Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Puriﬁcation

pharmaceutics

Article Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Puriﬁcation

Pete Gagnon *, Blaz Goricar, Nina Mencin, Timotej Zvanut, Sebastijan Peljhan, Maja Leskovec and Ales Strancar

BIA Separations, Sartorius Company, Mirce 21, 5270 Ajdovscina, Slovenia; [email protected] (B.G.); [email protected] (N.M.); [email protected] (T.Z.); [email protected] (S.P.); [email protected] (M.L.); [email protected] (A.S.) * Correspondence: [email protected]

Abstract: HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence Citation: Gagnon, P.; Goricar, B.; enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It Mencin, N.; Zvanut, T.; Peljhan, S.; does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of Leskovec, M.; Strancar, A. Multiple-Monitor HPLC Assays for empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These Rapid Process Development, enhancements support many opportunities to improve characterization of raw materials and process In-Process Monitoring, and intermediates, to accelerate process development, provide rapid in-process monitoring, and support Validation of AAV Production and process validation. Purification. Pharmaceutics 2021, 13, 113. https://doi.org/10.3390/ Keywords: adeno-associated virus; AAV; HPLC; intrinsic fluorescence; extrinsic fluorescence; light pharmaceutics13010113 scattering; empty capsids; full capsids; process development; in-process analysis; validation

Received: 17 December 2020 Accepted: 13 January 2021 Published: 17 January 2021 1. Introduction Rapid analytical feedback is a critical element for timely development of processes that Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- yield clinical quality biological drugs. It plays an equally important role in documenting ms in published maps and institutio- that established processes remain within specified control limits during validation and nal affiliations. manufacturing. HPLC monitored by UV absorbance at 280 nm has contributed substantially to fulfillment of these roles with recombinant proteins like monoclonal antibodies. Relatively speaking, however, recombinant proteins are compositionally and structurally simple, typically present at high concentrations, and are the dominant components in cell Copyright: © 2021 by the authors. Li- culture harvests. censee MDPI, Basel, Switzerland. Development of HPLC assays for adeno-associated virus (AAV) must accommodate a This article is an open access article more challenging reality. Individual AAV capsids comprise 60 capsid proteins of 3 types, distributed under the terms and con- ideally but often not in a ratio of 1:1:10; which may lack encapsidated DNA or contain ditions of the Creative Commons At- DNA of varying lengths, with varying proportions of single-stranded and double-stranded tribution (CC BY) license (https:// sequences [1–3]. Post-translational modifications impose additional layers of heterogene- creativecommons.org/licenses/by/ ity [3–5]. AAV cell harvests and lysates bear contaminant species and levels similar to those 4.0/).

Pharmaceutics 2021, 13, 113. https://doi.org/10.3390/pharmaceutics13010113 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 113 2 of 14

encountered with recombinant protein cell culture harvests but AAV levels are orders of magnitude lower, leaving AAV virtually a trace component in an overwhelming excess of contaminants [6]. Monitoring HPLC assays simultaneously for multiple parameters provides more sensitive, more accurate, and more insightful characterization of sample composition without compromising the ability to rapidly process large numbers of samples. The simplest case exploits dual wavelength monitoring. Nucleic acids absorb UV maximally at 260 nm and proteins at 280 nm. The ratio of absorbance at 260 nm and 280 nm is generally accepted as being about 1.8 for double-stranded DNA and about 2.1 for single-stranded species. The 260/280 ratios for empty and full AAV capsids in a non-chromatographic study were reported to be 0.59 and 1.44, respectively [7]. AEC has been shown to separate empty and full capsids into separate peaks for serotypes 1, 2, 4, 5, 6, 8, and 9 [6,8–17]. Empty capsid peaks separated from full capsid peaks by anion exchange (AEC) commonly exhibit ratios in the range of 0.6 to 0.7, while full capsids peaks commonly exhibit ratios in the range of 1.3–1.4. Intermediate ratios are often interpreted to indicate co-elution of empty and full capsids but they can also result from variation in the amount of encapsidated DNA among different capsid subpopulations, and/or from coelution of contaminating host cell proteins and/or nucleic acids. UV profiles are unsuitable for judging the relative areas of dominantly-empty capsid peaks and dominantly-full capsid peaks directly from chromatograms. DNA absorbs about three times more UV per unit mass than capsid proteins and causes the proportion of full capsids to be overestimated [7]. Calculations incorporating extinction coefficients can be used to derive the amount of protein in each peak and then compare them [7,18], but this suspends the simplicity of estimating empty and full capsid proportions directly from relative peak areas. Even after adjustment for extinction coefficients, the dominance of UV absorbance by DNA reduces relative sensitivity for detection of empty capsids, especially if they are present at low levels [7]. Wavelength ratios are also used to estimate the distribution of host cell DNA contamination across chromatograms [6] but they tend to overestimate it. RNA also persists in cell harvests and has nearly identical spectral properties [19]. In-line monitoring of intrinsic fluorescence increases sensitivity over UV detection of proteins and minimizes the error factor imposed by nucleic acids. Intrinsic protein fluorescence derives mostly from tryptophan residues, with a much lesser contribution by tyrosine and still less by phenylalanine [20–22]. Tryptophan is remarkably abundant in AAV capsids proteins, constituting 2.2% of complete capsids [23]. VP1 from AAV2 contains 15 tryptophan residues. VP2 and VP3 each contain 12. The excitation and emission wavelengths of DNA overlap with tryptophan but intrinsic fluorescence emission of DNA is comparatively negligible [24–26]. This enables protein-based estimation of relative empty and full capsid peak areas directly from AEC chromatograms [6]. As with UV, intrinsic fluorescence-based estimates are potentially influenced by co-elution of empty and full capsids and/or by co-elution with contaminating proteins. The high tryptophan content of AAV capsids also elevates their intrinsic fluorescence above the majority of cell culture and host cell proteins. Bovine serum albumin, which is only 7% larger than VP3, contains only two tryptophan residues (0.3%) [27]. Histone proteins have none [28–33]. Low histone fluorescence aids detection of AAV against high backgrounds of chromatin contamination across chromatography profiles of AAV lysates [6]. Tryptophan fluorescence is quenched to a degree by interactions with DNA [34]. The magnitude of such effects with full AAV capsids remains to be quantified. Intrinsic fluorescence also varies as a result of protein conformational changes [35]. This could alter intrinsic fluorescence to mass ratios under the extreme conditions commonly employed by affinity and ion exchange chromatography of AAV. Sensitivity of nucleic acid measurement can be amplified by monitoring extrinsic fluorescence conferred by intercalating dyes [36–41]. These dyes pass through lipid membranes and provide a tool to assess titer according to the amount of DNA or RNA Pharmaceutics 2021, 13, 113 3 of 14

fluorescence [42–45]. Data are divided on their ability to enter proteins capsids. They are reported to do so with adenovirus [46] but not with AAV [47]. Results can be obtained quickly, but co-stained host cell DNA in the form of degraded chromatin inflates the fluorescence signal [6,18,43–47]. Chromatin interference can be factored out by measuring dye fluorescence before and after capsid lysis but with a net result of low sensitivity [47]. HPLC reduces chromatin interference by separating the virus from most of the chromatin and increases sensitivity by concentrating the virus [6,44,45]. Accurate determination of DNA mass by extrinsic fluorescence requires experimental controls to accommodate many interfering influences [37–40]. Single-stranded DNA sequences bind intercalating dies differently than double-stranded sequences. Co-staining of RNA imposes potential error. Nucleic acid-binding proteins interfere with dye access, depress uptake kinetics, and reduce sensitivity. Slow dye uptake can be overcome with extended incubation, but differing salt and divalent metal cation concentrations may intro- duce further variation. Photobleaching of the dye during staining and analysis can reduce fluorescence yield. Chromatographic monitoring by light scattering represents an emerging area of interest. Early studies with protein characterization have focused on SEC [48–52] for two reasons. The first is that the chemical environment is constant across the elution profile. That ensures a constant refractive index, which is important for precision measurements [53]. The second is that light scattering by itself cannot discriminate size diversity within mixed samples. SEC separates by size and makes it possible to compare the amount of light scattering among bodies of different sizes. Larger particles produce more scatter than an equal mass of smaller particles. More recent publications show that light scattering detection combined with ion exchange chromatography produces useful information beyond the ability of SEC, despite the chemical environment changing across gradient elution profiles [52,54,55]. In either case, light scattering enables selective detection of AAV capsids from complex chromatograms [6]. Their 20–25 nm diameter provides good sensitivity. The size similarity of empty and full capsids makes it possible to estimate the relative proportions of dominantly-empty peaks and dominantly-full capsid peaks. However, full capsid peak area is inflated by the greater mass per full capsid [1,54,55]. Interference by proteins and other small host contaminants is negligible but chromatin contamination interferes with quantitative estimation in unpurified samples. Unfiltered cell cultures contain large amounts of chromatin remnants ranging in size from a few nm up to several µm [56]. Filtration to 0.45 µm truncates the top end of the range to about 400 nm but the remainder still overlaps with AAV [6,57–60]. Beyond simple monitoring of light scattering intensity to identify AAV peaks, light scattering offers potential to accurately determine AAV capsid size and mass in purified preparations. This report documents the practical value of HPLC-based AAV analysis with multiple monitors. Examples include AEC, CEC, and SEC with samples at different process stages monitored by UV absorption at 260 nm and 280 nm by intrinsic fluorescence, extrinsic Picogreen fluorescence, and light scattering. Discussion follows, addressing interpretation and utility of these assays for qualification of raw materials, process development, process validation, and in-process monitoring of AAV manufacturing operations.

2. Materials and Methods 2.1. Samples and Sample Preparation Triton X-100 lysates of sf9/BEV and HEK293 cells producing AAV2/8 were obtained from The University of Nantes, Center for Translational Therapy for Genetic Diseases, INSERM UMR 1089, Nantes, France. AAV8 was chosen particularly because AEC is known to separate empty and full capsids into separate peaks [6,9,10,16]. All were membrane filtered to 0.45 µm. Host cell DNA (chromatin) contamination was reduced in some by tangential flow filtration (TFF, 300 kDa membrane) into 20 mM Tris, 500 mM sodium chloride, 5 mM magnesium chloride, 1% sucrose, 0.1% Poloxamer 188, pH 8.0; they were then digested with a salt-tolerant nuclease enzyme (Kryptonase, 50 unit/mL, BIA Separations, Pharmaceutics 2021, 13, 113 4 of 14

Ajdovscina, Slovenia) at ambient temperature for 16 h in the TFF unit, and then processed further by TFF to remove nucleotides and nucleoproteins liberated by DNA lysis. Chro- matin contamination was alternatively reduced by addition of prototype positively charged particles (CIMasphere H-Bond, BIA Separations) at a proportion of 2% (v:v), incubated mixing for 60 min, then filtered through a 0.45 µm membrane to remove solids. Initial AAV purification was performed by CEC on 1 mL or 8 mL CIMmultus SO3 monoliths (BIA Separations). CEC columns were equilibrated to 50 mM formic acid, 200 mM sodium chloride, 1% sucrose, 0.1% Poloxamer 188, pH 3.5, eluted with a linear gradient to 50 mM formic acid, 2 M sodium chloride, pH 3.5, then cleaned with 2 M sodium chloride plus 1 M sodium hydroxide. Volumetric flow rate on 1 mL monoliths was 5 mL/min (5 column volumes (CV)/min). Volumetric flow rate for 8 mL monoliths was 40 mL/min. Picogreen incubation time was evaluated on CEC-purified AAV with 2 µL of dye per 100 µL of sample at time intervals up to 24 h. In a separate series of experiments, the effect of Picogreen concentration was evaluated with 10 min staining at dye dilutions up to 500-fold. Samples were analyzed by SEC in 50 mM MES, 150 mM sodium chloride, 0.05% Poloxamer 188, pH 6.5, or with a variant buffer substituting 1 M sodium chloride in place of 150 mM. Total Picogreen signal was expressed as fluorescence peak area in mV*sec. Based on the results from these experiments, Picogreen staining of experimental samples was subsequently performed by adding 2 µL of Picogreen per 100 uL of sample, then incubating for 24 h at 4 ◦C in the dark (foil-wrapped) to minimize photobleaching of the dye. Dark autosampler vials were used for the same purpose. Capsids were lysed in some experiments to release encapsidated DNA. CEC-purified AAV was diluted 5-fold in 50 mM MES, 150 mM NaCl, 0.05 M Poloxamer 188, pH 6.5. Proteinase K (Thermo Fisher Scientific, Waltham, MA USA) was added to a final concentration of 200 µg/mL and incubated at ambient temperature. Samples were analyzed at time intervals ranging from 1 h to 24 h. Relative intrinsic fluorescence of tryptophan and calf thymus DNA (Sigma-Aldrich, Saint Louis, Mo USA) were compared by injecting 100 µL of tryptophan at a concentration of 10 µg/mL (total 1 µg) onto an analytical SEC column and determining the peak area in mV*sec. That value was compared to the peak area from a 100 µL injection of calf thymus DNA (Sigma-Aldrich) at 100 µg/mL (total 10 µg). The different sample concentrations were required to keep the peaks on scale during SEC. Beyond the use of UV wavelength ratios, the respective identities of dominantly-full capsid peaks and dominantly-empty capsids peak separated by AEC were confirmed by PCR [61].

2.2. HPLC Analysis Chromatographic analyses were performed on a PATfix LPG HPLC system with integrated MWD UV–Vis detector (190–700 nm, 8 channel deuterium lamp, 50 mm flow cell path length), conductivity, and pH monitor (BIA Separations); further equipped with a Prominence RF-20A (200–650 nm, dual wavelength fluorescence detector (Shimadzu, Kyoto, Japan), and a DAWN HELEOS II multi-angle light scattering detector (Wyatt Technology, Dembach, Germany). UV absorbance was monitored at 260 nm and 280 nm. Light scattering was measured at an angle of 90◦. Intrinsic fluorescence was monitored at an excitation wavelength of 280 nm and an emission wavelength of 348 nm. Extrinsic (Picogreen) fluorescence was monitored at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Analytical SEC was conducted using a TSKgel GSW4000swxl column (7.8 mm × 300 mm, Tosoh Bioscience, Greisheim, Germany) with 50 mM MES, 150 mM sodium chloride, 0.05% Poloxamer 188, pH 6.5 at a volumetric flow rate of 0.5 mL/min. Analytical CEC was conducted on a 100 µL CIMac SO3 monolith (BIA Separations). The column was equilibrated with 50 mM acetic acid, pH 4.0; eluted with a linear salt Pharmaceutics 2021, 13, 113 5 of 14

gradient to 50 mM acetic acid, 3.0 M sodium chloride, pH 4.0; then cleaned with 2 M sodium chloride plus 1 M sodium hydroxide. Volumetric ﬂow rate was 1 mL/min. Analytical AEC separations of empty and full capsid samples were performed on a 100 µL quaternary amine monolith (CIMac AAV, BIA Separations). The column was equilibrated with 50 mM bis-tris-propane, 2 mM magnesium chloride, pH 9.0; eluted with a linear salt gradient to 50 mM bis-tris-propane, 2 mM magnesium chloride, 200 mM sodium chloride, pH 9.0; then cleaned with 2 M sodium chloride plus 1 M sodium hydroxide. Volumetric ﬂow rate was 1 mL/min. Analytical AEC analysis was alternatively performed using a 100 µL CIMac PrimaS monolith. The column was equilibrated with 10 mM bis-tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 8.0; eluted with a linear gradient to 10 mM bis-tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 10.0; then cleaned with 0.5 M phosphoric acid, 0.5 M acetic acid, and 0.05% benzyl alcohol.

3. Results 3.1. In-Line HPLC Monitoring of Empty and Full AAV Capsids Figure1 illustrates separation of empty and full capsids by AEC with a salt gradient, monitored for UV absorbance, light scattering, and intrinsic fluorescence. The ratio of UV absorbances at 260 nm and 280 nm enabled identification of an early peak populated dominantly by empty capsids (260/280 = 0.64), and a later peak populated dominantly by full capsid (260/280 = 1.31). Integration of the UV elution profile at 260 nm estimated that the full capsid peak represented 92% of the total peak area. Absorbance at 280 nm estimated a full capsid peak area of 85%. Light scattering estimated that the full capsid peak represented 78%, but that should be understood to be an overestimate due to the influence of higher mass in full capsids [1,54,55]. Intrinsic fluorescence gave the lowest estimate of Pharmaceutics 2021, 13, x FOR PEER REVIEW full capsid peak area at 76%. Even that should be understood to represent an overestimate,6 of 14

since the 260/280 ratio of the full capsid peak suggested that it contained a proportion of empty capsids. UV and light scattering-based estimates bear the same limitation.

FigureFigure 1. Separation 1. Separation of empty of and empty full capsids and full by anion capsids exchange by anion (AEC) withexchange different (AEC monitoring) with methods. different (a) monitoring UV absorptionmethods. at 260 (a nm) UV and absorption 280 nm. (b) Light at 260 scattering. nm and (c) Intrinsic 280 nm. fluorescence. (b) Light scattering. (c) Intrinsic fluorescence. In a separate fluorometric comparison of intrinsic fluorescence on SEC, DNA produced 3.2. In-Line HPLCa Monitoring peak area of 4.3of Nucleic mV*sec per Acids.µg. Tryptophan gave an area of 38,943 mV*sec per µg, producing a differential of 9057 versus DNA. To provide a rough estimation of how much Figure 2 illustratesDNA contributes the effects to the intrinsic of Picogr fluorescenceeen dilution of full capsids, and that incubation differential was time divided on byDNA staining of CEC-purified45.5 to account AAV for AAVin conjunction capsids containing with 2.2% SEC. tryptophan. Injection This of produced Picogreen a differential by itself of 199 for equivalent masses of DNA and capsid proteins. Protein mass per capsid was produced no fluorescencecalculated at peaks. 3.89 MDa A (5 dye× 87 dilution kDa (VP1) of + 20× 5 × 72gave kDA a (VP2) stained + 50 peak× 62 kDa area (VP3)). of 96% compared to the DNAundiluted mass per dye. capsid A wasdilution calculated of 50× at about gave 1.5 75% MDa and based 100× on 5000 gave bases 58%. of single-Dye uptake at a dilutionstranded of 50× DNA began with to an approach average molecular saturation weight after of 303.7 24 Da h per incubation. base. This yielded Picogreen a fluorescence at 1 h was 31% of the value observed at 24 h. Formulating the SEC buffer with 1 M sodium chloride reduced the Picogreen fluorescence peak area by about 60% (not shown). Figure 3 compares UV and Picogreen fluorescence for AAV capsids fractionated by two different AEC methods. The sample for both was CEC-purified AAV following nuclease-TFF treatment to minimize chromatin content. Both methods showed about 80% of the Picogreen signal associated with the empty capsid peak. The 260/280 ratio of the full capsid peak indicated the presence of empty capsids. This pointed to a likelihood that the Picogreen fluorescence in the full capsid peak was associated with co-eluting empty capsids, leading to a preliminary conclusion that Picogreen did not enter full AAV capsids.

Figure 2. Picogreen staining requirements for adeno-associated virus (AAV) capsids. (a) Dye concentration. (b) Incubation time.

Figure 3. Picogreen detection of capsid exterior-associated DNA. (a) AEC eluted with a salt gradient. (b) AEC eluted with a pH gradient.

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Figure 1. Separation of empty and full capsids by anion exchange (AEC) with different monitoring methods. (a) UV absorption at 260 nm and 280 nm. (b) Light scattering. (c) Intrinsic fluorescence.

3.2. In-Line HPLC Monitoring of Nucleic Acids. Figure Figure1. Separation 2 illustrates of empty the and effects full ca ofpsids Picogr by anioneen dilution exchange and (AEC incubation) with different time monitoringon DNA methods. (a) UV absorption at 260 nm and 280 nm. (b) Light scattering. (c) Intrinsic fluorescence. Pharmaceutics 2021staining, 13, 113 of CEC-purified AAV in conjunction with SEC. Injection of Picogreen by6 of 14itself produced no fluorescence peaks. A dye dilution of 20× gave a stained peak area of 96% 3.2.compared In-Line HPLC to the Monitoringundiluted dye. of Nucleic A dilution Acids. of 50× gave 75% and 100× gave 58%. Dye up- takeFigure at a dilution 2 illustratesfull capsidof 50× proteinthe began effects to DNAto approachof mass Picogr ratio eensaturation of 2.6 dilution to 1. Multiplyingafter and 24 incubation h the incubation. mass ratio time times Picogreen on the DNA stainingfluorescence of CEC-purified atintrinsic 1 h was fluorescence 31%AAV of in differential the conjunction value per obse unit massrvedwith produced atSEC. 24 h.Injection a per-capsidFormulating of intrinsic Picogreen the fluorescence SEC bybuffer itself producedwith 1 M no sodium fluorescencedifferential chloride of 517.peaks. reduced On this A basis,dyethe Picogreendilution capsid proteins of fluorescence 20× would gave account a stainedpeak for 99.8% area peak of by intrinsic aboutarea fullof 60% 96% capsid fluorescence. Encapsidated DNA would contribute 0.2%. compared(not shown). to the undiluted dye. A dilution of 50× gave 75% and 100× gave 58%. Dye uptake atFigure a dilution 3 compares3.2. of In-Line 50× HPLC UVbegan and Monitoring to Picogreen approach of Nucleic fluorescence saturation Acids forafter AAV 24 hcapsids incubation. fractionated Picogreen by fluorescencetwo different at AEC1 h Figure wasmethods. 231% illustrates ofThe the thesample value effects forobse of Picogreenbothrved was at dilution CEC-purified24 h. andFormulating incubation AAV time thefollowing on SEC DNA buffer nuclease-TFF treatmentstaining to of CEC-purifiedminimize chromatin AAV in conjunction content. with Both SEC. methods Injection showed of Picogreen about by itself 80% of with 1 M sodiumproduced chloride no fluorescence reduced peaks.the Picogreen A dye dilution fluorescence of 20× gave a peak stained area peak by area about of 96% 60% (notthe shown). Picogreen signalcompared associated to the undiluted with dye.the empt A dilutiony capsid of 50× peak.gave 75%The and 260/2 10080× gave ratio 58%. of the Dye full capsidFigure peak 3 indicatedcomparesuptake at the aUV dilution presence and of Picogreen 50× ofbegan empty to fluorescence approach capsids. saturation This for pointed afterAAV 24 capsidsto h incubation. a likelihood fractionated Picogreen that the by Picogreen fluorescencefluorescence in at the 1 h full was capsid 31% of the pe valueak was observed associated at 24 h. with Formulating co-eluting the SEC empty buffer cap- two different AECwith methods. 1 M sodium The chloride sample reduced for the both Picogreen was fluorescenceCEC-purified peak AAV area by following about 60% nuclease-TFFsids, leading treatment to(not a preliminary shown). to minimize conclusion chromatin that content. Picogreen Both did methods not enter showed full AAV about capsids. 80% of the Picogreen signal associated with the empty capsid peak. The 260/280 ratio of the full capsid peak indicated the presence of empty capsids. This pointed to a likelihood that the Picogreen fluorescence in the full capsid peak was associated with co-eluting empty capsids, leading to a preliminary conclusion that Picogreen did not enter full AAV capsids.

Figure 2.FigurePicogreen 2. stainingPicogreen requirements staining for requirements adeno-associated virusfor adeno-associated (AAV) capsids. (a) Dye virus concentration (AAV).(b capsids.) Incubation (a time.) Dye concentration. (b) Incubation time. Figure3 compares UV and Picogreen fluorescence for AAV capsids fractionated by two different AEC methods. The sample for both was CEC-purified AAV following nuclease-TFF treatment to minimize chromatin content. Both methods showed about 80% of the Picogreen signal associated with the empty capsid peak. The 260/280 ratio of the full capsid peak indicated the presence of empty capsids. This pointed to a likelihood that Figure 2. Picogreen staining requirements for adeno-associated virus (AAV) capsids. (a) Dye con- the Picogreen fluorescence in the full capsid peak was associated with co-eluting empty centration. (b) Incubationcapsids, leading time. to a preliminary conclusion that Picogreen did not enter full AAV capsids.

Figure 3. Picogreen detection of capsid exterior-associated DNA. (a) AEC eluted with a salt gradient. (b) AEC eluted with a pH gradient.

FigureFigure 3. Picogreen 3. Picogreen detection of detection capsid exterior-associated of capsid exterior-associated DNA. (a) AEC eluted with DNA. a salt gradient.(a) AEC (b eluted) AEC eluted with with a salt a gradi- pH gradient. ent. (b) AEC eluted with a pH gradient. Figure4 provides additional evidence that capsid-associated Picogreen staining did not represent encapsidated DNA. Proﬁle (a) illustrates the SEC Picogreen proﬁle of CEC-

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Figure 4 provides additional evidence that capsid-associated Picogreen staining did not represent encapsidated DNA. Profile (a) illustrates the SEC Picogreen profile of CEC- purified AAV from filtered lysate. The exclusion limit of this column was at about 10 min. The Picogreen signal from 10.0 min to 13.5 min represented high molecular weight chromatin remaining after the CEC step. The peak centered at 15.5 min represented AAV capsids. Digestion for 1 h with proteinase K (profile b) reduced the amount of capsids by about 32% but liberated a very large fraction of plasmid DNA. If the Picogreen fluorescence associated with the untreated capsids had been from encapsidated DNA, then the fluorescence area of the plasmid peak should have been 32% of the fluorescence peak area of the undigested capsids. Digestion also produced a late-eluting population of small pep- tides as expected, eluting immediately after a small proteinase K peak. Digestion for 24 h liberated much more plasmid but surprisingly reduced the amount of capsids by only 55% compared with the undigested sample (profile (c)). The 260/280 ratio for capsids in the undigested control was 1.10. The ratio after 1 h was 1.10 and after 24 h was 1.12. These data indicate that empty and full capsids were digested at about the same rate. They also provided a third line of evidence that encapsidated DNA did not Pharmaceuticscontribute2021, 13, 113 to Picogreen fluorescence. If it had done so, then the 260/280 ratio of the 7undi- of 14 gested capsid peak should have been reduced in proportion to the increase in the size of the plasmid peak. Comparison of plasmid fluorescence with capsid fluorescence also highlighted thatpuriﬁed the amount AAV from of ﬁlteredDNA lysate.associated The exclusion with capsid limit of thisexteriors column represents was at about 10a small min. The Picogreen signal from 10.0 min to 13.5 min represented high molecular weight chromatin fraction of the amountremaining of after DNA the inside CEC step. full The capsids. peak centered at 15.5 min represented AAV capsids.

FigureFigure 4. SEC 4. of cationSEC of exchange cation (CEC)-purified exchange (CEC)-purified AAV capsids stained AAV with capsids Picogreen. stained (a) Before with treatment Picogreen. with proteinase (a) Before K. (b)treatment After 1 h incubation. with proteinase (c) After 24 K. h. (b) After 1 h incubation. (c) After 24 h. Digestion for 1 h with proteinase K (profile b) reduced the amount of capsids by about 3.3. In-Line HPLC32% Characterization but liberated a very of Raw large and fraction In-Process of plasmid Materials. DNA. If the Picogreen fluorescence associated with the untreated capsids had been from encapsidated DNA, then the fluores- Figure 5 illustratescence area the of the utility plasmid of peakanalytical should have CEC been for 32% evaluation of the fluorescence of raw peakmaterials area of theand as a process developmentundigested capsids.guide. DigestionProfile (a) also sh producedows the a late-elutingelution profile population of filtered of small peptideslysate pre- as stained with Picogreen.expected, The eluting vertical immediately dotted after lin ae small on profile proteinase (a) Kindicates peak. where AAV eluted, based on profile (b),Digestion which was for 24 run h liberated on the much same more column plasmid under but surprisingly identical reduced conditions. the amount The of capsids by only 55% compared with the undigested sample (profile (c)). The 260/280 ra- general parallel tioof for260 capsids nm, 280 in the nm, undigested and Picogr controleen was fluorescence, 1.10. The ratio afterall rising 1 h was steeply 1.10 and at after the beginning of the24 gradient h was 1.12. then These gradually data indicate sloping that empty downwards, and full capsids showed were digestedthat chromatin at about dominated the contaminantthe same rate. population. They also provided a third line of evidence that encapsidated DNA did Chromatogramnot contribute (b) shows to Picogreen CEC analysis fluorescence. after CEC If it had capture done so, from then thefiltered 260/280 lysate. ratio ofChro- the undigested capsid peak should have been reduced in proportion to the increase in the size matogram (c) illustratesof the plasmid the peak.CEC Comparisonelution pro offile plasmid of AAV fluorescence purified with by capsid CEC fluorescenceafter the lysate also was treated withhighlighted nuclease thatand the TFF amount to preemptively of DNA associated reduce with chromatin capsid exteriors contamination. represents a small Pro- files (b) and (c) bothfraction show of the the amount 260 ofand DNA 280 inside profiles full capsids. virtually on top of each other. This is consistent with CEC3.3. In-Line eluting HPLC empty Characterization and full of ca Rawpsids and In-Processin the same Materials peak. The broader capsid peak on profile (c) isFigure the 5result illustrates of a the different utility of analyticalgradient CEC configuration for evaluation on of raw a 1 materials mL column. and Profiles (a) and (b)as awere process run development on a 100 µL guide. column. Profile (a) shows the elution profile of filtered lysate The ratio ofpre-stained Picogreen with fluorescence Picogreen. The to UV vertical absorbance dotted line at on 260 profile nm (a) in indicates profiles where (b) and AAV (c) eluted, based on profile (b), which was run on the same column under identical conditions. showed a clear Thebenefit general of parallelpreemptive of 260 nm,chromatin 280 nm, and reduction. Picogreen fluorescence,The ratio of all fluorescence rising steeply at to absorbance, measuredthe beginning as percen of the gradientt of full then scale, gradually provided sloping an downwards, indication showed of relative that chromatin DNA dominated the contaminant population. Chromatogram (b) shows CEC analysis after CEC capture from filtered lysate. Chro- matogram (c) illustrates the CEC elution profile of AAV purified by CEC after the lysate was treated with nuclease and TFF to preemptively reduce chromatin contamination. Pro- files (b) and (c) both show the 260 and 280 profiles virtually on top of each other. This is consistent with CEC eluting empty and full capsids in the same peak. The broader capsid peak on profile (c) is the result of a different gradient configuration on a 1 mL column. Profiles (a) and (b) were run on a 100 µL column. Pharmaceutics 2021, 13, x FOR PEER REVIEW 8 of 14

contamination. The ratio for the capsid peak in profile (b) was 0.47. The ratio for the capsid peak in profile (c) was 28% lower at 0.34. This could indicate that advance TFF-nuclease treatment reduced the amount of DNA bound to capsid exteriors but it could also indicate that the capsids in profile (b) co-eluted with chromatin. The latter hypothesis is favored Pharmaceutics 2021, 13, x FOR PEER REVIEW 8 of 14 by the additional observation that advance chromatin extraction (c) eliminated the DNA populations flanking the AAV peak (b).

contamination. The ratio for the capsid peak in profile (b) was 0.47. The ratio for the capsid peak in profile (c) was 28% lower at 0.34. This could indicate that advance TFF-nuclease treatment reduced the amount of DNA bound to capsid exteriors but it could also indicate that the capsids in profile (b) co-eluted with chromatin. The latter hypothesis is favored Pharmaceuticsby2021 ,the13, 113 additional observation that advance chromatin extraction (c) eliminated8 the of 14 DNA populations flanking the AAV peak (b).

Figure 5. CEC with Picogreen staining for characterization of DNA contamination. (a) Clarified lysate. (b) After CEC capture from clarified lysate. (c) After CEC capture from lysate treated with nuclease and TFF.

Figure 6 illustrates the utility of monitoring analytical SEC with Picogreen fluorescence for evaluation of raw materials and as a process development guide. Profile (a) was developed from an AAV8 lysate produced in sf9/BEV cells. Profile (b) was developed from an AAV8 lysate produced in HEK293 cells transfected with the same plasmids. Heavy FigureFigure 5. CEC with5. CEC Picogreen with stainingPicogreen for characterization staining for ofchar DNAacterization contamination. of (DNAa) Clarified contamination. lysate. (b) After (a CEC) Clarified chromatin contamination is implied in both by the dominance of the 260 nm profile and capturelysate. from clarified (b) After lysate. CEC (c) After capture CEC capture from fromclarified lysate treatedlysate. with (c) nucleaseAfter CEC and TFF. capture from lysate treated with confirmed by the Picogreen response. Panel (c) illustrates chromatin reduction by sample nuclease and TFF. The ratio of Picogreen fluorescence to UV absorbance at 260 nm in profiles (b) and treatment with positively(c) showed charged a clear benefit particles. of preemptive A well-defined chromatin reduction. AAV peak The ratiowas ofapparent fluorescence from the UV Figuretrace, largely 6 illustratesto absorbance, devoid the measuredof utility host cell as of percent monitoricont ofaminants fullng scale, analytical providedexcept for anSEC indication a persistentwith ofPicogreen relative fraction DNA fluores- of high molecular weightcontamination. chromatin. The ratio for the capsid peak in profile (b) was 0.47. The ratio for the capsid cence for evaluationpeak in of profile raw (c) materials was 28% lower and at as 0.34. a process This could development indicate that advance guide. TFF-nuclease Profile (a) was developedFigure 7 highlights fromtreatment an AAV8 the reduced value lysate the of amountproduced monitoring of DNA in boundanalyticalsf9/BEV to capsid cells. SEC exteriors Profile with butintrinsic (b) it couldwas fluorescence.developed also indicate from Thean UV AAV8 profile lysate of thatthe produced thefiltered capsids harvest inin profile HEK293 in (b) profile co-eluted cells (a) transfected with was chromatin. so heavily with The dominated latterthe same hypothesis plasmids. by is chromatin favored Heavy that no AAV peakby was the additionalapparent. observation Intrinsic that flu advanceorescence chromatin preferentially extraction (c) amplified eliminated the the DNA AAV chromatin contaminationpopulations flanking is implied the AAV in peak both (b). by the dominance of the 260 nm profile and signalconfirmed because by of theits highPicogreenFigure tryptophan6 illustrates response. the content utility Panel of monitoringand (c )made illustrates analytical it possible chromatin SEC with to Picogreenvisualize reduction fluorescence the capsidsby sample againsttreatment a high with backgroundfor positively evaluation ofcharged of contaminating raw materials particles. and proteinA as well-defined a process fluorescence. development AAV peak guide.Profile was Profile (b) apparent illustrates (a) was from overlaid intrinsic developedfluorescence from an profiles AAV8 lysate from produced a series in sf9/BEV of experiments cells. Profile (b)performed was developed on fromthe fil- the UV trace, largelyan AAV8 devoid lysate produced of host in cell HEK293 cont cellsaminants transfected except with thefor same a persistent plasmids. Heavyfraction of teredhigh lysate molecular shownchromatin weight in profile contaminationchromatin. (a). Capsids is implied were in bothtreated by the with dominance 4 M NaCl of the 260 at nmthe profile pH values and shown, Figurethen filtered 7 highlightsconfirmed to remove by the the value Picogreen solids of monitoring response.before SEC. Panel analytical (c)Panel illustrates (c) SECillustrates chromatin with reductionintrinsic results by fluorescence.when sample the filtered lysate wastreatment treated with with positively 4 M NaCl charged at particles. pH 3.5 A in well-defined the presence AAV peakof positively was apparent charged from The UV profilethe of UVthe trace, filtered largely harvest devoid in of hostprofile cell contaminants(a) was so heavily except for dominated a persistent fraction by chromatin of particles.that no AAV peakhigh molecularwas apparent. weight chromatin. Intrinsic fluorescence preferentially amplified the AAV signal because of its high tryptophan content and made it possible to visualize the capsids against a high background of contaminating protein fluorescence. Profile (b) illustrates overlaid intrinsic fluorescence profiles from a series of experiments performed on the filtered lysate shown in profile (a). Capsids were treated with 4 M NaCl at the pH values shown, then filtered to remove solids before SEC. Panel (c) illustrates results when the filtered lysate was treated with 4 M NaCl at pH 3.5 in the presence of positively charged particles.

FigureFigure 6. SEC 6. withSEC Picogreen with Picogreen staining. (a )staining. Filtered sf9/BEV (a) Filtered lysate. ( bsf9/BEV) Filtered HEK293lysate. lysate.(b) Filtered (c) Filtered HEK293 HEK293 lysatelysate. (c) afterFiltered treatment HEK293 with CIMasphere lysate after H-Bond treatment particles. with CIMasphere H-Bond particles. Figure7 highlights the value of monitoring analytical SEC with intrinsic fluorescence. The UV profile of the filtered harvest in profile (a) was so heavily dominated by chromatin that no AAV peak was apparent. Intrinsic fluorescence preferentially amplified the AAV signal because of its high tryptophan content and made it possible to visualize the capsids

Figure 6. SEC with Picogreen staining. (a) Filtered sf9/BEV lysate. (b) Filtered HEK293 lysate. (c) Filtered HEK293 lysate after treatment with CIMasphere H-Bond particles.

Pharmaceutics 2021, 13, 113 9 of 14

Pharmaceutics 2021, 13, x FOR PEER REVIEW 9 of 14 against a high background of contaminating protein fluorescence. Profile (b) illustrates overlaid intrinsic fluorescence profiles from a series of experiments performed on the filtered lysate shown in profile (a). Capsids were treated with 4 M NaCl at the pH values Pharmaceutics 2021, 13, x FOR PEER REVIEW shown, then filtered to remove solids before SEC. Panel (c) illustrates results when the9 of 14

ﬁltered lysate was treated with 4 M NaCl at pH 3.5 in the presence of positively charged particles.

Figure 7. SEC with intrinsic fluorescence. (a) Filtered lysate. (b) After treatment with 4 M NaCl at indicated pH values. (c) After treatment with CIMasphere H-Bond particles at 4 M NaCl, pH 3.5.

Figure 7.FigureSECFigure with 7. SEC intrinsic8 shows with fluorescence. intrinsic the ability (fluorescence.a) Filtered of light lysate. scattering(a ()b Filtered) After treatment tolysate. aid with (detectionb) 4After M NaCl treatment of at indicatedAAV with capsids pH 4 values. M NaClin fil- at (c) Afterteredindicated treatment lysates with pH applied CIMasphere values. to(c) H-Bond analyticalAfter particlestreatment HPLC at 4 with M NaCl,columns. CIMasphere pH 3.5. Empty H-Bond and particlesfull capsids at 4 M eluted NaCl, pHin the 3.5. same peak with SEC Figure(a) and8 shows CEC theat (b). ability AEC of lightseparated scattering them to aid (c). detection The high of light AAV capsidsscattering in backgroundFigure in 8 (a) showsfiltered and (c) lysatesthe was ability applied caused of to light by analytical high scattering molecular HPLC columns.to aid weight detection Empty chromatin, and of full AAV capsids also capsids indicated eluted in in filtered lysates appliedthe same to peak analytical with SEC HPLC (a) and CECcolumns. at (b). Empty AEC separated and full them capsids (c). The eluted high light in the by the dominancescattering of the 260 background nm profile in (a) acro and (c)ss wasthe causedchromatograms. by high molecular Background weight chromatin, light scat- also teringsame was peak reduced withindicated SEC across (a) by theand the salt dominance CEC gradient at (b). of the AECfor 260 CEC nmseparated profile(b) because across them the most (c). chromatograms. The of the high high light Background molecular scattering weightbackground chromatin inlight (a) remained and scattering (c) was bound was caused reduced during by across high the the molecularsalt salt gradient weight for and CEC chromatin, required (b) because removalalso most indicated of the by NaOHby the [6]. dominance This washigh also of molecular the reflected 260 weight nm in profile chromatin the do acrominance remainedss the of chromatograms. bound the 280 during nm the profile. salt Background gradient However, and required light DNA scattering was reducedremoval across by NaOH the salt [6]. gradient This was also for reflectedCEC (b) in because the dominance most of thethe 280 high nm molecular profile. contamination remainsHowever, substantial, DNA contamination as shown remains in panel substantial, (a) of as Figure shown in 5. panel (a) of Figure5. weight chromatin remained bound during the salt gradient and required removal by NaOH [6]. This was also reflected in the dominance of the 280 nm profile. However, DNA contamination remains substantial, as shown in panel (a) of Figure 5.

FigureFigure 8. Light 8. scatteringLight scattering identiﬁcation identification of AAV in chromatograms of AAV in chromatograms of ﬁltered lysate. (a )of SEC. filtered (b) CEC lysate. with salt (a) gradient SEC. (b) elution.CEC (c )with AEC withsalt saltgradient gradient elution. elution. (c) AEC with salt gradient elution.

4. Discussion and Conclusions 4. DiscussionFigure 8. Light and scattering4.1. Conclusions In-Line identification HPLC Monitoring of byAAV UV Absorbancein chromatograms and Intrinsic of Fluorescence filtered lysate. (a) SEC. (b) 4.1.CEC In-Line with HPLC salt gradient MonitoringMonitoring elution. by capsid( cUV) AEC Absorbance separation with salt by andgradient intrinsic Intrinsic fluorescenceelution. Fluorescence. enables more accurate determination of empty capsid peak size versus full capsid peak size than UV because DNA Monitoring capsid separation by intrinsic fluorescence enables more accurate deter- 4. Discussion andcontributes Conclusions only 0.2% of full capsid fluorescence (Figure1)[ 6,24–26]. This enables determi- mination of emptynation capsid of relative peak peak size sizes versus based full almost capsid exclusively peak onsize intrinsic than fluorescenceUV because of capsid DNA contributes4.1. In-Line only HPLC 0.2% Monitoring of full capsid by UV fluorescence Absorbance and(Figure Intrinsic 1) [6,24–26]. Fluorescence. This enables deter- minationMonitoring of relative capsid peak sizes separation based almost by intrinsic exclusively fluorescence on intrinsic enables fluorescence more accurate of capsid deter- proteins.mination As ofwith empty UV, capsidpartial co-elutionpeak size versusof empty full and capsid full capsidspeak size or thantheir UVco-elution because with DNA contaminatingcontributes only host 0.2%proteins of full could capsid affect fluorescence accuracy. (Figure 1) [6,24–26]. This enables deter- minationThe contribution of relative ofpeak DNA sizes could based be almostsignificantly exclusively less than on intrinsic 0.2%. With fluorescence tryptophan of pro-capsid ducingproteins. at least As 9000with timesUV, partial more fluorescenceco-elution of peemr ptyunit and mass full than capsids DNA, or even their trace co-elution levels withof proteincontaminating contamination host proteinsin a purified could DNA affect control accuracy. sample would substantially inflate apparent DNAThe contribution content. of DNA could be significantly less than 0.2%. With tryptophan pro- ducingMonitoring at least intrinsic9000 times fluorescence more fluorescence also enhances per unit sensitivity mass than of DNA, AAV even detection trace levels over of hostprotein cell proteins contamination by virtue in ofa purified capsid protei DNAns control containing sample an would unusually substantially high proportion inflate ap- (2.2%)parent of tryptophanDNA content. residues [23]. This particularly helps one visualize AAV in SEC chro- matogramsMonitoring of unpurified intrinsic lysates fluorescence (Figures also6–8). e Thenhances other sensitivity side of this of coin AAV is thatdetection intrinsic over fluorescencehost cell proteins underestimates by virtue contamination of capsid protei byns pr containingoteins with an lesser unusually tryptophan high proportioncontent. This(2.2%) extends of tryptophan to underestimating residues [23]. chromatin This particularly contamination helps one since visualize histone AAV proteins in SEC lack chromatograms of unpurified lysates (Figures 6–8). The other side of this coin is that intrinsic fluorescence underestimates contamination by proteins with lesser tryptophan content. This extends to underestimating chromatin contamination since histone proteins lack

Pharmaceutics 2021, 13, 113 10 of 14

proteins. As with UV, partial co-elution of empty and full capsids or their co-elution with contaminating host proteins could affect accuracy. The contribution of DNA could be significantly less than 0.2%. With tryptophan producing at least 9000 times more fluorescence per unit mass than DNA, even trace levels of protein contamination in a purified DNA control sample would substantially inflate apparent DNA content. Monitoring intrinsic fluorescence also enhances sensitivity of AAV detection over host cell proteins by virtue of capsid proteins containing an unusually high proportion (2.2%) of tryptophan residues [23]. This particularly helps one visualize AAV in SEC chromatograms of unpurified lysates (Figures6–8). The other side of this coin is that intrinsic fluorescence underestimates contamination by proteins with lesser tryptophan content. This extends to underestimating chromatin contamination since histone proteins lack tryptophan [28–32]. The high proportion of tryptophan in AAV capsid proteins also contributes to their unusually high UV extinction coefficient of 3.72 at 280 nm [7].

4.2. In-Line HPLC Monitoring by UV Absorbance and Light Scattering Light scattering enables easy detection and confirmation of capsids for all chromatography methods. With AEC of purified samples, the relative area of the dominantly-full capsid peak areas is slightly higher than that judged by intrinsic fluorescence but much more reasonable than the substantial overestimates derived from UV absorption (Figure1 ). Light scattering with AEC can also provide insight about the relative size of empty and full capsid peaks in harvests and lysates (Figure8). Co-elution with high molecular weight chromatin interferes with quantitative estimation, but light scattering can still be useful to evaluate large differences of empty and full capsids among different cell lines and transfection methods. Empty and full capsids co-elute in a single peak from SEC and CEC columns (Figures5–8). Measurement of light scattering intensity at a single angle is sufficient for capsid detection in crude and purified samples. Measurement of light scatter at multiple angles is necessary for accurate determination of particle size and mass in highly purified samples [47–55].

4.3. In-Line HPLC Monitoring by UV Absorbance and Extrinsic Fluorescence Monitoring extrinsic fluorescence from intercalating dyes opens a wide range of opportunities to characterize nucleic acid content in AAV samples, but many issues require careful attention. The first is the long incubation time required to stain unpurified samples (Figure2). Complete staining of purified DNA under optimal conditions requires only a few minutes, but longer incubation is required to overcome steric exclusion of the dye by strongly bound proteins in unpurified samples [36–41]. Protocols for determination of virus titer typically recommend an incubation period of 1 h [40–42]. This acknowl- edges suppression of dye uptake in unpurified samples but underestimates its magnitude. Figure2 shows that 1 h incubation produces only 32% of the signal produced at 24 h. It also warns that variations of a few minutes more or less than 1 h would produce major errors. The simplest application of extrinsic fluorescence is visualizing the distribution of host cell DNA in the form of chromatin across chromatograms of candidate purification methods (Figures5 and6)[ 6,19]. Chromatin is a greater burden for AAV purification than for recombinant proteins because low AAV titers make the chromatin-to-product ratio tens to hundreds of times higher [6,19,57–60]. Figures3–6 demonstrate that Picogreen is effective in conjunction with AEC, CEC, and SEC, but quantitative comparisons should be made with care. Even assuming uniform staining conditions, chromatography conditions can displace intercalated dye. This is demonstrated by SEC in the presence of 1 M sodium chloride reducing Picogreen fluorescence by 60% compared to peak area in 150 mM sodium chloride. CEC purification of AAV commonly employs pH 3.5 and sodium chloride elution gradients up to 2 M. AEC commonly employs pH 9.0 and sodium chloride gradients up to 200 mM. AEC often employs divalent metal cations that are known to enhance Picogreen fluorescence [41]. The Pharmaceutics 2021, 13, 113 11 of 14

same factors that affect Picogreen fluorescence also affect composition and conformation of chromatin [19,58,62–65], which can further affect comparability among chromatography methods. The ability of intercalating dyes to pass through lipid membranes makes them natural candidates to evaluate titers of lipid enveloped viruses [42–45], but they are apparently not able to enter AAV capsids [47]. Figures3 and4 confirm the inability of Picogreen to detect AAV-encapsidated DNA but reveal DNA associated with empty capsid exteriors. Given that the samples in Figure3 were treated in advance to remove chromatin, then partially purified by CEC, and finally fractionated by AEC, the association of empty capsids and DNA must be understood to be highly stable. Interactions of nucleic acids with nucleic acid binding proteins are known to exhibit such stability [57–60], but the phenomenon has not been previously reported for AAV capsids. The fact that empty and full capsids coelute in SEC (Figures6–8) suggests that exterior- associated DNA is likely in the form of small fragments tightly bonded to empty capsid surfaces. Otherwise, they would increase the hydrodynamic diameter of empty capsids and cause them to elute earlier than full capsids. It also seems apparent that DNA binding to capsid surfaces must alter their native charge characteristics by neutralizing capsid positive charges involved in DNA binding. Large amounts of DNA per capsid might be expected to cause empty capsids to elute earlier from CEC than full capsids. On the contrary, empty and full capsids generally coelute from CEC in a single peak (Figures5 and8). More investigation is warranted to fully characterize the phenomenon of DNA-binding to capsid exteriors, further including how broadly it occurs across serotypes. Evaluation of highly purified empty and full capsids prepared by ultracentrifugation would be desirable to prevent chromatin contamination from confusing interpretation. Proteolysis of empty capsids to release the external DNA for analysis by other means is an obvious goal, but it will first require development of protocols for their quantitative lysis.

4.4. Applications of Multi-Monitor HPLC Assays. HPLC assays with multiple monitors create many opportunities to improve the quality of information to guide development, validation, and monitoring of production processes for AAV. Beyond supporting purification process development [6], they offer potential to compare productivity of different host cells, compare and optimize cell culture formula- tions, optimize transfection protocols, determine when to harvest, and develop sample preparation methods. After basic processes have been established, these assays can be used to set specifications and document that all aspects of the production chain proceed in compliance with those specifications. HPLC also offers potential to document accuracy of other analytical methods or guide development of needed improvements. PCR is a good example. Accurate results require effective sample preparation methods. Sample preparation often involves treatment with proteolytic enzymes to liberate plasmid DNA from AAV capsids. However, Figure4 shows that barely 30% of the capsids are lysed after 1 h and barely 50% after 24 h. It stands to reason that deficient lysis must affect apparent titers. Nuclease enzymes are often used in PCR sample preparation with the intention to eliminate non-encapsidated plasmid, which is then followed by a step to remove or inactivate the enzyme to prevent digestion of the plasmids [61]. If that step is less than 100% effective, loss of plasmid DNA will depress the apparent titer. Multi-monitor HPLC can be applied either to obtain confidence that sample preparation steps perform as intended or provide a timely warning and the analytical tools to develop that confidence. The same is also true in reverse: other analytical methods can be used to interpret and validate the findings of HPLC-based assays. Co-characterization of empty and full capsid content by HPLC and cryo-TEM is an obvious example. HPLC characterization of full, partially full, and empty capsids fractionated by density gradient centrifugation is another. Either way, and whatever the analytical goal, complementarity among different analytical approaches increases the depth of insight they can provide. Pharmaceutics 2021, 13, 113 12 of 14

Author Contributions: Conceptualization, P.G., A.S.; methodology, B.G., S.P., N.M., T.Z. M.L., P.G.; original draft preparation, P.G.; review and editing, B.G., A.S, N.M., T.Z., M.L., P.G. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. Conﬂicts of Interest: The authors declare no conﬂict of interest. Sartorius had no role in the design of the study, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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