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Pharmaceutical Technology Regulatory Sourcebook October 2020 55 to-lot consistency. Various techniques have been developed and applied to characterize AAV vector capsid content (Table I). Initial methods indirectly determined the full and non-full (both partial and empty) AAV capsid ratio using quantitative polymerase chain reaction (qPCR) to quantitate viral genomes present and the total capsid quantified by capsid specific enzyme- linked immunosorbent assay (ELISA). Because the ELISA also quantitates capsid-free proteins, this approach has the potential to misrepresent the actual ratio of full: non-full capsid (7,9,10). Tr ansmi ssion e lec tr on mic r oscopy ( TEM). TEM al- lows direct visualization of full, empty and partial capsids. However, the method is highly laborious and subjective (7), making it less suitable for cur- rent good manufacturing practice (cGMP) testing. Spec trophotometr y. Spectrophotometr y can es- timate viral capsid content, based on charac- terizing the expected absorbance of the viral capsid and genome to derive their extinction coefficients, by applying the 260/280 ratio to quantify the DNA and proteins in a solution (8). The number of empty capsids in purified vec- tor preparations reduces the A260/A280 ratio in a predictable manner. The method is quick and easy to perform and utilizes common laboratory equipment. How- ever, it requires highly purif ied and concen- trated (> 5 x 1011 vg/mL) vector, and accuracy can be reduced by impurities associated with more complex compositions (8). As a result, this method is generally only applied for analytical testing during early product development (11). Anion-exchange high-performance liquid chromatography. Because full capsids have lower isoelectric point (pI) values than the empty capsids due to the nega- tive charge of DNA present in the capsid, anion-ex- change high-performance liquid chromatography (AEX–HPLC) and capillary isoelectric focusing (cIEF) allow for characterization of vector cap- sids. The AEX–HPLC method benefits from the native f luorescence of aromatic amino acids and avoids interference from UV absorption of light at 280nm by packaged viral DNA, improving the accuracy of quantification over other meth- Table I. Summary, capsid content methods. Method Capsid detection* Run time Throughput Comments ELISA/qPCR F,E Hours High Two separate methods, Indirect calculation TEM F,E ~ 1 day Low Subjective results requiring human interpretation UV Spec F,E Min High Requires very pure preparations along with knowledge of extinction coefficient of capsid AEX-HPLC F,E 30 min High Reproducible and robust, HPLC commonly used in regulatory environment cIEF F,E,P α <1 hour High Requires very pure preparations due to UV detection SEC-MALS F,E 30 min High Provides capsid concentration and degree of aggregation AUC F,E,P ~6 hours Low Fully characterizes partial capsids. Large samples size requirement CDMS F,E,P ~2 hours [15] Low Fully characterizes full, partial, empty capsids. Currently not commercially available * F=Full, E=Empty, P=Partial, α=Partial, not fully characterized. ELISA/qPCR is Enzyme-Linked Immunoassay/quantitative polymerase chain reac- tion; TEM is transmission electron microscopy; UV spec is ultraviolet spectroscopy; AEX-HPLC is Anion-exchange-high-performance liquid chroma- tography; cIEF is Capillary isoelectronic focusing; SEC-MALS is size-exclusion chromatography with multiple angle light scattering; AUC is analytical ultrcentrifugation;CDMS is charge detection mass spectrometry.