in vector lot composition at both the?genomic and proteomic levels. This was motivated by the fact that human and insect cells have different capacities to produce protein post-translational modifications (PTMs).10 Protein folding and PTMs can influence therapeutically administered proteins, including altering stability, targeting/trafficking, functional activity, and immunogenicity, all of which could affect rAAV potency. Additionally, a L-371,257 concern in generating recombinant proteins for humans within insect cells is usually potential immunotoxicity. Humans can have acute allergenic reactions to non-mammalian N-glycans, as well as any N-glycan with an 1,3-fucose or 1,2-xylose linkage around the innermost Production Platforms We sought to determine whether rAAV L-371,257 capsids were post-translationally altered, and whether capsid PTMs or any host cell protein (HCP) process impurities differed between production methods and lots. We used deep proteomic profiling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). To eliminate variables between human and baculovirus-platforms for comparative high-resolution MS/MS analysis, the following safeguards were implemented: (1) all vector productions were carried out at the same time in one facility using identical equipment by the same individuals; (2) vector lots were harvested, purified, and underwent quality control together using the same assays; (3) vector aliquots were frozen down identically and simultaneously; and L-371,257 (4) aliquots were thawed together and prepared for analysis from lots that had been frozen for the same amount of time to eliminate the effects of time spent frozen on potential PTM retention. One additional parameter that was important to control was the input DNA and backbone vector sequence itself. rAAV transfer vectors are encoded on plasmids for transfection in the human manufacturing platform and within a live baculovirus in the insect platform. To overcome this difference, we designed a custom transfer vector plasmid that has the necessary backbone components to be used in both systems (Physique?S1). Thus, the only differences remaining were those standard to each method: human HEK293 cells were produced in adherent cultures and transiently transfected with three production plasmids, while insect cells were grown in suspension cultures infected with two second-generation5 recombinant baculoviruses needed to produce rAAV. A set of rAAV8 productions was carried out and subdivided into four preparations for simultaneous purification: lots purified from cell lysates and media supernatant from each platform. LC-MS/MS analysis resulted in rAAV8 capsid protein protection of 98.8% (human) and 97.3% (baculovirus-Platforms are Post-Translationally Modified and Exhibit Differential PTM Profiles (A) PTM identities and residue positions along the length of the rAAV8 polypeptide from your N to C terminus in the baculovirus (baculo-)vector. PTMs are colored by type (acetylation, green; methylation, blue; phosphorylation, cyan; deamidation, orange; O-GlcNAcylation, magenta). Residues above the sequence are externally facing around the capsid. Residues below are lumenal or buried. Residues within the gray box from 1 to 220 represent the disordered region of AAV8 yet to be crystallized. The two regions for LamR binding (491C547 and 593C623) are highlighted L-371,257 in yellow boxes. (B) Cumulative capsid PTMs observed from all baculo-rAAV8 lots, purified from both cell lysates and media. Same color code as in (A). (C) Same as (A) but with human-produced rAAV8. (D) Same as (B) but with human rAAV8. (E) Shared and unique capsid PTMs for rAAV8 produced in the baculo-(yellow) and human (purple) platforms. Same color code as in (A). Excluded are deamidation degradation marks which are universal. (F) Unfavorable staining and TEM imaging of baculo-rAAV8 cell-purified vector. White arrow indicates full capsid; reddish arrow indicates vacant capsid; for reference for (F)C(I) (percent full capsids noted on left). Initial magnification, 20,000. (G) Same as (F) but media-purified vector. (H) Same as (F) but with human rAAV8 cell-purified vector. (I) Same as (H) but with media-purified vector. (J) Silver KRT20 stain of capsid VP species present in vector lots from (F)C(I). (K) 2D gel images from human-produced rAAV8 from pH 3 to pH 10..