Identification of Recombinant Chimpanzee Adenovirus C68 Degradation Products Detected by AEX-HPLC

Abstract

Physicochemical tests represent important tools for the analytical control strategy of biotherapeutics. For adenoviral modalities, anion-exchange high performance liquid chromatography (AEX-HPLC) represents an important methodology, as it is able to simultaneously provide information on viral particle concentration, product purity and surface charge in a high-throughput manner. During product development of an adenoviral-based therapeutic, an accelerated stability study was performed and showed changes in each of the AEX-HPLC reportable attributes. These changes also correlated with a decrease in product infectivity prompting a detailed characterization of the impurity and mechanism of the surface charge change. Characterization experiments identified the impurity to be free hexon trimer, suggesting that capsid degradation could be contributing to both the impurity and reduced particle concentration. Additional mass spectrometry characterization identified deamidation of specific hexon residues to be associated with the external surface charge modification observed upon thermal stress conditions. To demonstrate a causal relationship between deamidation and surface charge changes observed by AEX-HPLC, site-directed mutagenesis experiments were performed. Through this effort, it was concluded that deamidation of asparagine 414 was responsible for the surface charge alteration observed in the AEX-HPLC profile but was not associated with the reduction in infectivity. Overall, this manuscript details critical characterization efforts conducted to enable understanding of a pivotal physicochemical test for adenoviral based therapeutics.

Keywords: AEX-HPLC; adenovirus; deamidation; hexon; kinetics; mass spectometry; non-human primate.

FIGURE 1

Singlicate AEX-HPLC data from the AdC68 #1 accelerated stability time course. Peaks designated the “Main” and “Impurity” are labeled as such. The timepoints in the accelerated stability study are labeled on the figure, with blue representing the profile of the unstressed material. The retention times for each time point are shown in the figure.

FIGURE 2

AEX-HPLC and RP-HPLC profiles of the 12-weeks accelerated stability sample. The chromatographic profile of the unfractionated 12-weeks stability sample is shown in (A). After enrichment of the “Impurity” and “Main” peaks by the preparative AEX-HPLC assay, efficient enrichment was demonstrated of the “Impurity” (B) and “Main” (C) peaks by the analytical AEX-HPLC method. Analyses of fractionated “Impurity” and “Main” peaks by RP-HPLC are shown in (D,E), respectively.

FIGURE 3

Deamidation of selected hexon asparagine residues during the accelerated stability time course. (A) shows percent deamidation determined for N76, 414, 486 and 533 in AdC68 #1. (B) shows percent deamidation for N414 and the AEX-HPLC relative retention time shift both fit to a single exponential equation [Y=Y0 + (Plateau-Y0)*(1-exp (-K*x)], the calculated rate constants and the associated error for AdC68 #1.

FIGURE 4

Deamidation level of selected hexon asparagine residues from the 0- and 12-weeks accelerated stability unfractionated samples and the associated “Impurity” and “Main” peaks fractions.

FIGURE 5

AEX-HPLC of the N414D mutant overlaid with the singlicate AdC68 #1 accelerated stability time course. The timepoints in the accelerated stability study are labeled on the figure, with blue representing the profile of the unstressed material. The gray dashed line represents the N414D mutant. The retention times for each time point and for the mutant are shown in the figure.

FIGURE 6

Ribbon diagrams of the hexon AdC68 crystal structure (2obe.pdb) with selected asparagine residues highlighted. (A) demonstrates that N414 is on the surface and N76 is buried within the trimer. (B) demonstrates that N486 and N533 are buried within the hexon.