Residual cell penetrating peptide detection by LC-ms

Abstract

Disclosed herein are methods for separating, detecting, identifying, and quantifying cationic peptides in a sample. The invention relates to use Hydrophilic Interaction Chromatography (HILIC) for the separation of cationic peptides in a sample and mass spectrometry to detect, identify and quantitate the peptides at femtomole levels.

Description

Residual Cell Penetrating Peptide Detection by LC-MSRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 742,065 filed January 6, 2025.

[0002] BACKGROUND

[0003] Two classes of hydrophilic, basic polypeptides play critical biological roles. One class, cationic antimicrobial peptides, possess antibacterial activity, while a second class, cellpenetrating peptides, is capable of transporting cargo molecules across viable cell membranes. The cationic charge and hydrophilic nature of these polypeptides allows for them to bind cell membranes. Cell membrane binding causes these polypeptides to undergo conformational changes, which in turn, ultimately results in bacterial cell lysis or the delivery of cargo molecules within cells.

[0004] The unique capabilities of these polypeptides have prompted research efforts to understand their structure-activity relationships and develop novel therapeutic and delivery agents. But the hydrophilic nature of these polypeptides makes their study challenging. This is exemplified by the difficulties in purifying and examining these polypeptides by liquid chromatography (LC) combined with mass spectrometry (MS), a commonly used workflow for studying polypeptides.

[0005] In most instances, the LC portion of the LC-MS workflow is conducted using reverse-phase chromatography. But, because of their chemical nature, hydrophilic, basic polypeptides are poorly retained on standard LC columns such as Cl 8. Many strategies have been put forward to increase the retention behavior of hydrophilic, basic polypeptides, but their isolation remains difficult.

[0006] Aside from the LC challenges, hydrophilic, basic polypeptides suffer from poor MS response. Accordingly, a sensitive, selective and robust method to detect and quantify these positively charged polypeptides remains needed. Towards these ends, disclosed herein are LC-MS-based methods and compounds for studying, identifying, and quantifying positively charged polypeptides.BRIEF SUMMARY

[0007] Hydrophilic, basic polypeptides play critical roles in biology such as bacterial defense and cell penetration. The study of these fascinating polypeptides is notoriously difficult. These polypeptides are prone to non-specific binding, are not retained or show poor peak shapes under most chromatographic conditions, and challenging to resolve by mass spectrometry. Prior attempts to resolve these polypeptides using hydrophilic interaction chromatography (HILIC) have failed. This prompted us to develop unique methods and compositions to overcome these difficulties.

[0008] Towards these ends, disclosed herein are methods encompassing subjecting a sample to chromatography, and thereby separating the sample, applying the separated sample to mass spectrometry, and detecting one or more polypeptides with a negative Grand Average of Hydropathy score by mass spectrometry. In some instances, the chromatography is HILIC.

[0009] Further described herein, are methods encompassing subjecting a sample to chromatography, and thereby separating the sample, applying the separated sample to mass spectrometry, and detecting and quantifying one or more polypeptides with a negative Grand Average of Hydropathy score by mass spectrometry. In some instances, the chromatography is HILIC.

[0010] These and other methods and compositions will be described below.DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows the analytical workflow used for the LC-MS analysis of CPPs.

[0012] Figure 2 depicts TIC and SIM chromatograms (A and B) and both full mass and SIM spectra of NLS peptide (C and D) obtained using the developed HILIC -MS method described in Figure 1.

[0013] Figure 3 shows TIC and extracted ion chromatograms (X1C), raw mass spectrum and deconvoluted mass spectrum of NLS peptide obtained from the MS data processing using BiopharmaFinder 5.1.

[0014] Figure 4 depicts TIC and SIM chromatograms (A and B), and both full MS and SIM MS of NLS’ peptide (C and D) obtained using the developed analytical method described in Figure 1.

[0015] Figure 5 shows TIC, XIC, raw MS and deconvoluted MS of NLS’ peptide obtained from the MS data processing using BiopharmaFinder 5.

[0016] Figure 6 shows a series of SIM chromatograms obtained from the injection of NLS standards in the range 1.0 ng / mL to 11.4 ng / mL (top) and a calibration curve of NLS to NLS’ peak area ratio vs NLS concentration (bottom).

[0017] Figure 7 describes plots summarizing the %CV values (top) and % recovery (bottom) obtained for the analysis of NLS peptide using the developed LC-MS method.

[0018] Figure 8 illustrates the specificity of the method by comparing a blank chromatogram (top) against a SIM profile obtained from the injection of 1.0 ng / mL of NLS peptide.

[0019] Figure 9 highlights the high mass accuracy of the developed LC-MS when comparing the experimental isotopic distribution of 1.0 ng / mL NLS peptide (top) vs the theoretical isotopic distribution of charge state 3+ from NLS (bottom).

[0020] Figure 10 compares SIM chromatograms obtained from the injection of PPX at 450 ng / mL containing no NaCl in peptide diluent solution (top) with the one collected from the analysis of 50 ng / mL PPX containing 138 mM NaCl in peptide diluent solution (bottom).

[0021] Figure 11 shows TIC and SIM chromatograms obtained from the LC-MS discovery analysis of TAT, R9, and PPX peptides (left panel), and the ones collected for their corresponding TAT’, R9’, and PPX’ heavy labeled species (right panel).

[0022] Figure 12 depicts Full-MS and SIM-MS spectra obtained from the LC-MS discovery analysis of TAT, R9, and PPX peptides (left panel), and the ones collected for the corresponding TAT’, R9’, and PPX’ heavy labeled species (right panel).

[0023] Figure 13 shows TIC and XIC chromatograms, and raw MS and deconvoluted MS spectra of both TAT (panel A) and heavy labeled TAT’ (panel B) peptides obtained from the discovery data analysis performed in BiopharmaFinder 5.1.

[0024] Figure 14 shows TIC and XIC chromatograms, and raw MS and deconvoluted MS spectra of both R9 (panel A) and heavy labeled R9’ (panel B) peptides obtained from the discovery data analysis performed in BiopharmaFinder 5.1.

[0025] Figure 15 shows TIC and XIC chromatograms, and raw MS and deconvoluted MS spectra of both PPX (panel A) and heavy labeled PPX’ (panel B) peptides obtained from the discovery data analysis performed in BiopharmaFinder 5.1.

[0026] Figure 16 shows a series of SIM chromatograms obtained from the injection of TAT, R9, and PPX standards (left panel) and the corresponding calibration curves obtained from the light CPP to heavy labeled CPP’ peak area ratio vs light CPP concentration (right panel).

[0027] Figure 17 depicts bar plots highlighting the repeatability of the developed LC-MS method in terms of %CV obtained from the triplicate injections of TAT, R9, and PPX calibration and QC standards (left panel). The accuracy of the method, via %recovery of spiked vs calculated concentration of TAT, R9, and PPX calibration and QC standards, is also shown as a scatter plots (right panel).

[0028] Figure 18 displays a comparison of SIM chromatograms obtained from blanks and QC standards of TAT, R9, and PPX peptides injected at the lowest concentrations.

[0029] Figure 19 depicts a bar plot of SIM peak areas obtained from the HILIC-MS analysis of 700 ng / mL PPX peptide using both NaCl and LiCl at 25, 50-, 75-, 100-, and 138-mM concentrations in the peptide diluent solution.DETAILED DESCRIPTION

[0030] Two large families of polypeptides have drawn extensive research attention over the past two decades. The first family, cationic antimicrobial peptides (AMPs), have the capacity to kill microorganisms via selective membrane disruption. Cell-penetrating peptides (CPPs) is the second family. CPPs can penetrate cell membranes with little cytotoxicity and to ferry cargo into cells. AMPs and CPPs are both cationic polypeptides with some family members being hydrophilic and basic. These polypeptides can also be conjugated to a biologic, a medication that comes from living organisms, to increase their transport across cell membranes.

[0031] As mentioned, hydrophilic, basic polypeptides are difficult to isolate by chromatography and to resolve by mass spectrometry (MS). In reverse phase chromatography, a technique commonly used to isolate polypeptides, hydrophilic basic polypeptides pass through the column without significant retention. This is because reverse phase chromatography is designed to strongly interact with hydrophobic molecules. To ultimately detect and quantify,cationic, hydrophilic, basic polypeptides an alternate chromatographic technique, from reverse phase, is applied. This technique is hydrophilic interaction liquid chromatography (HILIC).HILIC

[0032] HILIC (hydrophilic interaction liquid chromatography) refers to a separation technique that combines a hydrophilic stationary phase with a hydrophobic organic mobile phase composed of a relatively higher percentage of organic solvent and a low percentage of aqueous buffer. The order of elution is reversed relative to reverse phase chromatography, with hydrophilic compounds being retained longer than hydrophobic compounds. Accordingly, in some embodiments, a method of detecting a polypeptide with a negative Grand Average of Hydropathy (GRAVY) score is disclosed, the method encompassing applying a chromatographic technique, the chromatographic technique being HILIC, in some instances, and the polypeptide is detected by MS. In other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score is disclosed, the method encompassing applying a chromatographic technique, the chromatographic technique being HILIC, in some instances, and the polypeptide is detected and quantified by MS.

[0033] Any polar chromatographic surface, the stationary phase, can be used for HILIC. Several stationary phases have emerged for HILIC, including bare silica, underivatized silica that contain functional groups such as siloxanes, silanols with (or without) a small quantity of metals, amide-bonded silica, derivatized silica, such as polysulfoethyl A, Polycat A, PolyWAX, TSKgel amide 80, (ZIC)-HILIC, and “click” saccharides. Each of these materials display different retention characteristics and separation selectivities and require distinct buffer constitutions. Type B silica gels generally provide better separations for basic polypeptides.

[0034] In some embodiments, the stationary phase is maintained as a column. In some embodiments, the column has a temperature of from 25°C to 45°C. In some instances, the column has a temperature of 45°C.

[0035] A typical mobile phase for HILIC chromatography includes water-miscible polar organic solvents such as acetonitrile with a small amount of water. A HILIC buffer typically contains more than 70% acetonitrile. To control the mobile phase ionic strength and pH, additives, such as ammonium acetate and ammonium formate can be added. Ammonium acetate and formate are chosen because of their greater compatibility with MS, but ammoniumbicarbonate, triethylamine phosphate (TEAP), sodium perchlorate and sodium methylphosphonate (Na-McPCh) can also be used in HILIC.

[0036] The buffer pH influences the interaction of the analyte with the stationary phase. Whether the buffer pH is above or below the p ?aof the analyte determines its charge state, which in turn affects the hydrophilicity of the analyte and interaction with the stationary phase.

[0037] HILIC separations are performed either in isocratic mode with a high percentage of organic solvent or with gradients starting with a high percentage of organic solvent and ending with a high proportion of aqueous solvent.

[0038] In some embodiments, the method of detecting a polypeptide with a negative GRAVY score will include HILIC separations using isocratic mode. In other embodiments, the method of detecting a polypeptide with a negative GRAVY score will include HILIC separations using gradients. In still other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score will include HILIC separations using isocratic mode. In further embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score will include HILIC separations using gradients.

[0039] A special type of HILIC called electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) specifically utilizes the electrostatic interactions in HILIC. In ERLIC, a stationary phase is chosen that has a similar charge to the analytes to be separated. Analytes are on the one hand repelled by the stationary phase but on the other they are retained in the aqueous layer around the stationary phase. These opposing interactions allow isocratic resolution.Sample

[0040] A sample is a material that ultimately will be subjected to the methods and techniques disclosed herein. A sample refers to a material containing or suspected of containing at least one analyte molecule or interest. A sample can undergo processing steps before being subjected to the methods and techniques disclosed herein. Such processing steps can increase or decrease the constituent components of the sample.

[0041] Accordingly, in some embodiments, a method of detecting a polypeptide with a negative Grand Average of Hydropathy (GRAVY) score is disclosed, the method encompassing subjecting a sample to a chromatographic technique, in some instances, the chromatographictechnique will be HILIC, and the polypeptide is detected by MS. In other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score is disclosed, the method encompassing subjecting a sample to chromatographic technique, the chromatographic technique being, in some instances, HILIC, and the polypeptide is detected and quantified by MS.

[0042] A starting sample refers to a material containing or suspected of containing at least one analyte molecule of interest, for instance, a polypeptide of interest. A starting sample can undergo processing steps before being subjected to the methods and techniques disclosed herein. Such processing steps can increase or decrease the constituent components of the sample. In some instances, the starting sample is a cell culture media. A cell culture media being a liquid or gel solution specifically designed to provide the necessary nutrients and growth factors required to grow cells, for instance, mammalian cells outside of an organism. Examples of cell culture media include Dulbecco’s Modified Eagle Medium, Roswell Park Memorial Institute 1640, Iscove’s Modified Dulbecco’s Medium and Minimal Essential Medium.

[0043] In some embodiments, a method of detecting a polypeptide with a negative Grand Average of Hydropathy (GRAVY) score is disclosed, the method encompassing subjecting a starting sample to a chromatographic technique, in some instances, the chromatographic technique will be HILIC, and the polypeptide is detected by MS. In other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score is disclosed, the method encompassing subjecting a starting sample to a chromatographic technique, the chromatographic technique being, in some instances, HILIC, and the polypeptide is detected and quantified by MS.

[0044] A separated sample is one that results from a starting sample being subjected to a partitive or purification technique. In some instances, the separated sample is a cell culture media in which the cells have been removed. In other instances, the separated sample is virus containing preparations, the virus containing preparation having undergone procedures to enrich for the virus, medium containing, or suspected of containing, cationic antimicrobial peptides or cell-penetrating peptides, or both.

[0045] Subjecting a sample to partitive or purification techniques such as chromatography results in a separated sample. HILIC is an example of a partitive or purification technique which results in a separated sample.

[0046] In some instances, the separated sample is a cell culture medium that has been subjected to processes associated with the purification of Adeno-associated virus, such as chromatography (size exclusion, isoelectric, ion exchange, and affinity), filtration (tangential flow filtration) and ultracentrifugation (density gradient). Such a separated sample can also be considered a processed cell culture medium.

[0047] In some embodiments, a method of detecting a polypeptide with a negative Grand Average of Hydropathy (GRAVY) score is disclosed, the method encompassing applying a chromatographic technique, the chromatographic technique being HILIC, in some instances, the product of the chromatographic technique being a separated sample, and the polypeptide is detected by MS. In other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score is disclosed, the method encompassing applying a chromatographic technique, the chromatographic technique being HILIC, in some instances, the product of the chromatographic technique being a separated sample, and the polypeptide is detected and quantified by MS.

[0048] A mass ion sample is a material that has been ionized by a mass spectrometer and contains, or is suspected of containing, at least one analyte molecule of interest.

[0049] In some embodiments, a method of detecting a polypeptide with a negative Grand Average of Hydropathy (GRAVY) score is disclosed, the method encompassing applying a chromatographic technique, the chromatographic technique being HILIC, in some instances, and the polypeptide is detected by MS from a mass ion sample. In other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score is disclosed, the method encompassing applying a chromatographic technique, the chromatographic technique being HILIC, in some instances, and the polypeptide is detected and quantified by MS from a mass ion sample.Grand Average of Hydropathy (GRAVY)

[0050] A Grand Average of Hydropathy (GRAVY) score, alternatively GRAVY index or scale, refers to a measure of a protein or peptide’s hydrophobicity or hydrophilicity. A GRAVY score is calculated by adding the hydropathy value of each amino acid in a protein or peptide and dividing by the number of amino acid residues in the sequence. A positive GRAVY score indicates that the protein or peptide is hydrophobic, or likely hydrophobic, while a negative score indicates that the protein or peptide is hydrophilic, or likely hydrophilic. The higher the score above 0, the relatively more hydrophobic the protein or peptide is, the lower the score below 0, the relatively more hydrophilic the protein or peptide is. An example of one way in which a GRAVY sore is arrived at follows.Hydropathy Index by Kyte-DoolittleAlanine 1.8Arginine -4.5Asparagine -3.5Aspartic acid -3.5Cysteine 2.5Glutamine -3.5Glutamic acid -3.5Glycine -0.4Histidine -3.2Isoleucine 4.5Leucine 3.8Lysine -3.9Methionine 1.9Phenylalanine 2.8Proline -1.6Serine -0.8Threonine -0.7Tryptophan -0.9Tyrosine -1.3Valine 4.2

[0052] Using the information above, a GRAVY score can be determined for the hypothetical polypeptide Arginine*Arginine*Asparagine*Aspartic acid. -4.5+-4.5+-3.5+-3.5= -16. -16 / 4 = -4.0. The GRAVY score for the hypothetical polypeptide RRNG is -4.0, which would mean it is hydrophilic.

[0053] A method of detecting a polypeptide with a negative Grand Average of Hydropathy (GRAVY) score is disclosed herein, the GRAVY score being -1.0 - -5.0. In other embodiments, the GRAVY score being -2.0 - -4.5. In still other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score is disclosed, the GRAVY score being -1.0 - -5.0. In other embodiments, the GRAVY score being -2.0 - -4.5. Polypeptides with negative GRACY scores include members of the cationic antimicrobial peptide (AMPs) and cellpenetrating peptides (CPPs) families.Antimicrobial Peptides (AMPs)

[0054] Cationic antimicrobial peptides are relatively short polypeptides with cytotoxic activity or capable of inhibiting viral replication. Most AMPs are from are 5 - 100 amino acids in length, and typically 12 - 50 amino acids in length. As of 2019, more than 5,000 AMPs have been identified. Of these, 70% are basic and 14% among those have extreme pl > 12 values. The GRAVY score for AMPs ranges from -2.6 to 0.9.

[0055] In some embodiments, a method is disclosed, the disclosed method encompassing detecting a polypeptide with a negative GRAVY score, the polypeptide being an AMP. In other embodiments, a method is disclosed, the disclosed method encompassing detecting and quantifying a polypeptide with a negative GRAVY score, the polypeptide being an AMP.Cell-penetrating peptides (CPPs)

[0056] Cell-penetrating peptides (CPPs), like AMPs, are generally short polypeptides (<30 amino acids), often cationic and can not only penetrate cell membranes with little cytotoxicity but also ferry cargo into cells. Examples of CPPs include amino acids 48 - 60 (GRKKRRQRRRPPQ) from transactivator of transcription (TAT) protein of HIV- 1, penetratin (RQIKIWFQNRRMKWKKGG) derived from the amphiphilic Drosophila Antennapedia homeodomain, and polyarginine (RRRRRRRR). More than 1,500 natural and synthetic CPPs have be identified or designed. A comprehensive collection of CPPs is available through the database CPPsite 2.0. Analysis of CPPs in the database CPPsite 2.0 shows that more than 90% of them are cationic and among those 64% have a pl >12. As stated by Wen et. al ., in their review article, “Quantitation of Super Basic Peptides in Biological Matrices by a Generic Perfluoropentanoic Acid-Based Liquid Chromatography -Mass Spectrometry Method,” the“extremely basic mature of these CPPs, such as (Arg)9 . . . creates an enormous challenge for their detection and analysis.”

[0057] In some embodiments, a method is disclosed, the disclosed method encompassing detecting a polypeptide with a negative GRAVY score, the polypeptide being a CPP. In other embodiments, a method is disclosed, the disclosed method encompassing detecting and quantifying a polypeptide with a negative GRAVY score, the polypeptide being a CPP.Mass Spectrometry

[0058] Mass spectrometry (MS) refers to an analytical technique used to detect, identify, and quantitate molecules by measuring the mass-to-charge ratio (m / z) of applied materials. The major components of a mass spectrometer are the ion source, mass analyzer, and detection unit. An ion plume is produced and introduced into the mass spectrometer through an ion source. The most used ion source techniques are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI), both of which are used widely. Several types of mass analyzers are available, including ion trap, orbitrap, time-of-flight (TOF), and quadrupole. These mass analyzers can be used independently or can be combined to achieve a tandem analysis.

[0059] In some embodiments, a method of detecting a polypeptide with a negative GRAVY score is disclosed, the method encompassing detecting the polypeptide by MS, the MS using electrospray ionization. In other embodiments, the polypeptide is detected by MS, the MS using MALDI.

[0060] In still other embodiments, a method of detecting and quantifying a polypeptide with a negative GRAVY score is disclosed, the method encompassing detecting the polypeptide by MS, the MS using electrospray ionization. In still further embodiments, the polypeptide is detected and quantified by MS, the MS using MALDI.Quantitative Mass Spectrometry

[0061] Current absolute quantitative strategies in MS can be divided into labeled and label-free. In labeled quantitative MS, an isotope-labeled polypeptide homologue is used as a quantification standard and is spiked into the material to be tested as well as the calibration standard. Absolute quantification is carried out through the MS ratio of the target polypeptide to its isotope-labeled polypeptide homologue.EXAMPLES

[0062] Aspects of the present invention will be illustrated by way of example only and with reference to the following experimentation.Example 1: LC-MS quantitation of NLS peptide in peptide diluent solution

[0063] A schematic of the LC-MS method is depicted in Figure 1.

[0064] LC settings were set as follows: column temperature 40°C; autosampler temperature 4°C; flow rate 0.2 mL / min, runt time 15 mins. Mobile phase A = 99.9% water (UHPLC-MS Grade water) and 0.1% DFA (LC-MS grade lonHance™ difluoroacetic acid). Mobile phase B = 99.9 % acetonitrile (UHPLC-MS Grade acetonitrile) and 0.1% DFA.

[0065] Blanks, calibration standards, and quality control standards were prepared in a peptide diluent solution containing 80% acetonitrile, 19.9% water, 0.1% tri fluoroacetic acid (LC-MS Grade trifluoroacetic acid), and 75 mM of sodium chloride (Sodium Chloride 99.5% for HPLC). Blanks and standards were injected (3 pL) in triplicate into the LC system. NLS peptide was separated using a gradient.

[0066] The gradient is formed by mixing mobile phase B and mobile phase A, varying the relative percentage of mobile phase B and mobile phase A over a specified period. The gradient also comprises three (3) isocratic holds for peptide loading, column washing and reequilibration.

[0067] During sample injection (column loading) mobile phase comprises 85% mobile phase B and 15% mobile phase A and an isocratic hold is maintained for 0.5 mins. The peptides are eluted by changing from 85% mobile phase B and 15% mobile phase A at the start to 15% mobile phase B and 85% mobile phase A at the end. The second isocratic hold of the mobile phase at 15% B for 1.5 mins is used to flush any remaining impurities from the column. The last isocratic hold executed after the linear gradient sets the mobile phase B at 85% for 3.5 mins for column re-equilibration. That is, the HILIC column (Thermo Fisher Accucore-150-Amide-H1LIC column, 2.6 pm, 2.1 m-mx 100 mm) is equilibrated under the initial conditions of the gradient for the next injection.

[0068] For the calculation of mobile phase volume / volume ratio, the following example can be used: By 85% v / v mobile phase B is meant for every 100ml of mobile phase, 85mlacetonitrile and 15ml aqueous mobile phase A. Where DFA is used: 85ml acetonitrile, 14.9ml aqueous mobile phase, e.g. water, and 0.1ml DFA.

[0069] The eluate of the HILIC separation that contains the CPP is injected into the electrospray ion source (ESI) of the mass spectrometer (Thermo Orbitrap™ MS Exploris™ 240). Ion generated in the source are then analyzed in the orbitrap section of the instrument. The developed method comprises two parallel scanning stages. In the first scanning step, a full mass spectrum is collected in a mass range of about 300 m z to 1000 m / z. The information obtained from the broadband mass scanning is used for peptide identification via comparison of experimental mass vs theoretical molecular weight. The main purpose of the second scanning stage, defined as Selected Ion Monitoring (SIM), is the CPPs quantitation. Briefly, peptide charged species generated in the source of the mass spectrometer are isolated in the quadrupole with a mass window of 1.6 m / z and further detected in the orbitrap component.

[0070] Full MS was collected in the mass range 300 m / z - 1000 m / z. NLS peptide ion [M+3H]3+(498.3 m / z) was isolated in the quadrupole with a 1.6 m / z window, and further detected in the orbitrap at 60,000 resolution. Peptide identification was performed in BiopharmaFinder 5.1 using Full MS data. Quantitation of peptide concentration was conducted in Chromeleon 7.3.1 via a calibration curve of peak areas extracted from SIM chromatograms.

[0071] Three microliters of a 700 ng / mL NLS peptide standard was injected into the LC-MS system to optimize both ionization and MS scanning conditions. The TIC described in Figure 2A showed the separation of multiple species in the RT range 2.0 to 10 mins. A closer look at the SIM chromatogram collected between 4.0 mins and 9.0 mins depicted a single sharp peak at RT 6.55 minutes attributed to NLS peptide (Figure 2B). A further inspection of the full mass projection of the peak at RT 6.55 mins (Figure 2C) showed a charge state distribution centered at the 3+ ion (497.94 m / z). An expanded view of the isotopic pattern for the ion [M+3H]3+confirmed the detection of NLS peptide (Figure 2D).

[0072] The identity of the targeted peptide is confirmed using three (3) fundamental components. The first identification layer is obtained from the deconvolution of the raw mass spectrum collected during full MS scan mode. BiopharmaFinder 5.1 (Thermo Fisher Scientific, Inc.) software is used to obtain the peptide deconvoluted molecular mass as well as the mass assignment error. The calculated molecular mass is compared against the theoretical molecularweight of the peptide determined from its amino acid sequence. The second layer of identity relies on the detection of a quantitation ion and at least one confirmation ion. For instance, a CPP peptide exhibiting charge states 3+, 4+ and 5+ in the full MS spectrum can be positively identified if at least a quantitation ion [M+5H]5+and a confirmation ion [M+4H]4+are both detected in the orbitrap component of the instrument with minimum signal-to-noise of 3. Finally, the quantitation peak extracted from the SIM chromatogram should be aligned (RT difference < 0.2 mins) with the ion signal detected in the Total Ion chromatogram (TIC).

[0073] A detailed inspection of Figure 3 deconvoluted mass profile evidenced the identification of NLS peptide via its accurate mass 1489.808 Da (200 ppb mass error).Furthermore, a clear matching of the peptide extracted ion chromatogram (XIC) with the corresponding ion signal detected in the TIC was also observed.

[0074] Equivalent results, in both the LC and MS domains, were obtained for the injection of 700 ng / mL of labeled NLS peptide (NLS’). TIC and SIM chromatograms showing NLS’ signals at RT 6.55 mins are described in Figure 4A and Figure 4B, respectively. Additionally, three charge state ions 2+, 3+, and 4+ of NLS’ were detected in the full mass spectrum depicted in Figure 4C. Details of NLS’ ion [M+3H]3+signals in the SIM-MS domain clearly illustrates the characteristic isotopic pattern of a compound labeled with13C and15N isotopes. That is, a lower abundant monoisotopic peak in a labeled molecule compared to the corresponding mass signal for an un-labeled compound (see Figure 2D vs Figure 4D).

[0075] A closer look at the deconvoluted mass spectrum of Figure 5 indicated that NLS’ peptide could be identified in the MS domain via its calculated mass 1523.857 Da. This molecular weight represents a mass difference of 34.049 Da, with respect to the mass of NLS peptide, that accounts for 24x12C atoms and 10x14N atoms replaced in three lysine and one arginine amino acids during13C and15N labeling. Confirmation of NLS’ peptide identity was also obtained from complementary TIC and XIC peptide ion signals (see Figure 5).

[0076] A set of seven NLS standards spiked with NLS’ at 25 ng / mL in peptide diluent containing 75mM of sodium chloride was injected in triplicates into the LC-MS system. A monotonic increase in SIM peak intensity was observed for NLS peptide in the concentration range 1.0 ng / mL to 11.4 ng / mL (see Figure 6 top). Inspection of the SIM-MS spectrum highlighted in the inset of Figure 6 revealed that NLS peptide can be detected and identified at1.0 ng / mL level. A calibration plot of concentration vs peak area ratio of NLS to NLS’ exhibited a linear trend (R2=0.998) indicating that the developed LC-MS method can detect and quantify NLS peptide in the concentration range 1.0 ng / mL - 11.4 ng / mL (Figure 6 bottom). A further inspection of Figure 7 revealed that NLS peptide can be accurately and precisely quantified at 1.0 ng / mL, which was established as the limit of quantitation (LOQ) of the method. The limit of detection (LOD) for the analysis of NLS peptide, determined using the Hubaux-Vox approach (LODH'V), was 0.6 ng / mL. It should be noted that, unlike the instrumental LOD, the LODH'Vindicates the minimum amount of an analyte that can be detected with a specific level of uncertainty based on a set of calibration data.

[0077] Examination of method variability for the calibration and QC standards, injected at low, medium, and high concentrations, revealed CV values below 15 % (Figure 7, top).Similarly, 80%-120% of the spiked NLS peptide in calibration and QC standards were recovered across the studied concentration range, thus demonstrating an acceptable method accuracy (Figure 7, bottom).

[0078] Table 4. Mass error obtained during the identification of NLS peptide by HILIC-MS (N=3)Spiked co measured LS theoretic Mass erro(ng / mL)(Da)mass (Da)(ppm)1.0 497.948 497.944* 9.43.0 497.948 497.944* 8.06.0 1489.82 1489.81 6.7* Theoretical isotopic peak mass of NLS charge state 3+

[0079] The comparison of a blank chromatogram (peptide diluent) with those obtained from the injection of NLS peptide at 1.0 ng / mL (Figure 8) revealed the absence of significant interferences in the targeted retention time range. Moreover, the relatively low mass errors calculated during the identification of NLS peptide, either via molecular mass assignment or monoisotopic peak match, confirmed the specificity of the method in the studied concentration range (Table 4 and Figure 9).

[0080] Overall results obtained from the evaluation of the method performance parameters indicate that the developed LC-MS workflow is reliable for quantifying residual amounts of NLSpeptide (LOQ: 1.0 ng / mL) in peptide diluent solution. The extended applicability of the CPP LC-MS method will be also illustrated in Example 2.Example 2: LC-MS quantitation of TAT, R9, and PPX peptides in peptide diluent solution.

[0081] LC settings were kept as described in Example 1. Blanks, calibration standards, and QC standards were prepared in a peptide diluent solution containing 80% acetonitrile, 19.9% water, 0.1% trifluoroacetic acid, and 100 mM -138 mM of sodium chloride depending on the analyzed CPP. Blanks and standards were injected (3 pL) in triplicate into the LC system. R9, TAT, and PPX peptides were separated using the HILIC gradient described above. Full mass spectra for each CPP peptide were collected in the mass range described in Table 3. Similarly, [M+5H]5+, [M+4H]4+’ and [M+9H]9+species were isolated (1.6 m / z window) in the quadrupole for the SIM analysis of TAT, R9, and PPX peptides, respectively. Final detection of the isolated peptide ions was conducted in the orbitrap at 60,000 resolution. Peptide identification was performed in BiopharmaFinder 5.1 following the same data processing approach utilized for NLS peptide. Quantitation of CPP peptides concentration was conducted in Chromeleon 7.3.1 via a calibration curve of peak areas extracted from SIM chromatograms and corrected by peak areas of known spiked amounts of labeled TAT, R9, and PPX peptides.

[0082] The LC-MS workflow described in Figure 1 was also utilized for the analysis of TAT, R9, and PPX peptides. While general LC conditions were kept constant, both full MS and SIM settings were tailored to the characteristics of each peptide. For example, full MS scan range, SIM quantitation ion, and center mass were all adjusted as a function of the observed background noise, MS sensitivity, and the presence of undesirable interferences (see Table 3).

[0083] Quantitation was conducted via the injection of several standards containing known amounts of the targeted peptide and an internal standard. The internal standard, which is the CPP labeled with heavy isotopes, for instance carbon- 13 (13C) and nitrogen- 15 (15N), is employed to correct for potential sample and instrument fluctuations. A heavy isotope is one that is heavier than the most common isotope of the element. Such elements can either be radioactive or non-radioactive.

[0084] A calibration curve plotting peptide concentration against the ratio of the peptide peak area to the internal standard peak area, obtained from SIM chromatograms, is used to determine the concentration of the targeted peptide.

[0085] Four CPPs with different molecular weights and GRAVY scores were used as positive control in this work. The grand average of hydropathy (GRAVY) value described in Table 1 is defined by the sum of hydropathy values of all amino acids in the sequence divided by the length of the peptide. The hydropathy of a peptide is an indication of its hydrophobic or hydrophilic character, calculated by the affinity between amino acid side chains and water molecules. Details of CPP properties used in the present study are described in Table 1.Table 1. Summary of general properties of the analyzed CPP peptidesNuclear Localization Signal (NLS) 490.77 -2.1PPX 4885.85 -2.7HIV-TAT (48-60) 1719.02 -3.5R9 1423.69 -4.5LC settings

[0086] Column temperature 40°; autosampler temperature 4°C; flow rate 0.2 mL / min, runt time 15 mins. Mobile phase A = acidified 100% water. Mobile phase B = acidified 100% acetonitrile. Acidification is due to adding DFA 0.1% v / v to Mobile phase A and Mobile phase B.MS settings

[0087] Settings utilized for the full and SIM mass spectrometry analysis of cationic peptides are described in Table 2.Table 2. Ionization and scanning settings for the MS analysis of CPPsSIM scan details for each peptide

[0088] Details of the scanning conditions used during the SIM-MS analysis if cationic peptides are described in Table 3.

[0089] Table 3. SIM MS scanning conditions for the analysis of CPPs*Denotes labeled peptide with13C and15N

[0090] A detailed analysis of Table 1 and Table 3 revealed that CPP behavior in the HILIC system is influenced by both the GRAVY score and molecular size. Increased retention of peptides in the HILIC column was observed as the GRAVY score of the CPP negatively increased. Furthermore, the molecular weight of the peptide significantly impacts its interaction with the column's stationary phase, regardless of the GRAVY score. For instance, although the PPX peptide appears more hydrophobic than R9, its larger size ultimately confers a superior hydrophilic character, as evidenced by a retention time of 8.55 minutes.

[0091] The concentration of sodium chloride in the peptide diluent was identified as a critical factor influencing the elution of CPPs from the HILIC system, thereby reducing potential carryover. Notably, the addition of 138 mM NaCl to the peptide diluent solution resulted in a tenfold increase in SIM sensitivity for the PPX peptide (Figure 10). Furthermore, the data demonstrated that a higher concentration of NaCl in the peptide diluent enhances the elution of more hydrophilic peptides. For example, R9, the peptide with the most negative GRAVY score, was efficiently eluted from the HILIC column with 138 mMNaCl. In contrast, NLS, the least hydrophilic peptide among the studied CPPs, was effectively removed from the stationary phase with 75 mM NaCl. This effect of sodium chloride on CPP elution efficiency may be linked to the chaotropic nature of sodium ions and their ability to compete with guanidinium ions, in arginine amino acids, for the amide functionalities of the HILIC stationary phase.

[0092] The SIM chromatograms obtained from the CPP discovery injections showed sharp peaks at 7.6 min, 7.8 mins, and 8.4 min, corresponding to TAT, R9, and PPX peptides, respectively (Figure 11, left panel). Equivalent results obtained for the TAT’, R9’, and PPX’ labeled species are highlighted in Figure 11, right panel. These results illustrate previous explanations about the crucial impact of GRAVY score on CPP peptides retention in HILIC columns.

[0093] A detailed examination of the full mass spectra presented in Figure 12, left panel, revealed a comparable charge state distribution for both TAT and R9 peptides, featuring the protonated species [M+3H]3+, [M+4H]4+, and [M+5H]5+. In contrast, the largest PPX peptideamong the studied CPPs exhibited a mass profile centered at the [M+9H]9+species, illustrating the impact of size and molecular weight on charging efficiency. Notably, the detection of adducts was more pronounced for PPX compared to both TAT and R9, likely due to the increased availability of binding sites and functionalities. Comparable mass profiles to those obtained for the lighter CPP species were observed for the heavily labeled species of TAT, R9, and PPX (Figure 12, right panel). The mass spectra collected during the SIM workflow also display the characteristic isotopic patterns for each CPP and its associated labeled species, enabling identification through spectral matching (Figure 12). Molecular mass comparison and retention time confirmation can be also used for the identification of the CPPs. For instance, Figures 13, 14 and 15 illustrate the BiopharmaFinder identification results from the discovery injections of TAT, R9, and PPX (panel A), and their labeled species (panel B). Notably, mass errors from the calculation of peptides molecular weight remained below 10 ppm, confirming the unambiguous identification of the studied CPPs.

[0094] Several performance parameters including linearity, Limit of Quantification, Limit of Detection, precision, accuracy, and specificity were evaluated for the developed LC-MS method.Linearity, LOQ and LOD

[0095] A series of SIM chromatograms depicted in the left panel of Figure 16 revealed a monotonic increase in ion intensity for both TAT and R9 peptides within the concentration range of 5 ng / mL to 57 ng / mL. Conversely, the ion intensity of PPX peptide exhibited tenfold lower sensitivity. A closer examination of the isotopic patterns highlighted in the insets, confirms the accurate identification of the CPPs at their lowest concentration level, potentially indicating the LOQ of the method. Inspection of the calibration plots of CPP / CPP’ peak area ratio versus concentration, shown in the right panel of Figure 16, demonstrated acceptable coefficient of determination (R2> 0.98) for each peptide, thereby confirming the linearity of the developed LC-MS method within the studied concentration range.

[0096] Further analysis of both precision and accuracy at the lowest calibration level for each peptide indicates that the LOQ of the developed LC-MS method is 5 ng / mL for both TAT and R9 peptides, and 50 ng / mL for PPX peptide. On the other hand, the LODH Vof the LC-MS method was 4 ng / mL for both TAT and R9, and 40 ng / mL for PPX peptide, respectively.Precision and accuracy

[0097] The bar plots in the left panel of Figure 17 show CV values below 15% (N=3) for both peptide calibration and QC standards across the studied concentration range. These results demonstrate that our developed LC-MS method for quantifying TAT, R9, and PPX peptides is repeatable within the evaluated concentration range. As anticipated, higher intra-assay variability was observed at the lowest CPP concentrations, while greater consistency was achieved as peptide concentrations increased. It is important to note that a comprehensive evaluation of the method’s precision, which was not conducted in this study, should also include inter-assay variability, reproducibility, and intermediate precision.

[0098] The accuracy of the developed LC-MS method was assessed by determining the recovery of CPPs spiked at different concentrations. Examination of the scatter plots in the right panel of Figure 17 indicates that calibration and QC samples of TAT and R9 peptides, spiked at low, medium, and high levels were quantitatively recovered (% recovery values between 80% and 120%). Similarly, adequate recovery values were observed for PPX standards within the 50 ng / mL to 570 ng / mL concentration range. Noteworthy, a higher bias (15-17% deviation) in the determination of TAT and R9 concentrations was observed for the lower spiked levels, in good agreement with the precision results obtained in the same concentration range. Nevertheless, both the precision and accuracy of the developed LC-MS are acceptable within the studied concentration range of the analyzed CPPs.Specificity

[0099] The mass accuracy obtained from determining the molecular weight of TAT, R9, and PPX peptides at low, medium and high concentrations was utilized to assess the specificity of the developed HILIC-MS method. Inspection of Table 5 reveled that TAT, R9, and PPX peptides were accurately identified across the spiked concentration range with mass error < 10 ppm, thereby demonstrating the specificity of the method. Note that, due to the low intensity observed for R9 ion signals in the full MS of 6 ng / mL and 12 ng / mL QC standards, the monoisotopic mass of its [M+4H]4+ion, detected during the SIM workflow, was used for mass accuracy determination.

[0100] The comparison of chromatograms from blank injections with those obtained from the analysis of spiked standards can also be used to confirm specificity. An examination of SIMchromatograms collected for blanks and low-concentration QC standards revealed no significant ion signal interferences at the retention times for TAT, R9, and PPX (Figure 18). Therefore, the developed HILIC-MS method can be considered specific for the analysis of TAT, R9, and PPX peptides within the evaluated concentration ranges and experimental conditions.

[0101] Table 5. Mass error obtained during the identification of TAT, R9, and PPX peptides in QC standards using the developed HILIC-MS (N=3)* Theoretical mass of R9 monoisotopic peak at charge state 4+

[0102] An extensive examination of the performance parameters evaluated in this study indicates that the developed HILIC-MS method can be reliably used for the analysis of NLS, TAT, R9, and PPX peptides in samples containing 80% acetonitrile, 20% water, and 0.1% trifluoracetic acid. The addition of sodium chloride to peptide diluent solutions, within the concentration range of 75 mM to 138 mM, was found to be crucial for significantly enhancing the elution and MS sensitivity of the studied CPPs. Other more chaotropic cations from the Hofmeister series (e.g., Li1, Ca21, Mg21, etc.) may potentially yield similar or even better results. For instance, substituting NaCl with LiCl in the peptide diluent solution resulted in comparable MS sensitivities for the analysis of the PPX peptide (Figure 19). This innovative method is significantly advantageous, as it overcomes the challenges associated with the weak interaction and low recovery of highly hydrophilic peptides in most columns used in analyticalchromatography.

Claims

1. CLAIMSWE CLAIM:

1. A method comprising, subjecting a starting sample to hydrophilic interaction chromatography (HILIC), thereby forming a separated sample, subjecting the separated sample to mass spectrometry, thereby forming a mass ion sample, detecting a polypeptide within the mass ion sample, the polypeptide has a negative Grand Average of Hydropathy (GRAVY) score.

2. The method of claim 1, wherein the GRAVY score is -1.5 to -5.0.

3. The method of claim 2, wherein the GRAVY score is -2.0 to -4.5.

4. The method of claim 1, wherein the HILIC has a column temperature is from 25 - 45°C.

5. The method of claim 1, wherein the polypeptide is a cell-penetrating peptide (CPP).

6. The method of claim 1, wherein the starting sample is a cell culture medium.

7. The method of claim 6, wherein the cell culture medium is a processed cell culture medium.

8. The method of claim 1, wherein the sample comprises a virus.

9. The method of claim 8, wherein the virus is a therapeutic virus.

10. The method of claim 9, wherein the therapeutic virus is adeno-associated virus.

11. The method of claim 5, wherein the CPP is selected from R9, NLS, TAT, and PPX.

12. A method comprising, subjecting a sample to hydrophilic interaction chromatography (HILIC), thereby forming a separated sample, subjecting the separated sample to mass spectrometry, thereby forming a mass ion sample, quantifying a polypeptide within the mass ion sample, the polypeptide has a negative GRAVY score.

13. The method of claim 12, wherein the mass ion sample comprises a labeled polypeptide.

14. The method of claim 13, wherein the labeled polypeptide comprises a heavy isotope.

15. The method of claim 14, wherein the labeled polypeptide comprises a CPP.

16. The method of claim 15, wherein the CPP is selected from R9, NLS, TAT, and PPX.

17. The method of claim 12, wherein quantifying the polypeptide is a component of a virus preparation quality control regime.

18. The method of claim 17, the polypeptide is a CPP.

19. A composition comprising a labeled polypeptide and an unlabeled polypeptide in a buffer suitable for mass spectrometric analysis with both the labeled and the unlabeled polypeptides being a CPP.

20. The composition of claim 19, wherein the labeled polypeptide comprises a heavy isotope.

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