ONLINE SUPPLEMENT Lubich et al.
Supplement Figure 1
Supplement Figure 1: General gating strategy Presented is a representative gating strategy of the flow-cytometry-based technology to analyze sub-visible particles in protein-based therapeutics. Size range gates were controlled and if needed corrected using Fluoresbrite® YG Carboxylate Size Range Kit I & II [A]. CountBright™ Absolute Counting Beads were gated out using the 660/20 band-pass filter in the red detector array [B]. Gating on protein sub-visible particles in the Bis-ANS channel (violet detector array, 450/50 band-pass filter) was done by acquiring a non-protein sub-visible particle control (silicon oil particles, preparation Non-Prot) as a negative control reference [C] and protein sub-visible particle controls (biotinylated BSA-avidin complex, preparation Protand aggregated amyloid beta 1-40 peptide, preparation Prot-Crossß) as positive controls [E and G]. Protein sub-visible particles bearing a cross-β-sheet structure were analyzed in the DCVJ channel (blue detector array, 530/30 band-pass filter) using preparation Non-Prot and preparation Prot as negative reference controls [D and F] for cross-β-sheet structures and preparation Prot-Crossß as a positive control [H]. All size range gates were appliedfor each staining gate.
SupplementTable 1A
SupplementTable 1B
Supplement Table 1C
SupplementTables 1A-1C: Precision and accuracy of the flow-cytometry based technology
The precision of the method was evaluated by assessingthe inter- and intra-assay variations. Aggregated amyloid beta 1-40 peptide (preparation Prot-Crossß) was used as a positive control for analyzing concentrations (in particles/ml)of total sub-visible particles, protein sub-visible particles and cross-β-sheet protein particles. We analyzed preparation Prot-Crossß in 6 independent experiments for inter-assay variation [Table 1A] and in 6 parallel experiments on the same day for intra-assay variation [Table 1B]. Each sample was measured in 3 replicates in each experiment and mean numbers of sub-visible particles contained in 1 ml were calculated taking the dilution factor into account. This approach was used to calculate numbers of total sub-visible particles, protein and protein containing sub-visible particles and cross-β-sheet protein particles. All mean values for the inter-assay and for the intra-assay were used to calculate the coefficient of variation (CV %) for each property. We observed that the CV % of all the properties tested ranged between 10.32 % and 16.32 % for theinter-assay variations and between 2.05 % and 3.92 %for the intra-assay variations. In addition, we tested the accuracy of the method regarding the size range gates by calculating the CV% of total sub-visible particle counts per ml for each size gate [Table 1C]. We met the acceptance criteria that were set to a maximum of 25 %for each CV which complies with regulatory guidelines [38, 39].
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