Methods for processing and analyzing viral capsid proteins

Abstract

The present disclosure provides methods for preparing digested viral proteins, including adenovirus and adeno-associated virus capsid proteins, from a sample of viral proteins, and methods for analyzing such digested viral proteins via liquid chromatography-tandem mass spectrometry. The methods include the use of a mixture of sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to rapidly and easily prepare digested viral proteins.

Description

[Technical field]

[0001] The present disclosure provides methods for preparing digested viral proteins, including adenovirus and adeno-associated virus capsid proteins, from a sample of viral proteins, and methods for analyzing such digested viral proteins via liquid chromatography-tandem mass spectrometry. The methods include the use of a mixture of sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to rapidly and easily prepare digested viral proteins. [Background technology]

[0002] Recombinant adeno-associated virus (AAV) vectors have become an excellent choice for gene therapy applications with high safety and efficacy due to low toxicity, availability of viral serotypes, and stable gene expression. See, e.g., Flotte, TR, Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Ther 2004, 11(10), 805-10.

[0003] AAV consists of a single-stranded DNA enclosed in an icosahedral protein capsid shell. The capsid is composed of 60 subunits of three viral proteins (VPs) (VP1, VP2, and VP3) in an approximate molar ratio of 1:1:10 that share a common C-terminal amino acid sequence.

[0004] A robust, convenient analytical approach to determine capsid viral protein heterogeneity is needed to complement recently developed methods for the production of recombinant AAV from producer cell lines, including Sf9 insect and human embryonic kidney HEK293 cells. For example, the U.S. Food and Drug Administration (FDA) recommends the identification of gene therapy products (e.g., AAV serotypes) from other products in the same facility. The most common current techniques for identifying AAV serotypes are enzyme-linked immunosorbent assays (ELISAs) and immunoblotting. However, both techniques lack sufficient sensitivity for products with a high degree of similarity, and highly specific antibodies must be generated for each type of AAV.

[0005] Several sample preparation strategies have been developed that allow efficient protein extraction and digestion prior to mass spectrometry analysis in proteomics studies, involving the use of detergents or chaotropic agents, followed by desalting or dilution necessary to preserve the protease activity of the digestive enzymes. However, the multiple work-up steps inevitably result in substantial sample loss, generating low-concentration, high-volume digested VP material that cannot be loaded onto an LC column in a single injection for detailed LC-MS / MS characterization of trace VPs. AAV analysis also requires additional sample handling steps (and resulting losses) relative to those in standard proteomics studies, including denaturation of the capsid with acetic acid to release the VPs, followed by exchange of buffers to those compatible with proteomics sample work-up. Although protocols involving such multi-step sample preparation can provide accurate structural information, they are time-consuming, lack robustness, and thus are not suitable for routine quantitative analysis.

[0006] Protein precipitation has been widely used to isolate proteins from diverse matrices, but its optimization remains a major challenge due to the variability of variable parameters, such as the target protein (chemistry and concentration), the matrix (especially the ionic strength of the formulation buffer), as well as the optimal incubation time, temperature, and type of organic solvent. All of these parameters can affect the performance of the method and may lead to low or inconsistent protein recovery. According to a recent study, optimized conditions for acetone precipitation of proteins from yeast lysates, resulting in 98 ± 1% recovery, include the addition of sodium chloride (1-100 mM) and a short incubation time (2 min) at room temperature (RT). However, an optimal procedure for precipitating proteins from a defined sample may result in substantial loss of protein in other samples with different matrices. Thus, specific optimization is especially important for VPs, since they are present at very low concentrations in complex matrices.

[0007] Detailed characterization of the three capsid viral proteins (VP1, 2, and 3) of adeno-associated virus is desperately needed to ensure the consistency of gene therapy products and processes. These proteins are typically present at very low concentrations in matrices containing high concentrations of excipients and salts. Thus, there is a need for a convenient method for sample preparation prior to proteomic analysis.

[0008] Similarly, adenoviruses (AdVs) have recently become widely used therapeutic vectors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines. AdVs are large, non-enveloped viruses with an icosahedral capsid formed from several proteins that enclose double-stranded DNA. Modifications in the type of cell line used or the scale of production and purification can affect the composition of AdVs, affect the interaction of viral particles with cells, and thus the biological activity and potency of the product. As VPs are major components and key players in the early stages of infection by viral particles, their heterogeneity and content must be assessed to ensure product and process consistency. Peptide mapping can provide detailed information about these proteins, e.g., their amino acid sequences and post-translational modifications (PTMs), which are important for the development and optimization of manufacturing processes. However, sample preparation remains the main bottleneck for successful proteomic analysis of viral proteins (VPs) of AdVs due to their low concentration and vast stoichiometric range.

[0009] The present invention provides a fast, reproducible VP sample preparation approach that involves protein precipitation followed by redissolution in sodium deoxycholate (SDC) / N-dodecyl-beta-D-maltoside (DDM), allowing the generation of low-volume tryptic digests without further clean-up steps. The suitability of this precipitation method was further evaluated by dissolving the resulting protein pellet in guanidine hydrochloride (Gu-HCl) followed by Asp-N digestion. Using this approach with trypsin and Asp-N digestion, respectively, 100% and 99.2% sequence coverage of AAV VP1 was obtained. In addition, the N- and C-terminal amino acid sequences of AAV VP1, VP2, and VP3 were fully characterized along with their PTMs.

[0010] As described herein, the use of lower SDC / DDM concentrations avoided the removal of SDC and allowed the identification of all major structural proteins of AdV5 with high amino acid sequence coverage (on average 92% of the amino acids in Adv5 VP) and the quantification of 53 PTMs in a single LC-MS / MS experiment using trypsin protease.

[0011] The presented method is highly reproducible, robust, and suitable for proteomic studies of viruses, such as AAV and AdV serotypes. Moreover, it is not labor-intensive and can be easily adapted to both large and small amounts of starting material. Summary of the Invention

[0012] In some embodiments, provided herein is a method of preparing digested viral proteins, the method comprising precipitating viral proteins from a sample containing the viral proteins, dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to generate a solution, and digesting the viral proteins with a protease.

[0013] In further embodiments, provided herein is a method of analyzing digested viral proteins, the method comprising precipitating viral proteins from a sample containing the viral proteins, dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to generate a solution, digesting the viral proteins with a protease, removing the SDC from the solution, and analyzing the digested viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0014] In embodiments, the step of removing SDC from the solution can be omitted, since it has been found that lower concentrations of SDC do not interfere with LC-MS / MS analysis.Therefore, also provided herein is a method of analyzing digested viral proteins, comprising precipitating viral proteins from a sample containing the viral proteins, dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to generate a solution, digesting the viral proteins with a protease, and analyzing the digested viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0015] In an embodiment, the viral protein is an adeno-associated virus capsid protein (AAV capsid protein), an adenovirus protein, a lentivirus protein, a retrovirus protein, or a herpes simplex virus protein. Preferably, the viral protein is an AAV capsid protein. In a further embodiment, the viral protein is an adenovirus protein, such as an adenovirus 5, 26, 35, or 48 protein. Preferably, the adenovirus protein is an adenovirus 5 protein. In an exemplary embodiment, the viral protein is a lentivirus protein.

[0016] Preferably, the viral proteins are dissolved in a mixture comprising about 0.01%-1.5% (w / w) SDC and about 0.01%-1.0% (w / w) DDM. For example, the mixture comprises about 0.5%-1.5% (w / w) SDC and about 0.01%-1% (w / w) DDM. In embodiments, the mixture comprises about 0.5%-1.5% (w / w) SDC and about 0.2%-1.0% (w / w) DDM, or the mixture comprises about 0.75%-1.25% (w / w) SDC and about 0.5%-0.8% (w / w) DDM. Preferably, the mixture comprises a ratio of about 1:0.5 w / w (SDC:DDM). The mixture may also include lower detergent concentrations, for example, about 0.01%-0.6% (w / w) SDC and about 0.01%-1% (w / w) DDM. In embodiments, the mixture may include about 0.01%-0.6% (w / w) SDC and about 0.01%-0.6% (w / w) DDM, or the mixture may include about 0.2%-0.4% (w / w) SDC and about 0.05%-0.2% (w / w) DDM. Suitably, such a mixture may include a ratio of about 3.5:1 w / w (SDC:DDM).

[0017] In embodiments, dissolution in solution occurs at about pH 6.0 to about pH 9.0. Preferably, digestion is performed at about 30° C. to 40° C. for a period of about 2 to 12 hours. In an exemplary embodiment, precipitation comprises precipitation with chloroform / methanol / water and centrifugation. Preferably, digestion comprises digestion with trypsin. In embodiments, digestion is performed at a viral protein:trypsin ratio of about 20:1 to about 100:1 w:w.

[0018] Suitably, the digested viral proteins are between about 3 and 70 amino acids in length.

[0019] In an exemplary embodiment, the analysis involves injecting the digested viral proteins into a liquid chromatography mass spectrometer without first performing a buffer exchange or desalting step. Preferably, the solution volume analyzed by LC-MS / MS is less than 50 μL. In a preferred embodiment, the sample containing viral proteins has a concentration of viral proteins of about 0.001 mg / mL to about 0.10 mg / mL. [Brief description of the drawings]

[0020] [Figure 1] 1 shows an overview of the viral protein extraction and analysis protocol described herein. [Diagram 2] 2A-2G show total ion current (TIC) chromatograms of monoclonal antibody A (mAb A) with the indicated percentages of SDC / DDM. [Figure 3-1] FIG. 3A shows the results of LC-UV-MS analysis of Anc80 capsid viral protein. [Figure 3-2] Figures 3B and 3C show the results of LC-UV-MS analysis of Anc80 capsid viral protein. [Figure 4] The extracted ion chromatogram (upper panel) and MS / MS spectrum (lower panel) of the tryptic peptide of T23 are shown. [Diagram 5] The extracted ion current (XIC) and MS / MS spectrum of tryptic peptide T33 are shown. [Figure 6] 6A to 6C show the MS / MS spectra of the N- and C-terminal peptides of VP1. [Figure 7] 7A to 7D show MS / MS spectra of the N-terminal amino acids of VP2 and related PTMs after Asp-N digestion. [Figure 8] The MS / MS spectrum of the N-terminal amino acid sequence of VP3 is shown. [Figure 9] As described herein, a modified viral protein extraction and analysis protocol is outlined that does not require removal of SDC. [Figure 10] Total ion current (TIC) chromatograms of monoclonal antibody A (mAb A) are shown. (A) TIC of the supernatant after SDC removal and (B) TIC of the SDC pellet washed with water. [Figure 11] TIC chromatograms of trypsin-digested mAb A are shown after treatment with 0.1%-0.4% w / v SDC and 0.1% w / v DDM without SDC removal (top seven panels) or with 1% w / v SDC and 0.5% w / v DDM with additional SDC removal. [Figure 12] Extracted ion currents (XICs) of eight selected peptides of monoclonal antibody A (mAb A) after treatment with 0.1%-0.4% w / v SDC and 0.1% w / v DDM without SDC removal (top seven panels) or with 1% w / v SDC and 0.5% w / v DDM with additional SDC removal are shown. [Figure 13] MS / MS spectra of the N-terminal amino acid sequences of pIX and pVI are shown. (A) Acetylated peptide from the N-terminus of pIX, containing no methionine and acetylated serine. (B) Acetylated methionine from the N-terminal peptide of pVI, and (C) Acetylated and oxidized methionine. Expanded mass spectra of the b1 ion are shown in B and C, with the observed mass shift (+16 Da) in the corresponding insets. [Figure 14] Representative MS / MS spectra of (A) phosphorylated peptide and (B) deamidated peptide of AdV5 VP are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The use of the words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, may mean "one," but may also be consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0022] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device used to determine the value. Typically, the term is meant to encompass a variation of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or less, depending on the context, e.g., the accuracy of the measurement method.

[0023] Although use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0024] As used in this specification and the claims, the terms "comprising" (and any form of "comprising", e.g., comprise and comprise), "having" (and any form of having, e.g., have and has), "including" (and any form of "including", e.g., includes and include), or "containing" (and any form of "containing", e.g., contains and contain) are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps.

[0025] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is a powerful technique to study the structure of VPs and their post-transcriptional modifications (PTMs). However, preparation of VP samples for characterization by LC-MS / MS is challenging because these proteins are typically present at very low concentrations in matrices that often contain excipients and high concentrations of salts. Thus, efficient sample preparation is important for precise and accurate results.

[0026] The methods described herein involve a sample preparation approach involving protein precipitation followed by redissolution in a mixture of sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM), allowing for the generation of low volume digests without further clean-up steps for viral protein analysis via liquid chromatography-tandem mass spectrometry.

[0027] In an exemplary embodiment, a method for preparing digested viral proteins (VPs) is provided, comprising: a) precipitating viral proteins from a sample containing viral proteins; b) dissolving viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to produce a solution; c) digesting the viral proteins with a protease.

[0028] Suitably, steps a) to c) of the method described herein are performed consecutively in this order: a) to c). As described herein, the result of step a) provides a precipitate containing precipitated viral proteins. The precipitate of step a) is dissolved in step b). In step c) the viral proteins present in the solution of step b) are digested with a protease to provide a solution of digested viral proteins, i.e. a solution of peptides.

[0029] 1. A method for analyzing digested viral proteins, comprising: a) precipitating viral proteins from a sample containing viral proteins; b) dissolving viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to produce a solution; c) digesting viral proteins with a protease; d) removing SDC from the solution; e) analyzing the digested viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0030] Suitably, steps a) to e) of the analytical method described herein are performed consecutively in this order: a) to e). As described herein, the result of step a) provides a precipitate containing precipitated viral proteins. The precipitate of step a) is dissolved in step b). In step c) the viral proteins present in the solution of step b) are digested with a protease to provide a solution of digested viral proteins, i.e. a solution of peptides. In step d) the SDC is removed from the solution of digested viral proteins to provide a solution ready for analysis. In step e) the digested viral proteins are analyzed.

[0031] Step d) can be omitted if the SDC concentration is low enough so as not to interfere with the LC-MS / MS analysis. Thus, a method for analyzing digested viral proteins comprising: a) precipitating viral proteins from a sample containing viral proteins; b) dissolving viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to produce a solution; c) digesting viral proteins with a protease; e) analyzing the digested viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0032] Advantageously, steps a) to e) of the analytical method may be performed consecutively in this order: a) to c) and e). As described herein, the result of step a) provides a precipitate containing precipitated viral proteins. The precipitate of step a) is dissolved in step b). In step c) the viral proteins present in the solution of step b) are digested with a protease to provide a solution of digested viral proteins, i.e. a solution of peptides. In step e) the digested viral proteins are analyzed.

[0033] As used herein, the term "viral protein" refers to a protein that forms a part of a virus, for example a protein that forms a structural component of a virus, such as a viral capsid protein.

[0034] Examples of viral proteins or viral proteins that can be prepared and analyzed according to the methods described herein include, but are not limited to, adeno-associated virus capsid proteins (AAV capsid proteins), adenovirus proteins, lentivirus proteins, retrovirus proteins, and herpes simplex virus proteins.

[0035] In a preferred embodiment, the viral protein is an adeno-associated virus (AAV) protein, in particular an AAV capsid protein. AAV is composed of a single-stranded DNA enclosed in an icosahedral protein capsid shell. The capsid is composed of 60 subunits of three viral proteins (VP1, VP2, and VP3) in an approximate molar ratio of 1:1:10, which share a common C-terminal amino acid sequence.

[0036] In further embodiments, the viral protein is an adenovirus protein adenovirus containing double-stranded DNA inside an icosahedral capsid with a total molecular weight of about 150 MDa. Human adenovirus capsid is composed of 13 different proteins, herein referred to as viral proteins "VP", classified as major proteins (hexon, penton base, and fiber), cement / minor proteins (pIIIa, pVI, pVIII, and pIX), and core proteins (pV, pVII, pTP, pμ, AVP, and pIVa2). The major and minor proteins account for the highest and lowest percentages (about 64.1% and about 15.6%, respectively) of the total weight of AdV5 proteins. In an exemplary embodiment, the adenovirus protein is an adenovirus 5, 26, 35, or 48 protein.

[0037] Lentiviruses contain a single-stranded RNA genome with reverse transcriptase. Exemplary lentiviral proteins are known in the art.

[0038] It is understood that "AAV capsid protein" means to include two or more AAV capsid proteins, including three viral proteins from AAV in different ratios and amounts. Similarly, "AdV VP" means to include two or more AdV proteins, including thirteen viral proteins from AdV in different ratios and amounts.

[0039] As used herein, "precipitation" refers to a method in which proteins from a virus, including AAV capsid proteins, are removed from solution to allow for further analysis and characterization, including through various instruments, such as mass spectrometry. In an embodiment, the method preferably includes precipitating viral proteins from a sample containing the viral proteins. "Sample" refers to the product of any biological reaction that produces a virus, including adenovirus, lentivirus, retrovirus, herpes simplex virus, or AAV, and preferably refers to the production of viruses from one or more cell lines, including human embryonic kidney (HEK) cells, including, for example, HEK-293, Sf9 cell lines, HeLa cells, and the like.

[0040] Various methods of precipitating viral proteins are known in the art. For example, viral proteins can be precipitated using a chloroform / methanol / water precipitation technique, which involves centrifugation to form a protein pellet. Such a method involves the sequential addition of methanol, chloroform, and water to a sample, with a short vortex mixing and high-speed centrifugation step (10 seconds at 14,000 g) after each addition. Protein precipitates generally appear at the interface as a white layer between the upper and lower phases. The upper phase can be removed and discarded, and then cold methanol is added to the remaining mixture. After centrifugation, the supernatant is removed. Finally, the pellet is dried by vacuum centrifugation.

[0041] Additional precipitation methods include the use of cold acetone followed by storage in a freezer (eg, for about 60 minutes), centrifugation, removal of the supernatant, and then drying of the protein pellet.

[0042] After precipitation of the viral proteins, the viral proteins are dissolved in a mixture containing sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to produce a solution. As used herein, "dissolving" viral proteins refers to the production of a working solution of viral proteins in a mixture containing SDC and DDM, but does not necessarily require complete dissolution of viral proteins. Instead, dissolution may also include the production of a suspension, or near suspension, of viral proteins in the mixture (i.e., while a clear solution may be formed, a cloudy or slightly precipitated solution / suspension may also result when using an SDC / DDM mixture).

[0043] As described herein, it has surprisingly been found that the use of a mixture of SDC and DDM as part of a viral protein digestion significantly reduces sample processing steps, making it accessible to a wide range of biology laboratories and eliminating the need for high expertise in proteomics workflows. In an exemplary embodiment, viral proteins are dissolved in a mixture comprising about 0.01%-1.5% (w / w) SDC and about 0.01%-1.0% (w / w) DDM.

[0044] In an embodiment, the viral proteins are dissolved in a mixture comprising about 0.5%-1.5% (w / w) SDC and about 0.01%-1.0% (w / w) DDM, preferably about 0.5%-1.5% (w / w) SDC and about 0.2%-1.0% (w / w) DDM, more preferably about 0.5%-1.5% (w / w) SDC and about 0.4%-1.0% (w / w) DDM. The mixture is an aqueous mixture. For all percentages expressed as w / w, the percentage refers to the weight of the component relative to the total weight of the mixture. For example, the amount of SDC in the mixture can be about 0.4%-1.6%, or about 0.5%-1.5%, or about 0.6%-1.5%, about 0.75%-1.25%, about 0.8%-1.2%, about 0.9%-1.1%, or about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, or about 1.2% (w / w). The amount of DDM in the mixture can be about 0.01% to 1.0% (w / w), or about 0.02% to 1.0% (w / w), or about 0.05% to 1.0% (w / w), or about 0.1% to 0.6%, or about 0.2% to 1.0% (w / w), or about 0.3% to 1.1% (w / w), or about 0.4% to 1.0%, about 0.5% to 0.9%, about 0.5% to 0.8%, about 0.5% to 0.7%, about 0.5% to 0.6%, or about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, or about 0.9% (w / w).

[0045] In a preferred embodiment, the mixture comprises about 0.75%-1.25% (w / w) SDC and about 0.5%-0.8% (w / w) DDM, more preferably the mixture comprises a ratio of about 1:0.5 (w / w) (SDC:DDM).

[0046] The mixture of SDC and DDM is prepared in a suitable buffer, for example a bicarbonate buffer, such as ammonium bicarbonate, for example, about 30 mM to 100 mM, or about 30 mM to 70 mM, preferably about 50 mM ammonium bicarbonate, and having a pH of 6 to 9, or about pH 8. Additional buffers known in the art, for example Tris-HCl buffers, can also be used to prepare the mixture. Preferably, the mixture is prepared at a pH of about pH 6.0 to about pH 9.0, such that dissolution of viral proteins occurs at a pH of about pH 6.0 to about pH 9.0, preferably at a pH of about pH 7 to about pH 9, more preferably at about pH 8.

[0047] The method further comprises digesting the viral proteins with a protease. As used herein, "protease" refers to an enzyme that degrades proteins, specifically an enzyme that can degrade viral proteins. Exemplary proteases for use in the methods described herein include, for example, trypsin, Asp-N, Lys-C, Lys-N, chymotrypsin, or Glu-C proteases, preferably trypsin or Asp-N. Additional proteases that can be used in the methods described herein are known in the art.

[0048] The time and conditions for digestion with proteases will vary based on the protease selected. However, when trypsin is utilized, digestion is generally carried out at about 30°C to 40°C (preferably about 37°C) for a period of about 2 to about 4 hours, for example, about 2 to 12 hours, including about 3 hours. The amount of protease used will also vary based on the enzyme selected. For trypsin digestion, the protease is preferably used in a ratio of about 20:1 to about 100:1 w:w, or about 20:1 to about 40:1 w:w, more preferably about 30:1 w:w, the ratio being the weight ratio of viral protein to trypsin (viral protein:trypsin). Preferably, the protein is dissolved at about pH 6.0 to about pH 9.0 and at about 30°C to 40°C. Lysis is a fast step, usually in the range of a few minutes, but can extend over a period of about 2-12 hours, such as about 2-4 hours, for example, up to about 3 hours.

[0049] Protease digestion can be stopped using any suitable method, including, for example, the addition of acid, such as trifluoroacetic acid (TFA) or difluoroacetic acid (DFA) in the case of trypsin, and formic acid for Asp-N.

[0050] In an embodiment, after digestion of viral proteins with proteases, SDC is preferably removed from the solution. This solution can then be utilized in an analytical protocol, including via LC-MS / MS, as described herein, or can be stored as desired for subsequent analysis. SDC can be removed from the digest, for example, by oxidation (i.e., acid precipitation). Methods for removing SDC from solution preferably include separating SDC (which may appear as a slight turbidity) via centrifugation and removing the supernatant containing the viral proteins. However, this step also results in loss of peptides due to handling issues and because peptides, especially long hydrophobic ones, may co-precipitate with SDC. For example, experiments described herein show that washings of SDC precipitates still contain about 20% of the peptides previously present in the mixture. This is particularly undesirable when analyzing proteins present in low amounts, as may occur in the analysis of gene therapy vectors, e.g., AdV.

[0051] As described further herein, it has surprisingly been found that the use of a mixture of SDC and DDM at lower concentrations avoids the need for SDC removal (which may otherwise suppress electrospray ionization and interfere with chromatographic separation of peptides), thus reducing sample loss and eliminating another time-consuming processing step, while still allowing for excellent protein solubilization and denaturation for subsequent protease (e.g., trypsin) cleavage. As an added advantage, such mixtures can be directly analyzed using LC-MS / MS after the digestion step without stopping the protease digestion reaction. Thus, in other exemplary embodiments, the viral proteins are dissolved in a mixture comprising about 0.01%-0.6% (w / w) SDC and about 0.005%-1.0% (w / w) DDM, preferably about 0.01%-0.6% (w / w) SDC and about 0.005%-1.0% (w / w) DDM, more preferably about 0.01%-0.5% (w / w) SDC and about 0.01%-1.0% (w / w) DDM. More specifically, the mixture may comprise about 0.01%-0.5% (w / w) SDC and about 0.01%-0.6% (w / w) DDM. In such embodiments, the method may suitably not include a step d) of removing SDC from the solution. The mixture is an aqueous mixture. For all percentages expressed as w / w, the percentage refers to the weight of the component relative to the total weight of the mixture. For example, the amount of SDC in the mixture can be about 0.01%-0.6%, or about 0.02%-0.5%, or about 0.05%-0.5%, about 0.1%-0.5%, about 0.2%-0.4%, about 0.3%-0.4%, or about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, or about 0.4% (w / w). The amount of DDM in the mixture can be about 0.005%-1.0% (w / w), about 0.01%-1.0% (w / w), or about 0.01%-0.8% (w / w), or about 0.01%-0.6%, about 0.02%-0.6%, about 0.05%-0.6%, about 0.05%-0.5%, about 0.05%-0.2%, or about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, or about 0.5% (w / w).

[0052] In a preferred embodiment, the mixture comprises about 0.2%-0.4% (w / w) SDC and about 0.05%-0.2% (w / w) DDM. Preferably, the mixture comprises a ratio of about 3.5:1 (w / w) (SDC:DDM).

[0053] Thus, the modified method using lower concentrations of SDC and DDM represents a significant improvement in terms of required sample processing steps and reduced sample loss while maintaining sequence coverage due to the ability of the SDC / DDM mixture to increase potential protease (e.g., trypsin) cleavage sites and enhance the solubility of hydrophobic peptides.

[0054] As described herein, it has surprisingly been found that the use of a mixture of SDC and DDM as part of the viral protein digestion process does not require any clean-up steps after digestion.

[0055] It is well known that detergents can significantly interfere with reversed-phase (RP) chromatography and MS analysis of viral proteins, so detergent removal significantly improves the analysis of viral proteins via LC-MS / MS. Examples of detergents that can be removed via the methods described herein include various poloxamers (non-ionic triblock copolymers) and other non-ionic detergents such as Triton X-100, NP-40, Tween 20, and Tween 80.

[0056] The methods described herein allow the preparation, and ultimately the analysis, of digested viral proteins having an amino acid length of up to 100 amino acids. For example, the viral proteins extracted, digested with different proteases, and ultimately analyzed preferably comprise peptides having a length of 3-100 amino acids, including 3-90 amino acids, 3-80 amino acids, 3-70 amino acids, 3-60 amino acids, 3-50 amino acids, or 3-40 amino acids.

[0057] In embodiments, the methods described herein further comprise analyzing the viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS) after digestion of the viral proteins with a protease. In such embodiments, the method may further comprise removing SDC from the solution after digestion but prior to analysis. Removal may be accomplished by centrifugation with subsequent separation of the protein-containing fraction for subsequent analysis by LC-MS / MS.

[0058] In additional embodiments, methods for analyzing viral proteins are provided herein. As described herein, the methods for analyzing viral proteins allow for characterization of the amino acid sequences of the N- and C-terminal regions of viral proteins, along with full sequence confirmation of proteins and information on their post-translational modifications. In embodiments where the analyzed viral protein is a viral capsid protein (including AAV capsid protein), the analysis methods described herein provide high confidence confirmation of capsid identity and can distinguish serotypes with mass differences of less than 10 Da in capsid proteins.

[0059] The method of analysis preferably includes precipitating viral proteins from a sample containing the viral proteins, dissolving the viral proteins in a mixture containing sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to produce a solution, digesting the viral proteins with a protease, and optionally, preferably removing the SDC from the solution, and analyzing the viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0060] As described herein, the mixture for use in lysing viral proteins preferably comprises about 0.01%-1.5% (w / w) SDC and about 0.01%-1.0% (w / w) DDM. In an exemplary embodiment, the mixture comprises about 0.5%-1.5% (w / w) SDC and about 0.2%-1.0% (w / w) DDM, or the mixture comprises about 0.75%-1.25% (w / w) SDC and about 0.5%-0.8% (w / w) DDM, preferably the mixture comprises a ratio of about 1:0.75 w / w (SDC:DDM). The mixture may also comprise a lower detergent concentration, for example, about 0.01%-0.6% (w / w) SDC and about 0.01%-1.0% (w / w) DDM. Such a mixture may also comprise about 0.01%-0.6% (w / w) SDC and about 0.01%-0.6% (w / w) DDM, or the mixture may comprise about 0.2%-0.4% (w / w) SDC and about 0.05%-0.2% (w / w) DDM. Suitably, the mixture may comprise a ratio of about 3.5:1 w / w (SDC:DDM). In embodiments using lower detergent concentrations, the method may suitably not include step d) of removing SDC from the solution.

[0061] Various methods for precipitating viral proteins are described herein, including precipitation with chloroform / methanol / water and the use of centrifugation. Suitably, viral proteins are digested with a protease, including, for example, trypsin. Exemplary methods for trypsin digestion are described herein and include digestion with trypsin at a ratio of about 20:1 to about 100:1 w:w (viral protein:trypsin).

[0062] As described herein, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is a powerful technique for studying the structure and PTMs of viral proteins. However, preparation of viral protein samples (including viral capsid proteins) for characterization by LC-MS / MS is challenging because these proteins are typically present at very low concentrations in matrices that often contain excipients and high concentrations of salts. Non-ionic detergents, such as poloxamers, are frequently used to improve manufacturing processes to increase yields in gene therapy. Even when present at very low concentrations (e.g., 0.001%, w / v), poloxamers can strongly interfere with reversed-phase (RP) chromatography and MS analysis of VPs. Thus, efficient sample preparation is important for precise and accurate results, and as described herein, methods substantially remove any detergents present in viral samples, including poloxamers.

[0063] Methods for analyzing viral proteins are described throughout. Advantageously, the analysis method involves injecting digested viral proteins into a liquid chromatography mass spectrometer, but first performing a buffer exchange or desalting step. The use of a mixture of SDC and DDM as described herein allows the omission of such buffer exchange and desalting steps, significantly reducing the time and cost of viral protein analysis methods and reducing the complexity of such methods. Furthermore, as pointed out above, it has been found by the inventors that the lower concentrations of the SDC / DDM mixture described herein are fully compatible with LC-MS / MS analysis and therefore can remain in the sample.

[0064] The analytical methods described herein suitably utilize solution volumes for analysis via LC-MS / MS that are less than 50 μL. In embodiments, the solution volumes used for analysis via LC-MS / MS are from about 3 μL to about 50 μL, or from about 10 μL to about 50 μL, or from about 20 μL to about 50 μL.

[0065] The extraction and analysis methods can be used with samples having a viral protein concentration of about 0.001 mg / mL to about 0.10 mg / mL. As described throughout, the methods are suitable for analysis of digested viral proteins having a length of about 3 to 70 amino acids. EXAMPLES

[0066] Example 1: Extraction and analysis of AAV capsid proteins Figure 1 shows an overview of the extraction and analysis protocol outlined in the Examples below. The workflow involves the concentration of Anc80 AAV samples with a cut-off filter to reduce their volume. Capsid proteins are precipitated, the protein pellet is dissolved in a selected denaturing reagent, and the proteins are digested using either trypsin or Asp-N. Finally, the generated peptides are analyzed by LC-MS / MS. The SDC / DDM ratio was optimized for trypsin digestion and two common precipitation approaches were compared.

[0067] Materials and Methods Chemicals Dithiothreitol (DTT), Tris 2-carboxyethylphosphine (TCEP), ammonium bicarbonate (ABC), iodoacetamide (IAA), ultrapure formic acid, acetic acid, guanidine-HCl (Gu-HCl), Tris-HCl, acetone, methanol, acetonitrile (ACN), water, trifluoroacetic acid (TFA), sodium deoxycholate (SDC), and Tris base were purchased from Sigma-Aldrich (St. Louis, MO). Amicon Ultra-4 filters (10 kDa MWCO) were purchased from Millipore (Billerica, MA). Sequencing grade trypsin was purchased from Promega (Milwaukee, WI). Asp-N protease was purchased from Roche Diagnostics (Indianapolis, IN). Zeba spin desalting columns (7K MWCO, 0.5 mL) and N-dodecyl-beta-D-maltoside (DDM) were purchased from Thermo Fisher Scientific (Waltham, Mass.), and difluoroacetic acid (DFA) was purchased from Waters (Milford, Mass.).

[0068] Vector production and purification Anc80 samples were produced and purified on a scalable manufacturing platform at Lonza Houston, Inc. Transient triple transfection of suspension HEK293 cells was used to produce Anc80 in a 250 L single-use bioreactor as described in Bingnan Gu et al., Cell & Gene Therapy Insights 2018, 4(S1), 753-769, DOI: 10.18609 / cgti.2018.080, and an aliquot of recombinant Anc80 was purified using affinity chromatography followed by ion exchange chromatography to provide a sample of purified Anc80.

[0069] Sample preparation A sample of Anc80 AAV capsid protein was prepared as described below. A recombinant human monoclonal IgG4 antibody (designated mAb A) was also produced and purified at Lonza using standard manufacturing procedures.

[0070] 100 μL samples of purified Anc80 prepared as described above containing approximately 1 μg of Anc80 AAV capsid protein (VP) were subjected to intact protein analysis as described below after denaturing them by reducing their volume from 100 μL to 20 μL, adding 2 μL of acetic acid, and incubating the resulting mixture at room temperature for 10 min prior to LC-MS analysis for intact protein analysis as described below.

[0071] Peptide mapping was performed after reducing the volume of a 500 μL sample of purified Anc80 prepared as described above (containing approximately 5 μg of Anc80 AAV capsid protein (VP)) to 100 μL before protein precipitation as described below to provide the so-called "100 μL concentrated sample." The volume was reduced by centrifuging the sample through a 10 kDa cut-off filter, also known as a membrane filter, at 10,000 g and 20° C.

[0072] Chloroform / methanol / water protein precipitation (approach 1): VPs were precipitated with chloroform / methanol / water. Briefly, 400 μL of methanol, 100 μL of chloroform, and 300 μL of water were added successively to 100 μL of concentrated sample (prepared as described above), with a short vortex mixing and high-speed centrifugation step (10 s at 14,000 g) after each addition. The protein precipitate appeared at the interface as a white layer between the upper and lower phases. The upper phase was carefully removed and discarded, and then 300 μL of cold methanol was added to the remaining mixture. After centrifugation, the supernatant was removed. Finally, the protein pellet was dried by vacuum centrifugation.

[0073] All precipitation steps used cold solvents and centrifugation was performed at 14,000 g and 4° C. for 10 min.

[0074] Cold acetone protein precipitation (approach 2): Ice-cold acetone (400 μL) was added to 100 μL of concentrated sample (prepared as described above) and the solution was stored in a −20° C. freezer for 60 min. The sample was then centrifuged at 14,000 g and 4° C. for 10 min, the supernatant was removed and the protein pellet was dried by vacuum centrifugation.

[0075] Lysis with SDC and DDM and enzymatic digestion of capsid proteins For trypsin digestion, the protein pellet prepared as described above was dissolved in a 35 μL mixture of SDC (1% w / w) and DDM (0.5% w / w) in 50 mM ammonium bicarbonate (pH 8.0) and then vortex mixed at room temperature. The protein was reduced by adding 1 μL of 500 mM TCEP and incubating at 50° C. for 30 min, then subjected to trypsin digestion at 37° C. for 3 h (at a protein to protease ratio of 30:1 w / w). The digestion was stopped by adding 1 μL of TFA followed by careful mixing. The SDC, which appears as a slight turbidity, was separated by centrifugation (as above) and the supernatant was transferred to HPLC vials for LC-MS / MS analysis as described below, thereby removing the SDC.

[0076] For Asp-N digestion, protein pellets prepared as described above were dissolved in 6M Gu-HCl / 0.1M Tris and then subjected to reduction and alkylation with DTT and IAA, respectively. Samples were then desalted using Zeba spin filters according to the manufacturer's recommendations and subjected to protein digestion with Asp-N (protein to protease ratio of 30:1 w / w) for 12 h at 37° C. Enzyme activity was stopped by adding 5 μL of formic acid and the peptides generated were then analyzed by LC-MS / MS as described below.

[0077] Liquid chromatography-mass spectrometry Peptide mapping analysis A system consisting of a Vanquish UPLC coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for all analyses. For peptide mapping analysis, peptides in digested samples were separated using an Acquity UPLC peptide BEH C18 column (300 Å, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation) with a mobile phase consisting of 0.1% v / v formic acid in water (A) and acetonitrile (B). Peptides were eluted with a linear gradient from 1% v / v B to 60% v / v B over 80 min at a flow rate of 0.25 mL / min. The column was then washed with 98% v / v B for 10 min and conditioned with 1% v / v B for 8 min before the next injection.

[0078] The mass spectrometer was operated data-dependently in the m / z range of 200–2000. Full scan spectra were recorded at 2.0 e with a maximum injection time of 100 ms. 5 The ions were recorded at a resolution of 120,000 using an automatic gain control (AGC) target value of 0.25 nm. A maximum of 20 of the most intense ions with charge states between 2 and 8 were selected for high-energy c-trap dissociation (HCD) with a normalized collision energy of 35%. Fragment spectra were recorded at a resolution of 0.25 nm using an automatic gain control (AGC) target value of 0.25 nm. 4 The data were recorded at an isolation width of 2.5 Da and a resolution of 15,000, using an AGC target of 0.01 and a maximum injection time of 200 ms. Peaks were dynamically excluded from precursor selection for 5 s within a 10 ppm window. Selected peptides were subjected to electrospray ionization at a spray voltage of 3.5 kV and a heated capillary temperature of 320 °C.

[0079] Intact protein analysis Intact VP was separated using a MAbPac RP column (4 μm, 3.0 mm × 100 mm, Thermo Fisher Scientific) operated at 60 °C with a mobile phase consisting of 0.1% v / v DFA in water (A) and acetonitrile (B) at a flow rate of 0.25 mL / min. VP was eluted with a linear gradient from 10% v / v B to 38% v / v B over 54 min, followed by 2 min at 100% v / v B. The column was conditioned at the end of the 14 min gradient with 10% v / v B before the next injection was started. The UV signal from the eluate was recorded at 280 nm.

[0080] The mass spectrometer was operated with the following settings: capillary voltage 3.5 kV, surface-induced dissociation (SID) voltage 50%, resolution 17,500, and capillary temperature 320° C. Mass spectra were acquired in the positive mode in the range of m / z 800–3,500.

[0081] Data analysis Raw mass spectral data were processed with Protein Metrics (San Carlos, CA). For peptide mapping, database searches against the Anc80 viral capsid protein sequences (VP1, VP2, and VP3) were performed with tolerances of 6 and 20 ppm for peptides detected by MS and MS / MS analysis, respectively. N-terminal acetylation, methionine oxidation, phosphorylation, and asparagine deamidation were included in the search as variable modifications.

[0082] Intact mass spectra were deconvoluted with the following settings: mass range 55,000-85,000 Da, minimum difference between mass peaks 15 Da, maximum number of mass peaks 10, and peak sharing disabled.

[0083] Results and Discussion Optimization of SDC / DDM ratio for trypsin digestion Trypsin is one of the main proteases currently used in bottom-up proteomics research. However, regardless of the protease used, it is important to ensure that the target protein is denatured, that the digestion buffer sufficiently preserves the activity of the protease, and that the matrix containing the digest is compatible with LC-MS / MS analysis. This often requires multiple buffer exchange or desalting steps. The approach described herein shortens the sample preparation process that involves the use of SDC, since it can effectively denature proteins without compromising protein digestion prior to LC-MS / MS analysis. It should be noted that trypsin is active in solutions with up to 10% SDC, but increasing its concentration increases the loss of hydrophobic peptides through co-precipitation with the degraded detergent. Such losses are particularly undesirable in tryptic digestion of VPs, which may generate several long peptides that may co-precipitate during SDC precipitation or may elute poorly from the desalting column, causing loss of information. In an effort to minimize these potential problems, DDM is used as a combinatorial detergent to increase the solubility of hydrophobic peptides and avoid their co-precipitation with SDC. DDM is a non-ionic detergent that is compatible with tryptic digestion and chromatographic separation of peptides. A high concentration (≧80%) of ACN is required to elute from the reversed-phase chromatography column, which does not alter the performance of the column during peptide separation. In addition, the peak intensity of DDM eluting at the end of the reversed-phase gradient is low due to its weak ionization in electrospray ionization.

[0084] To maximize the recovery of hydrophobic peptides, different concentrations of DDM were first investigated. As described in Materials and Methods, a monoclonal antibody (mAb A) was denatured using 1% SDC (w / w) with different concentrations of DDM (0.0, 0.5, 0.75, 1.0, 1.5, and 2.0% w / w) and then subjected to trypsin digestion. A tryptic peptide with 49 amino acids was selected to assess the recovery. The peak intensity first increased with increasing DDM concentration and was maximum when the DDM concentration was 0.5-0.75% (see Figures 2A-2G). The similarity of the peaks in the total ion current (TIC) chromatograms of the tryptic digest of mAb A indicated that the addition of DDM to SDC did not modify the digestion performance of trypsin. The TIC of the tryptic digest of mAb A with only DDM revealed incomplete digestion related to the inability of DDM to denature the protein. However, the use of higher percentages of DDM is not recommended as it may reduce the loading capacity of the reversed-phase column, therefore 1% SDC with 0.5% DDM was utilized in the following experiments.

[0085] Performance of tested protein precipitation approaches Protein precipitation has two major advantages for VP sample preparation over other commonly used approaches. First, the capsid of AAV (such as Anc80) can already be denatured immediately after the addition of the organic solvent added for the purpose of precipitation, and precipitated simultaneously with or following this addition of the organic solvent VP. It therefore allows the omission of the capsid acid denaturation step and subsequent buffer exchange into a neutral buffer applied in common proteomics workflows. Second, it can separate VP from a wide range of detergents, and the precipitated VP can be easily dissolved in a low volume of denaturing reagent. Thus, workflows applying protein precipitation allow the analysis of all digested material in a single LC-MS / MS run, as well as detailed characterization of VP, which is typically available in process development or downstream processing samples at low concentrations and / or amounts.

[0086] In addition to the complexity of the matrix, the difference in the amount of VP1 and VP2 relative to VP3 (due to their approximately 1:1:10, VP1:VP2:VP3 molar ratio) poses a major challenge for proteomics studies. Inadequate sample preparation can result in sample loss, reducing the signal-to-noise threshold of VPs (especially VP1), and therefore loss of information. The performance of two frequently applied protein precipitation approaches was compared: precipitation with chloroform / methanol / water (approach 1) and precipitation with acetone (approach 2). Both approaches require organic solvent volumes larger than the sample volume and are not suitable for samples with volumes larger than 300 μl. Thus, samples require concentration by filter membranes with appropriate molecular weight cutoffs before analysis, as described in Materials and Methods.

[0087] Although the protein pellets obtained using both precipitation approaches applied in this study were digested under the same conditions, the results of the subsequent LC-MS / MS runs were substantially different. Approach 1 yielded stronger signal intensities of detected peptides and much richer information about amino acid sequences than approach 2, with >98% coverage. Another difference between the two approaches was in the solubility of the protein pellets in the SDC / DDM solution. Although the protein pellets obtained using both approaches were dissolved in the same amount of SDC / DDM, the pellets obtained with approach 1 disappeared completely, while the solutions obtained with approach 2 became slightly turbid. It has been shown that biphasic precipitation can remove DNA more efficiently than acetone precipitation before proteomic analysis, and we hypothesize that the insolubility of the pellets obtained using approach 2 could be related to the co-precipitation of DNA with VPs. The removal of unwanted cellular material, such as lipids and DNA, is important, as it can substantially interfere with enzymatic digestion and chromatographic separation.

[0088] Our results show that precipitation applying approach 2 does not work well for VP in the sample matrix used in this study, whereas approach 1 gives good results. Therefore, only data obtained using approach 1 is presented in the peptide mapping section below.

[0089] Intact protein analysis Since the main objective was to develop a fast, efficient, and robust procedure for preparing samples of VPs, intact mass spectrometry was applied in an initial screening to detect all VP species present in the samples. As shown by the UV chromatogram in Figure 3A, VP1 and VP2 eluted first and formed a shoulder of the major (VP3) peak. UV (280 nm) chromatograms (Figure 3A) and deconvoluted mass spectra of peaks eluting at retention times of 46.5 min (Figure 3B) and 47.2 min (Figure 3C). Zoomed-in deconvoluted mass spectra from the most intense signals from each peak are displayed in the corresponding insets.

[0090] Four major peaks were detected at masses of 81,194.6 Da, 66,001.5 Da, 59,411.0 Da, and 59,395.0 Da. The measured mass of 66,001.5 Da matches the amino acid sequence 139–736 of VP2, whereas the masses of 81,194.6 Da and 59,395.0 Da correspond to amino acids 2–736 of VP1 and amino acids 204–736 of VP3, respectively, with a mass shift of 42 Da in both cases, indicating one acetylation in each VP.

[0091] The other major peak, with a mass of 59,411.0 Da, corresponds to an oxidized variant of the acetylated 204-736 sequence of VP3. In addition, a weak signal with a mass of 59,350.0 Da was detected, corresponding to amino acids 204-736 of VP3. Several low intensity signals with different modifications of VP2 (acetylation and phosphorylation) were also detected. Since no evidence of disulfide bonds was reported, the theoretical mass of each VP was calculated assuming the absence of reduced disulfide bonds. The most plausible assignments of all detected signals are shown in Table 1. [Table 1]

[0092] Peptide mapping analysis: Amino acid sequence coverage Multiple digestion strategies have been applied to LC-MS / MS analysis of VPs to ensure complete coverage of their amino acid sequences, confirm the N- and C-terminal amino acid sequences, and quantify and localize the positions of PTMs. Trypsin digestion typically does not provide complete sequence coverage due to the low frequency of arginine (R) and lysine (K) in the VP sequence, resulting in a high frequency of R, which causes the generation of long hydrophobic tryptic peptides, or small hydrophilic tryptic peptides that may be missed in subsequent LC-MS / MS analysis.

[0093] Peptides generated by electrospray ionization after trypsin digestion have multiple charge states (≥2), mainly due to their C-terminal amino acids (K and R). Therefore, MS / MS spectral acquisition in this study was designed to detect ions with multiple charge states, as described in Materials and Methods. Three hydrophilic peptides could not be identified in the VP1 amino acid sequence, and thus there were gaps in the resulting amino acid sequence. The tryptic peptide ANQQK (aa 34–38) was not detected in any of the repeats, whereas peptide TAPGK (aa 138–142) ​​and peptide QQRVSK (QQR / VSK, aa 486–491) were not detected in one of the repeats. A manual search for these short peptides confirmed their elution in the flow-through (2.0–2.7 min) of the reversed-phase column, mainly in the form of singly charged ions. These ions were rejected due to fragmentation during MS / MS acquisition and therefore were not identified in the amino acid sequence of VP1 by the Protein Metrics software due to the lack of MS / MS information. The use of columns with lower particle pore sizes in the mobile phase (130 Å instead of 300 Å) or stronger ion-pairing agents such as DFA may allow better retention of these peptides on reversed-phase columns.

[0094] We next investigated the problem of detection and recovery of two large tryptic peptides (T23: aa171-238 and T33: aa323-390) generated by tryptic digestion of VP1. These peptides are very hydrophobic and therefore difficult to keep in solution or elute from the reversed-phase column. Both peptides were successfully detected with adequate signal intensity. The extracted ion chromatograms (XIC) and MS / MS signals of T23 and T33 are presented in Figure 4 and Figure 5, respectively. The T33 peptide was hydrophobic enough to elute in the 1-60% solvent B gradient (81 min) immediately after the end of 80 min, but no carryover to the next run was observed.

[0095] All hydrophilic and hydrophobic peptides of VP1 were detected after trypsin digestion, yielding 100% sequence coverage from a single LC-MS / MS run. These observations clearly demonstrate that SDC / DDM enables simple and efficient trypsin digestion of VP.

[0096] To assess the compatibility of protein precipitation by approach 1 with current proteomics workflows, the protein pellet was subjected to Asp-N digestion and LC-MS / MS analysis as described in Materials and Methods. Searching of the obtained peptides against the VP1 amino acid sequence showed >97% sequence coverage.

[0097] Asp-N digestion also produces several short peptides due to the high aspartic acid frequency in the VP1 amino acid sequence, which may result in singly charged ions and thus gaps in the resulting VP1 amino acid sequence. Four such gaps were detected in the sequence obtained after Asp-N digestion. The N-terminal sequence (aa1-12) contains several aspartates and can provide the following short peptides under optimal conditions: MAA (aa1-3), DGYLP (aa4-8), and DWLE (aa9-12). The first methionine residue of cellular proteins is often cleaved by methionine peptidases after protein synthesis, and then the second amino acid residue is acetylated. This modification was confirmed by intact mass spectrometry (Table 1). However, neither this peptide (AA) nor other dipeptides (530-531 and 609-610) were detected by LC-MS / MS analysis after Asp-N digestion.

[0098] A manual search for other amino acids missed in the Asp-N digest (DGYLP, DWLE, and DFAV) confirmed the presence of three singly charged ions that were strongly retained on the reversed-phase column due to high hydrophobicity (retention times: 33.6, 35.0, and 32.2 min, respectively). In total, six amino acids were not identified by the protocol with Asp-N digestion, which thus provided 99.2% coverage. Since the trypsin digestion provided full sequence coverage, the main advantage of using the additional Asp-N digestion is to identify MS / MS fragments missed by the trypsin digestion (e.g., peptides T23 and T33).

[0099] Characterization of the N- and C-terminal sequences of VP1, VP2, and VP3 VP1, VP2, and VP3 share the same C-terminal region and differ only in their N-terminus. The entire VP3 amino acid sequence (aa203-736) is wrapped around VP2 (aa138-736), and the entire VP2 amino acid sequence is wrapped around VP1 (aa1-736). The N-terminal amino acid sequences and PTMs in VP play important roles in endosomal escape and cellular trafficking of viral particles. Therefore, detailed characterization of VPs used as vectors in gene therapy (or other applications) is important not only to expand product knowledge but also to ensure product and process consistency.

[0100] Detection and identification of the N- and C-terminal regions of VPs are provided to highlight the efficiency of the presented approach for detailed VP structural characterization.

[0101] The amino acid sequences of both the N- and C-terminal regions of VP1 were confirmed by MS / MS spectra of tryptic peptides (Figures 6A and 6B), whereas the Asp-N digest only provided this information for the C-terminus (Figure 6C).

[0102] Acetylation of the first alanine (A) in the N-terminal amino acid sequence of VP1 (Figure 6A) was confirmed by the mass shift (+42 Da) observed in the b fragment ions (b2-b6). In addition, the detection of the complete y ion series in the MS / MS spectrum of the Asp-N peptide (Figure 6C) gives unequivocal confirmation of the amino acid sequence in the C-terminal peptide of VP1. However, this C-terminal peptide is shared in VP1, VP2, and VP3. This finding also confirms the assignment of VP1 by intact mass analysis, as described in Table 1.

[0103] The major signal (66,001.5 Da) obtained from the intact mass analysis of VP2 was assigned to amino acids 139-736, which contains low levels of acetylation and phosphorylation (Table 1). In addition, a weak signal at 66,101.7 Da was assigned to the complete sequence of VP2 (138-736). To confirm these assignments and to quantify and localize the modifications, peptide mapping data were evaluated. Analysis of a tryptic digest of VP2 revealed that its N-terminus starts with threonine (TAPGK), whereas peptides with and without threonine originate from Asp-N digestion. The reason for this discrepancy is that the tryptic peptide without threonine (APGK) produced a singly charged ion and was therefore not identified in the set of amino acid sequences, as already described. In contrast, analysis of the Asp-N digest detected both peptides, with the peptide without threonine providing the major signal. In addition, low levels of acetylation (0.3%) and phosphorylation (3.3%) of alanine and serine, respectively, were detected when threonine was absent at the N-terminus (Figure 7A-D) (VP2 N-terminus APGKKRPVEQSPQEP without threonine (A), VP2 N-terminus A(Ac)PGKKRPVEQSPQEP without threonine and containing acetylation of the first alanine (B), VP2 N-terminus APGKKRPVEQS(Phos)PQEP without threonine and containing phosphorylation of serine (C), and VP2 N-terminus TAPGKKRPVEQSPQEP with threonine (D)). Phosphorylation in VP2 was identified via a neutral loss of 98 Da observed in the MS / MS spectrum (Figure 7C).

[0104] Similar to VP1, the data showed that a methionine was cleaved from the N-terminus of VP3, and the MS / MS data confirmed that in most cases the first alanine in the amino acid sequence was acetylated (Figure 8). However, as shown in Figure 8, low levels of non-acetylated peptides were also detected (0.7%). These data are consistent with the results of intact mass analysis of VP3 (Table 1). In addition, several PTMs such as deamidation, phosphorylation, and methionine oxidation in VP1 were quantified by this approach.

[0105] conclusion The results provided herein clearly show that methanol / water / chloroform protein precipitation is a robust and convenient method to isolate VPs from their complex matrix and other capsid impurities. The procedure is simple and applicable to various AAV-based vaccines and serotypes, regardless of matrix type and sample amount. It can also be easily adapted for use in different proteomic workflows involving different enzymatic digestions and provide important complementary information, such as MS / MS fragmentation patterns and PTMs of VPs.

[0106] To circumvent the problems in detecting long hydrophobic peptides, a trypsin digestion strategy based on SDC / DDM-enabled detection of long hydrophobic peptides was developed, achieving 100% VP1 amino acid sequence coverage in a single LC-MS / MS run. This approach has been found to significantly reduce sample processing steps, making it accessible to a wide range of biology laboratories and eliminating the need for high expertise in proteomics workflows. The workflow can also be applied without a second enzymatic digestion, which is advantageous when a large number of samples have to be rapidly characterized. Furthermore, the use of a rapid and robust LC-MS / MS combined with fine-tuning, e.g., optimization of digestion time, can further expand the capabilities of the method for high-throughput analysis and accelerate both the development and processing of gene therapy products.

[0107] Example 2: Extraction and analysis of adenovirus capsid proteins Figure 9 shows an overview of the extraction and analysis protocol outlined in the Examples below. The workflow involves concentration of AdV5 samples on an appropriate cut-off filter (10 kDa) to reduce their volume, followed by precipitation of the VPs, their solubilization in SDC / DDM solution, reduction, and digestion with trypsin. Finally, the generated peptides are analyzed by LC-MS / MS. The method has two objectives: to confirm the identity of the major VPs of AdV5 (high amino acid sequence coverage) and to quantify their PTMs.

[0108] Materials and Methods Chemicals Tris 2-carboxyethylphosphine (TCEP), ammonium bicarbonate (ABC), ultrapure formic acid, acetic acid, guanidine-HCl, chloroform, methanol, acetonitrile (ACN), water, trifluoroacetic acid (TFA), and sodium deoxycholate (SDC) were purchased from Sigma-Aldrich (St. Louis, MO). Vivaspin 500 (10 kDa MWCO) was purchased from Cytiva (Marlborough, MA). Sequencing grade trypsin and N-dodecyl-beta-D-maltoside (DDM) were purchased from Promega (Milwaukee, WI) and Thermo Fisher Scientific (Waltham, MA), respectively.

[0109] Vector production and purification One vial of HEK293 RCB was thawed and cultured in shake flasks of various sizes every 3 or 4 days. Once sufficient cell mass was reached, the culture was used to inoculate a WAVE20 perfusion bioreactor at the target seeding density. The culture was medium exchanged and infected with wild-type AdV5. The culture was then lysed, filter clarified, and stored as the column load. Two batches of the column load were thawed and purified using an HR16 column packed with Source 15Q resin. The purified AdV5 viral particles were pooled and sterile filtered using a 0.2 μm filter. The filtered sample was aliquoted for LC-MS analysis.

[0110] Sample preparation A sample of recombinant AdV5 was prepared as described below: A recombinant human monoclonal IgG4 antibody (designated mAb A) was also produced and purified at Lonza using standard manufacturing procedures.

[0111] Peptide mapping was performed after the 300 μL volume of the AdV5 samples prepared as described above (containing approximately 40 μg of AdV5 VP) was reduced to 200 μL prior to protein precipitation as described below: The volume was reduced by centrifuging the samples through 10 kDa cut-off Vivaspin filters, also known as membrane filters, for 20 min at 10,000 g and 20° C.

[0112] Chloroform / methanol / water protein precipitation VPs were precipitated with chloroform / methanol / water. Briefly, 800 μL of methanol, 200 μL of chloroform, and 600 μL of water were added successively to 200 μL of concentrated sample (prepared as described above), with a short vortex mixing and high-speed centrifugation step (10 s at 14,000 g) after each addition. The protein precipitate appeared at the interface as a white layer between the upper and lower phases. The upper phase was carefully removed and discarded, and then 200 μL of cold methanol was added to the remaining mixture. After centrifugation, the supernatant was removed. Finally, the protein pellet was dried by vacuum centrifugation.

[0113] All precipitation steps used cold solvents (4°C) and centrifugation was performed at 14,000g and 4°C for 10 min.

[0114] Lysis with SDC and DDM and enzymatic digestion of mAB-A and capsid proteins For trypsin digestion, a portion of mAb A (60 μg) was dissolved in a 5 μL mixture of SDC (0.5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0% w / v) and DDM (0.5% w / v) in 50 mM ammonium bicarbonate (pH 8.0). After vortex mixing at room temperature, the sample was then diluted to 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, and 0.4% w / v SDC and 0.1% w / v DDM by adding 20 μL of 50 mM ammonium bicarbonate (pH 8.0). Proteins were reduced by adding 1 μL of 500 mM TCEP and incubating the resulting mixture at 50° C. for 30 min. Samples were subjected to trypsin digestion (with a protein to protease ratio of 30:1 w / w) for 3 hours at 37° C., and then digests were transferred directly to HPLC vials for LC-MS / MS analysis, as described below.

[0115] Alternatively, the viral protein pellet prepared as described above was dissolved in 5 μL of a mixture of SDC (1.75% w / w) and DDM (0.5% w / w) in 50 mM ammonium bicarbonate (pH 8.0). After vortex mixing at room temperature, the sample was then diluted to SDC and DDM concentrations of 0.35% and 0.1% w / v, respectively, by adding 20 μL of 50 mM ammonium bicarbonate (pH 8.0). Proteins in the sample were reduced and digested as described above. The digests were then directly transferred to HPLC vials for LC-MS / MS analysis, as described below.

[0116] Liquid chromatography-mass spectrometry A system consisting of a Vanquish UPLC instrument coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for all analyses. For peptide mapping analysis, peptides in digested samples were separated using an Acquity UPLC peptide CSH C18 column (130 Å, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation) with a mobile phase consisting of 0.1% v / v formic acid in water (A) and acetonitrile (B). Peptides were eluted with a linear gradient from 1% v / v B to 30% v / v B over 140 min at a flow rate of 0.25 mL / min, followed by 30% v / v B to 40% v / v B over 15 min. The column was then washed with 98% v / v B for 10 min and conditioned with 1% v / v B for 8 min before the next injection.

[0117] The mass spectrometer was operated in data-dependent mode in the range of 200-2000 m / z with a spray voltage of 3.5 kV and a heated capillary temperature of 320 °C. Full scan spectra were recorded at a resolution of 120,000 (full width at half maximum resolution at 400 m / z) using an automatic gain control (AGC) target value of 2.0e5 with a maximum injection time of 100 ms. A maximum of 20 of the most intense ions with charge states from 2 to 8 were selected for high-energy c-trap dissociation (HCD) with a normalized collision energy of 35%. Fragment spectra were recorded at an isolation width of 2.5 Da and a resolution of 15,000 using an AGC target value of 5.0e4 and a maximum injection time of 200 ms. Dynamic exclusion was activated for 5 s within a 10 ppm window for precursor selection. Fragment ions were recorded by the Orbitrap analyzer.

[0118] Data analysis Raw mass spectral data were processed with Protein Metrics (San Carlos, CA). For peptide mapping, database searches against the AdV5 viral capsid protein sequence were performed with tolerances of 6 and 20 ppm for peptides detected in MS and MS / MS analyses, respectively. For this, FASTA protein sequence files were downloaded from the UniProt database, which contains the complete curated entry for "human adenovirus C serotype 5" (31). N-terminal acetylation, methionine and tryptophan oxidation, phosphorylation, and asparagine deamidation were included in the search as variable modifications.

[0119] Results and Discussion Developing a robust and reproducible sample preparation workflow Intense efforts have been made to develop universal, robust and reproducible sample processing strategies for proteomic analysis. Important factors to consider when selecting a procedure to minimize sample loss and maximize detection of peptides by MS include sample concentration and matrix type. However, there is little consensus on optimal sample processing protocols, and this step remains the main bottleneck for successful proteomic analysis. Sample loss during processing steps is inevitable, which does not significantly affect the performance of most methods if a sufficiently large amount of starting material (at least 50-150 μg protein) is available. However, sample loss can cause major problems in proteomic analysis of gene therapy products (GTPs), since typically only limited amounts of material are available during the development stage (e.g., early clinical phase). The complex sample matrix and wide molecular weight range of VPs in GTPs compared to those in recombinant protein therapeutics also complicate their proteomic analysis.

[0120] Thus, it is difficult to apply common classical sample processing workflows for proteomic analysis of GTP, and new approaches are needed. Such an approach must be able to overcome these issues, efficiently remove interferences in the sample matrix, and degrade capsid proteins without the need for any further processing. It should also involve minimal liquid-liquid handling steps, without protein- or peptide-level clean-up steps, and cause minimal artificial modifications in sample processing to allow accurate quantification of PTMs. Furthermore, it should require the use of a single protease that can digest all VPs in the low abundance AdVs available in the sample from early development steps and provide high amino acid sequence coverage of them.

[0121] Example 1 described a sample preparation method for proteomic analysis of adeno-associated virus (AAV) that avoids many of the challenges associated with common classical approaches. Although the structural composition (e.g., capsid proteins and double-stranded DNA) and matrix complexity of AdV are similar to those of AAV, the throughput of the method described in Example 1 may be limited by the low relative abundance (0.1-0.3% of total protein mass) of some components of the AdV5 proteome. Thus, the AAV method required further optimization to reduce potential sample loss. Due to the limited available amount of AdV5 material, a monoclonal antibody (mAb A) was used initially for proof-of-concept in all optimization steps of the workflow, as described above.

[0122] The method essentially involves the concentration of virus particles using a centrifugal filter with an appropriate cut-off membrane (10 kDa). This is followed by the use of an organic solvent to break down the particles into their structural proteins and DNA, precipitating the VPs at the interface between the organic and aqueous phases. Those skilled in the art will appreciate that the composition of the organic solvent and aqueous phase can be adjusted to yield optimal results, depending on the protein of interest, the sample matrix components, and other factors. The sample matrix components and DNA are partitioned into either the aqueous or organic phase and are removed prior to lyophilization of the protein precipitate. Importantly, in this new approach, the tertiary structure of the proteins is disrupted after precipitation, so no further denaturation step with common chaotropic agents (e.g., urea or guanidine hydrochloride) is necessary.

[0123] To minimize the number of sample processing steps, precipitated proteins are redissolved in a low volume of aqueous SDC / DDM solution. Dissolving proteins in SDC or DDM solution can increase the potential cleavage sites by trypsin and enhance the solubility of hydrophobic peptides, thereby improving amino acid sequence coverage.

[0124] Direct introduction of samples into the MS system after protein digestion can be quite problematic, since high concentrations of SDC (1%) significantly suppress electrospray ionization and interfere with chromatographic separation of peptides. Previously published protocols therefore involve precipitation of SDC after addition of TFA and removal by centrifugation (i.e., acid centrifugation) or extraction with ethyl acetate (i.e., phase transfer). This step is a significant bottleneck when low volumes of material are available. Under these conditions, the supernatant cannot be completely separated from the pellet, resulting in significant sample loss (up to 21%), as shown by the results of precipitating SDC in mAb A solution, washing the pellet with water, and subjecting both the supernatant and the washed pellet to LC-MS / MS analysis (see Figure 10). Washing the pellet with water or organic solvents and combining the washing solution with the previously removed supernatant can improve peptide recovery, but requires an additional sample processing step to reduce the large sample volume, e.g., by vacuum centrifugation, prior to LC-MS / MS. In this study, the workflow was further optimized by reducing the percentage of SDC to allow omission of the SDC removal step and proceed directly to LC-MS / MS analysis.

[0125] To assess the feasibility of direct LC-MS / MS analysis of digested VP, the following variables had to be evaluated: solubility of SDC in the acidic mobile phase (risk of precipitation), peak shape and signal intensity of peptides in the presence of SDC during chromatographic separation and MS detection, respectively, as well as tryptic digestion of proteins in the presence of low concentrations of SDC.

[0126] Precipitation of SDC after injection of digested samples into the acidic mobile phase was a likely scenario that could lead to column blockage. However, surprisingly, there was no evidence of SDC precipitation during incubation of mixtures of SDC / DDM with mobile phase A (with ratios varying from 1:1 to 1:10) at room temperature for 30 min. In addition, no increase in column pressure was observed during several consecutive LC-MS / MS analyses of digested mAB A samples (with and without SDC removal).

[0127] Chromatographic separation and MS detection were further evaluated by digesting mAb A in the presence of 0.1% w / v DDM and various concentrations of SDC (0.1, 0.15, 0.2, 0.25, 0.3, 0.35, and 0.4% w / v), as described above, and then directly introducing the resulting peptides into the LC-MS / MS system. The signal intensities and peak shapes of eight identified peptides (herein referred to as P1-P8) were compared with those obtained with the classical approach (digestion in 1% SDC, followed by SDC precipitation and removal after addition of 2 μL of TFA). The data obtained showed no differences in chromatographic separation and peak shapes of selected peptides throughout the gradient (see Figures 11 and 12). The signal intensities of selected peptides tended to increase with increasing amounts of SDC up to 0.4% w / v (see Table 2). [Table 2]

[0128] It was found that higher amounts of SDC could enhance the denaturation / solubilization of the protein (here mAb A) and enhance the protease performance, but even at the lowest concentration tested, the overall digestion was still significantly more efficient. Notably, the signal intensities of selected peptides in digested samples were significantly higher than those obtained in the approach with SDC removal for SDC concentrations ≥ 0.35% w / v. For P7 and P8, even the signal intensities of samples with SDC ≥ 0.25% w / v were higher than those obtained after SDC removal (Table 2). This may be related to the hydrophobicity of these peptides and partial precipitation that may occur during the SDC removal step. Although the signal intensities of selected peptides with 0.35% and 0.4% w / v SDC were comparable, 0.35% w / v SDC was chosen as the optimal concentration for further experiments to avoid potential contamination in the source of the mass spectrometer during long-term use.

[0129] In Example 1, DDM was used as a combinatorial detergent to increase the solubility of hydrophobic peptides and avoid their precipitation after SDC removal. Although the SDC removal step was omitted in the workflow presented here, due to the lower SDC percentage (0.35% w / v) than the standard workflow (1% w / v), DDM was added to enhance protein solubility and thus increase the performance of the protease. To avoid mass overloading of the RP column after injection of all digested material, the effect of lower levels of DDM (0.1, 0.2, 0.3, 0.4, and 0.5% w / v) on the signal intensity of selected peptides was evaluated. Results showed that similar signal intensities were obtained with all these DDM concentrations (data not shown), therefore 0.1% w / v of DDM was utilized in further experiments. In summary, a combination of SDC (0.35% w / v) and DDM (0.1% w / v) in 50 mM ammonium bicarbonate (pH 8.0) solution was found to be the optimal buffer to dissolve and digest AdV5 VPs for characterization studies.

[0130] The results obtained with mAb A demonstrate that the methods described herein are useful for a range of proteins, including but not limited to AAV / AdV VP. It should also be noted that the model compound selected, mAb A, has a more rigid structure than VP due to several disulfide bridges, and thus even lower concentrations of SDC may be sufficient for effective denaturation and solubilization of VP.

[0131] AdV5 capsid protein amino acid sequence coverage Digested VPs of AdV5 were analyzed according to the developed method, as illustrated in Figure 9 and briefly described above. Due to the large differences in the relative abundance of AdV5 structural proteins (e.g., hexon and pTP account for 59.5% and 0.1% of the total protein mass, respectively), several modifications of the LC-MS / MS settings were required. First, silicotrypsin digestion of VPs of AdV5 resulted in the generation of more than 350 peptides with four or more amino acids, so a longer gradient was required to separate and identify the peptides. The highest sequence coverage was obtained when a longer gradient (150-180 min instead of 100 min) was used. In addition, to enhance the retention and detection of small peptides, the column with 300 Å pores used in Example 1 was replaced by one with smaller pores (130 Å).

[0132] Average amino acid sequence coverage (two technical replicates) of the major, cement, and core proteins of 93.2, 97.7, and 85.0%, respectively, were obtained (see Table 3). The highest sequence coverage (≥99%) was obtained for the penton, pIIIa, and pIX proteins, while the lowest sequence coverage (55%) was obtained for pTP. The sequence coverage of pTP was also the lowest in previous studies (36% with trypsin digestion and 55% when three different proteases were used). These results indicate that it is very difficult to obtain high sequence coverage of pTP. There are several reasons for this. First, it represents the lowest percentage (0.1%) of the total mass of the VPs of AdV5, and is therefore close to the detection limit of the applied UHPLC-MS / MS system. Second, the covalent binding of pTP to DNA may complicate its isolation by the presented approach. However, because precipitation with chloroform / methanol / water can generally remove contaminating DNA, it was hypothesized that either the pTPs were not fully precipitated or that precipitation of pTPs with DNA could reduce the ability of trypsin to properly digest it. The use of a nanoLC-MS / MS system in the sample processing steps and / or treatment with DNAase could be used to further enhance pTP sequence coverage. Despite these complications, a 55% sequence coverage of pTPs was obtained, clearly demonstrating that the presented approach is a convenient method for the characterization of VPs with low abundance in complex matrices. [Table 3]

[0133] The lower sequence coverage of some proteins of AdV5 (e.g., pV and pVII) is related to the generation of small peptides by trypsin digestion that are not retained on the RP column. These proteins have a high frequency of arginine and lysine in their amino acid sequences, and the resulting tryptic peptides (1-3 amino acids) cannot be retained even by columns with small (130 Å) pores. However, for pV and pVII, respectively, 94% and 92% amino acid sequence coverage was still obtained in this study.

[0134] A recent LC-MS / MS-based study confirmed the presence of additional VPs, classified as "nonstructural proteins", that may be present in AdV5 viral particles. To further investigate the VPs, peptides identified from MS / MS data were used in a search against the list of human adenovirus C serotype 5 proteins in the UniProt database (31 scrutinized proteins), and an additional 14 nonstructural viral proteins were found using the approach presented herein. They are expected to have a lower abundance compared to the structural viral proteins of AdV5, and their presence may be related to the type of purification step used. To the best of our knowledge, there is insufficient information on the details and role of these proteins in viral infection. However, the use of LC-MS / MS systems with lower flow rates and higher sensitivity (e.g., micro- or nano-LC-MS / MS systems) can further enhance their detection and identification, if necessary.

[0135] The amino acid sequences of some of the major structural proteins can differ among AdV serotypes, and peptide mapping analysis can be used to identify them. For example, the hexon and fiber proteins of AdV5 and AdV2 are significantly different and can be used as markers for serotype identification. In contrast, other VPs have much more constant amino acid sequences (e.g., pVII and pμ in AdV5 and AdV2).

[0136] The data obtained show that the method described herein significantly increases the amino acid sequence coverage of major structural proteins and is able to distinguish between different adenovirus serotypes, and therefore can be used to confirm the identity of viral vectors.

[0137] Quantitative analysis of AdV5 PTMs Quantifying PTMs of VPs at site-specific levels during the progression of infection can provide important new information on the biological and pathogenic mechanisms of viral infection. So far, limited information on these mechanisms has been obtained from quantitative proteomic approaches, and there is a need for further characterization of proteins in model viral systems such as AdV5. The results of this study highlight the sensitivity of the presented approach to identify multiple PTMs in a single LC-MS / MS run, and thus its potential use to correlate PTMs with biological functions. The analysis revealed a total of 53 PTMs in the primary structural VP of AdV5 (with a relative abundance of at least 0.5%), including phosphorylation, acetylation, deamidation, and oxidation (see Table 4). These modifications are known to affect the properties (e.g., stability) of GTP and are influenced by multiple variables of the production process, including cell line and purification technique.

[0138] These modifications are discussed below with an example of an MS / MS spectrum. Although PTMs of viral proteins are known from the intracellular maturation of capsid proteins, some of these modifications (e.g., deamidation and oxidation) can also be induced during purification, storage, or sample processing in proteomics workflows. Analysis of several therapeutic proteins with the presented approach confirmed that the applied workflow is unlikely to increase deamidation and oxidation levels (data not shown). [Table 4-1] [Table 4-2] [Table 4-3]

[0139] Protein acetylation is recognized as an important regulatory event during diverse infections with human viruses. Acetylation of N-terminal amino acids is the most frequently detected type of modification of a given amino acid of VPs. It has been shown that N-terminal acetylation of VPs may play an important role in their intracellular transport and entry into the nucleus. It is therefore important to confirm their N-terminal sequences and to quantify their acetylation in GTP. This modification is confirmed by the associated mass shift (+42.01 Da) in the total peptide mass and its localization is confirmed by data on the b-fragment ions generated in MS / MS analysis. Acetylation occurs mainly when the methionine at the N-terminus of VPs is cleaved by methionine aminopeptidase and then the resulting N-terminal amino acid residue is acetylated. For example, in the present study, it was found that ≥98.0% of the N-terminus of pIX was acetylated after methionine removal (Figure 13A). The MS / MS spectrum of the corresponding peptide showed a series of b fragment ions (b1-b8) with a mass shift of +42.01 Da on the serine residue, confirming acetylation at the N-terminus. However, in the absence of N-terminal methionine removal, the retained methionine can also be acetylated, as demonstrated by a series of b fragment ions (b1-b7) for the N-terminus of pVI with a mass shift of 42.01 Da (Figure 13B). Some of the amino acids can undergo additional modifications, for example, the N-terminal acetylated methionine of pVI can also be oxidized. This modification was confirmed by MS / MS analysis of the b1 fragment ion, as shown in the expanded mass spectrum in Figures 13B and C.

[0140] Acetylation at the N-terminus of some VPs (e.g., penton base and fiber) cannot be quantified by the approach presented here because these proteins have high arginine and lysine frequencies in their N-terminal amino acid sequences, and therefore these peptides also cannot be retained and detected, as discussed above. However, the use of a second protease (e.g., Asp-N) may provide the necessary information (see Example 1).

[0141] Serine, threonine, and tyrosine phosphorylation is another important type of PTM that is involved in the stability of the viral capsid and thus likely to affect the infection process. Phosphorylation is identified by the neutral loss of H3PO4 (97.98 Da) from the proteolytic peptide molecular ion in the MS / MS spectrum (Figure 14A). The relative abundance of phosphotyrosine (pY), phosphothreonine (pT), and phosphoserine (pS) in normal growing cells is approximately 2, 12, and 86%, respectively. The phosphopeptides have low intensity and for comprehensive analysis, additional chromatographic fractionation and enrichment steps of phosphopeptides are required before LC-MS / MS analysis. Since such steps were absent in the present workflow, a lower number of phosphorylation sites was observed than in previous studies. Overall, seven serine phosphorylation sites were identified according to their expected stoichiometric ratios, and no tyrosine or threonine phosphorylation sites were identified.

[0142] Oxidation and deamidation are common PTMs in biopharmaceuticals, where they affect both biological activity and efficacy. These modifications are expected to be critical quality attributes (CQAs) and therefore need to be evaluated throughout the development of viral vectors as pharmaceutical products. Nevertheless, the effects of these protein modifications on the efficacy and safety of viral vectors have been understudied, despite their potential importance. For example, deamidation of amino acids on the surface of the AAV capsid has been reported to result in valency heterogeneity and changes in vector function. The effects of oxidation and deamidation on the stability and properties of viral particles can be simulated by appropriate exposure to hydrogen peroxide and solutions with high pH, ​​respectively. Chemical modifications in viral particles can be monitored by several methods, e.g., capillary zone electrophoresis (CZE), dynamic light scattering (DLS), and electrophoretic light scattering (ELS). However, these methods only provide global information on the viral particle level and cannot quantify or localize these modifications at the protein or amino acid level.

[0143] RP chromatography is the only currently available method that can provide information about the level of oxidation in different VPs through changes in protein retention time or the appearance of new peaks, but cannot localize these modifications at specific methionines or tryptophans of the VPs.

[0144] Using the novel approach presented here, a total of 12 oxidation sites were identified in the major VP of AdV5, with the level of oxidation being less than 1% at all of these sites except M48pμ (5.9%).

[0145] Deamidation of asparagine residues (resulting in a mass shift of +0.98 Da) is a common, irreversible modification, whose mechanism has been studied in great detail by LC-MS / MS. It has been shown that the deamidation rate depends on the primary and higher order structure of the protein, on the pH, and on the temperature. In addition, asparagines in SNG, ENN, LNG, and LNN amino acid motifs have been reported to be most prone to deamidation. An example of the MS / MS spectrum of the corresponding deamidated peptide showed a series of y10-y15 fragment ions with a mass shift of +0.98 Da on the corresponding asparagine (Figure 14B). Due to the short sample processing and digestion times in the presented workflow, the detected deamidation levels (see Table 4) are highly unlikely to be artifacts. This was confirmed by the detection of low levels of deamidation in mAb A during the method optimization steps, as mentioned above. 25 deamidation sites were detected, mainly with NG, NN, and NS motifs in the major VP of AdV5 (Table 4). The highest level of deamidation in VP was associated with the NG motif. Similar studies found that the main deamidation of AAV8 was in the hypervariable region (HVR) that contains the NG motif. HVR is highly involved in the interaction with target cells and the immune system, and therefore plays an important role in reducing transduction by altering receptor binding.

[0146] As discussed above, the method presented herein allows for simultaneous quantification of multiple PTMs. Its applicability can be expanded by modifying the search parameters to be suitable for quantification of other PTMs (e.g., N- and O-glycosylation) if necessary. However, even without such enhancements, it can provide more information than previous approaches about the amino acid sequence and associated PTMs, thereby improving the GTP production process.

[0147] conclusion The novel workflow for AdV VP analysis presented here results in substantially shorter analysis times than conventional peptide mapping methods, which require the use of several proteases and extensive sample processing for sufficient detailed characterization. We provide here the first demonstration that a solution containing SDC and DDM can be used for simultaneous VP denaturation and digestion, without any further clean-up steps, directly prior to LC-MS / MS analysis. Surprisingly, despite the lower detergent concentration used for solubilization of the precipitate, analysis of the major VPs with the developed approach allowed a substantially higher average sequence coverage than before (up to 92%), and the quantification of 53 PTMs in a single LC-MS / MS run.

[0148] Minimization of sample preparation steps, along with increased analytical performance, reduces the risk of protein or peptide loss and enables high-throughput analysis when large numbers of samples must be rapidly characterized to support GTP development, stability testing, and characterization.

[0149] The comprehensive information provided by this approach can provide invaluable mechanistic insights into viral infection that are difficult to obtain by other approaches. In addition, it is envisioned that the approach will motivate future studies to monitor and control host cell proteins of GTP produced using different vectors and manufacturing processes.

[0150] Exemplary embodiments Embodiment 1 is a method of preparing digested viral proteins, comprising precipitating viral proteins from a sample containing the viral proteins, dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to produce a solution, and digesting the viral proteins with a protease. Embodiment 2 is a method of analyzing digested viral proteins, comprising precipitating viral proteins from a sample containing the viral proteins, dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to generate a solution, digesting the viral proteins with a protease, and analyzing the digested viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS). Embodiment 3 is a method of analyzing digested viral proteins, comprising precipitating viral proteins from a sample containing the viral proteins, dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to generate a solution, digesting the viral proteins with a protease, removing the SDC from the solution, and analyzing the digested viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS). Embodiment 4 includes the method of any one of embodiments 1-3, wherein the viral protein is an adeno-associated virus capsid protein (AAV capsid protein), an adenovirus protein, a lentivirus protein, a retrovirus protein, or a herpes simplex virus protein. Embodiment 5 includes the method of any one of embodiments 1 to 3, wherein the viral protein is an AAV capsid protein. Embodiment 6 includes the method of any one of Embodiments 1-3, wherein the viral protein is an adenoviral protein. Embodiment 7 includes the method of embodiment 6, wherein the adenovirus protein is an adenovirus 5, 26, 35, or 48 protein. Embodiment 8 includes the method of embodiment 6, wherein the adenovirus protein is an adenovirus 5 protein. Embodiment 9 includes the method of any one of embodiment 1 or embodiment 2, wherein the viral protein is a lentiviral protein.

[0033] Embodiment 10 includes the method of any one of embodiments 1 to 9, wherein the viral proteins are dissolved in a mixture comprising about 0.01% to 1.5% (w / w) SDC and about 0.01% to 1.0% (w / w) DDM. Embodiment 11 includes the method of embodiment 10, wherein the viral proteins are dissolved in a mixture comprising about 0.5% to 1.5% (w / w) SDC and about 0.01% to 1.0% (w / w) DDM. Embodiment 12 includes the method of embodiment 12, wherein the viral proteins are dissolved in a mixture comprising about 0.5% to 1.5% (w / w) SDC and about 0.2% to 1.0% (w / w) DDM. Embodiment 13 includes the method of embodiment 12, wherein the mixture comprises about 0.75% to 1.25% (w / w) SDC and about 0.5% to 0.8% (w / w) DDM. Embodiment 14 includes the method of embodiment 10, wherein the mixture comprises about 0.01% to 0.6% (w / w) SDC and about 0.01% to 1.0% (w / w) DDM. Embodiment 15 includes the method of embodiment 14, wherein the mixture comprises about 0.01% to 0.6% (w / w) SDC and about 0.01% to 0.6% (w / w) DDM. Embodiment 16 includes the method of embodiment 15, wherein the mixture comprises about 0.2% to 0.4% (w / w) SDC and about 0.05% to 0.2% (w / w) DDM. Embodiment 17 includes the method of any one of embodiments 1-3, wherein the mixture comprises a ratio of about 1:0.5 w / w or about 3.5:1 w / w (SDC:DDM). Embodiment 18 includes the method of any one of embodiments 1-17, wherein dissolution in solution occurs at about pH 6.0 to about pH 9.0. Embodiment 19 includes the method of any one of embodiments 1-18, wherein the digestion is carried out at about 30° C. to 40° C. for a period of about 2 to 12 hours. Embodiment 20 includes the method of any one of embodiments 1-19, wherein the precipitation comprises precipitation with chloroform / methanol / water and centrifugation. Embodiment 21 includes the method of any one of embodiments 1 to 20, wherein the digestion comprises digesting with trypsin. Embodiment 22 includes the method of embodiment 21, wherein the digestion is carried out at a ratio of viral protein:trypsin of about 20:1 to about 100:1 w:w. Embodiment 23 includes the method of any one of embodiments 1 to 22, wherein the digested viral protein is about 3 to 70 amino acids in length. Embodiment 24 includes the method of any one of embodiments 2 to 23, wherein the analysis includes injecting the digested viral proteins into a liquid chromatography mass spectrometer without first performing a buffer exchange or desalting step. Embodiment 25 includes the method of any one of embodiments 2 to 24, wherein the solution volume analyzed by LC-MS / MS is less than 50 μL. Embodiment 26 includes the method of any one of embodiments 1 to 25, wherein the sample containing viral protein has a concentration of viral protein from about 0.001 mg / mL to about 0.10 mg / mL.

[0151] Although specific embodiments have been illustrated and described herein, it should be understood that the claims should not be limited to the specific forms or arrangements of parts described and illustrated. Although exemplary embodiments are disclosed and specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It should be understood, therefore, that the embodiments may be practiced otherwise than as specifically described.

[0152] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [ka]

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Claims

1. 1. A method for preparing digested viral proteins, comprising: a. precipitating viral proteins from a sample containing said viral proteins; b. dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to form a solution; c) digesting the viral proteins with a protease.

2. 1. A method for analyzing digested viral proteins, comprising: a. precipitating viral proteins from a sample containing said viral proteins; b. dissolving the viral proteins in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) to form a solution; c. digesting the viral proteins with a protease; e. analyzing the digested viral proteins via liquid chromatography-tandem mass spectrometry (LC-MS / MS).

3. d. further comprising the step of removing the SDC from the solution; 3. The method of claim 2, wherein step d) is performed after step c) and before step e).

4. 4. The method of claim 1, wherein the viral protein is an adeno-associated virus capsid protein (AAV capsid protein), an adenovirus protein, a lentivirus protein, a retrovirus protein, or a herpes simplex virus protein.

5. The method of any one of claims 1 to 3, wherein the viral protein is an AAV capsid protein.

6. The method according to any one of claims 1 to 3, wherein the viral protein is an adenoviral protein.

7. 7. The method of claim 6, wherein the adenovirus protein is an adenovirus 5, 26, 35, or 48 protein.

8. The method of claim 6, wherein the adenovirus protein is an adenovirus 5 protein.

9. The method according to any one of claims 1 to 3, wherein the viral protein is a lentiviral protein.

10. 4. The method according to claim 1, wherein the viral proteins are dissolved in a mixture comprising about 0.01% to 1.5% (w / w) SDC and about 0.01% to 1.0% (w / w) DDM.

11. 11. The method of claim 10, wherein the viral proteins are dissolved in a mixture comprising about 0.5% to 1.5% (w / w) SDC and about 0.01% to 1.0% (w / w) DDM.

12. 12. The method of claim 11, wherein the viral proteins are dissolved in a mixture comprising about 0.5% to 1.5% (w / w) SDC and about 0.2% to 1.0% (w / w) DDM.

13. 13. The method of claim 12, wherein the mixture comprises about 0.75% to 1.25% (w / w) SDC and about 0.5% to 0.8% (w / w) DDM.

14. 11. The method of claim 10, wherein the mixture comprises about 0.01% to 0.6% (w / w) SDC and about 0.01% to 1% (w / w) DDM.

15. 15. The method of claim 14, wherein the mixture comprises about 0.01% to 0.6% (w / w) SDC and about 0.01% to 0.6% (w / w) DDM.

16. 16. The method of claim 15, wherein the mixture comprises about 0.2% to 0.4% (w / w) SDC and about 0.05% to 0.2% (w / w) DDM.

17. 11. The method of claim 10, wherein the mixture comprises a ratio of about 1:0.5 w / w or about 3.5:1 w / w (SDC:DDM).

18. 4. The method of any one of claims 1 to 3, wherein step b. dissolving in a solution occurs at about pH 6.0 to about pH 9.

0.

19. 4. The method of any one of claims 1 to 3, wherein the digesting step c. is carried out at about 30°C to 40°C for a period of about 2 to 12 hours.

20. 4. The method of any one of claims 1 to 3, wherein the precipitating step a. comprises precipitation with chloroform / methanol / water and centrifugation.

21. 4. The method of any one of claims 1 to 3, wherein the digesting step c. comprises digesting with trypsin.

22. 22. The method of claim 21, wherein the digestion is carried out at a viral protein:trypsin ratio of about 20:1 to about 100:1 w:w.

23. The method of any one of claims 1 to 3, wherein the digested viral proteins are about 3 to 70 amino acids in length.

24. 4. The method of claim 2 or 3, wherein analyzing step e. comprises injecting the digested viral proteins into a liquid chromatography mass spectrometer without first performing a buffer exchange or desalting step.

25. 4. The method of claim 2 or 3, wherein the solution volume analyzed by LC-MS / MS is less than 50 μL.

26. 4. The method of claim 1, wherein the sample containing the viral protein has a concentration of viral protein of about 0.001 mg / mL to about 0.10 mg / mL.

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