Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography

Abstract

To better understand host cell protein (HCP) retention in adeno-associated virus (AAV) downstream processes, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to quantitatively profile residual HCPs for four AAV serotypes (AAV2, -5, -8, and -9) produced with HEK293 cells and purified using POROS CaptureSelect AAVX affinity chromatography. A broad range of residual HCPs were detected in affinity eluates after purification (N _total = 2,746), and HCP profiles showed universally present species (N _universal = 1,117) and species unique to one or more AAV serotype. SWATH-MS revealed that HCP persistence was dominated by high-abundance conserved species (HACS), which appeared across all serotype conditions studied. Due to the notable contribution of these species to overall residual HCP levels, physical and functional characteristics of HACS were examined to determine trends that coincide with persistence. Subnetwork interaction mapping and Gene Ontology function enrichment analysis revealed extensive physical interactions between these proteins and significant enrichment for biological processes, molecular functions, and reactome pathways related to protein folding, nucleic acid binding, and cellular stress. The abundant and conserved nature of these HCPs and their functions offers a new perspective for mechanistic evaluations of impurity retention for AAV downstream processes.

Keywords: AAV; AAVX affinity chromatography; HCP; LC-MS/MS; adeno-associated virus; host cell protein; liquid chromatography-tandem mass spectrometry; proteomics.

Graphical abstract

Figure 1

POROS CaptureSelect AAVX elution pool total protein and AAV capsids POROS CaptureSelect AAVX affinity chromatography elution pool total protein amounts measured by Bradford and total AAV capsid content measured by AAVX Octet for AAV2, -5, -8, and -9, B1 and B2. Total protein (bars) is plotted on the left y axis on a normal scale. Elution pool total AAV capsid data (magenta triangles) is plotted on the right y axis on a log₁₀ scale. Error bars for protein concentration measurements correspond to SDs of technical replicates (n = 3).

Figure 2

SYPRO Ruby-stained protein gel of AAV lots purified with POROS CaptureSelect AAVX resin SYPRO Ruby-stained protein gel showing AAV2 (blue), AAV5 (orange), AAV8 (magenta), AAV9 (charcoal), and EGFP control (green) lysates after purification by POROS CaptureSelect AAVX affinity chromatography. Neutralized eluates were 4-fold concentrated and an equal volume of concentrated eluate was loaded per gel lane (2.5 μL). Viral capsid protein banding corresponding to VP1 (∼80–81 kDa), VP2 (∼65–66 kDa), and VP3 (∼59–60 kDa) can be seen for each AAV-containing condition.

Figure 3

Residual host cell protein identifications by serotype condition after POROS CaptureSelect AAVX purification All identified residual host cell proteins (HCPs) (A) and HCPs present in AAV-containing conditions but absent in the EGFP control lysate conditions (B) after POROS CaptureSelect AAVX affinity purification. Protein detection in all three triplicate LC-MS/MS injections for both biological duplicates was required for inclusion in each serotype group. For control-corrected conditions, HCPs detected in at least one EGFP control biological duplicate were removed. HCPs detected across all AAV serotypes are shown in red circles. A notable overlap in residual protein species was observed between AAV8 and AAV9 samples, shown in blue dashed circles. Proteins that appeared unique to each AAV serotype are shown in black dotted circles.

Figure 4

Percentage of total residual HCP content by abundance group Residual HCP profiling for the top 25, 25–100, and 100–500 most-prevalent species in each sample by normalized abundance (ng/μg HCP). Low-abundance HCPs outside of the top 500 are shown in turquoise. Total identified HCPs in each sample are shown at the top of each bar.

Figure 5

HCP abundance profiling by mean, median, and weighted ranking Individual HCP abundance profiling for the 251 species that appear within the 500 most-abundant subgroup across all AAV-containing samples (top-500∩). These species were ranked by mean abundance (ng/μg HCP) across all samples (A) and median abundance (ng/μg HCP) (B), shown in log₂ space. An additional ranking system based on weighted abundance scoring was devised, which was compared to the mean and median abundance ranking for the 150 most-abundant HCPs by median rank (C). Several HCPs appeared in particularly high abundance, which can be visualized more clearly from the log₂ transformed HCP mean abundance violin plot (D).

Figure 6

Physical subnetwork interaction mapping for high-abundance conserved HCP species HCP species that appeared within the 100 highest-abundance subgroup for all AAV-containing samples are shown (top-100∩). Blue arrows denote physical subnetwork interactions between proteins, with thicker, darker lines corresponding to higher-confidence scores. Protein nodes are sized proportional to the log₂ of the protein’s median normalized abundance (ng/μg HCP) across AAV-containing conditions (n_AAV = 8).

Figure 7

Isoelectric point and molecular weight boxplots for all identified and highly abundant HCPs within each serotype group pI (A) and molecular weight (MW) (B) distributions for all identified HCPs across biological duplicates of each serotype condition (“All”) and HCPs that ranked within the top 100 highest abundance for both biological duplicates of each serotype condition (top-100∩). Lines within the boxes indicate medians, and plus symbols indicate means. Boxes span 25^th–75^th percentiles and bars show data ranges. Upper ends for data ranges of MW are cut off, and full data ranges are shown in Figure S11.