Systems and methods for treating virus preparations to reduce heterogeneity

Abstract

A method for reducing heterogeneity of a virus preparation can include generating virus ions from the virus preparation; iteratively increasing at least one of the temperature and incubation period at increasing temperatures of at least one of the virus preparation and the generated virus ions; measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in the temperature and incubation period; determining a mass spectrum at each increase in the temperature and incubation period based on the respective mass-to-charge ratio and charge magnitude; and determining, based on the mass spectrum, an optimal temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international published application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 969,323, filed February 3, 2020, the disclosure of which is expressly incorporated herein by reference in its entirety. Government Rights

[0002] This invention was made with government support under GM131100 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0002]

[0003] The present disclosure relates generally to mass spectrometry, and more particularly to instruments and methods for measuring and analyzing the mass of biological mixture particles, including but not limited to viral particles, over different ranges of temperatures, incubation periods, heating profiles and / or cooling profiles. [Background technology]

[0003]

[0004] Adeno-associated virus (AAV) is an example of a widely accepted gene therapy vector due to its lack of pathogenicity, low immunogenicity, and the existence of many serotypes with different tropisms. It has been found that there is a possibility of dose-related immunotoxicity that may be related to sample preparation and packaging techniques. It may be beneficial to process viral preparations, such as, but not limited to, AAV, in a way that reduces the heterogeneity of such preparations. Summary of the Invention [Means for solving the problem]

[0004]

[0005] The present disclosure may include one or more of the features recited in the appended claims and / or one or more of the following features and combinations thereof: In a first aspect, a method for reducing heterogeneity of a virus preparation may include generating virus ions from a virus preparation, repeatedly increasing at least one of a temperature and an incubation period at increasing temperatures of at least one of the virus preparation and the generated virus ions, measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in the temperature and the incubation period, determining a mass spectrum at each increase in the temperature and the incubation period based on the respective mass-to-charge ratio and charge magnitude values, and determining, based on the mass spectrum, an optimal temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0005]

[0006] A second aspect may include the features of the first aspect and may further include the steps of varying the cooling profile to correspond to a manner in which increased temperatures are decreased after each incubation period, and determining, based on the mass spectra, an optimal temperature and incubation period that, together with the optimal cooling profile, minimizes or at least reduces heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0006]

[0007] A third aspect may include the features of the first aspect and may further include the steps of varying the heating profile to correspond to the manner in which the temperature of at least one of the virus preparation and the generated virus ions is increased, and determining, based on the mass spectrum, an optimal temperature and incubation period that, together with the optimal heating profile, minimizes or at least reduces heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0007]

[0008] A fourth aspect may include the features of the third aspect and may further include the steps of varying the cooling profile to correspond to a manner in which increased temperatures are decreased after each incubation period, and determining, based on the mass spectra, an optimum temperature and incubation period that, together with the optimum cooling profile, minimizes or at least reduces heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0008]

[0009] A fifth aspect may include the features of any of the first to fourth aspects, wherein the step of measuring the mass to charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of temperature and incubation period is performed using a charge detection mass spectrometer.

[0009]

[0010] A sixth aspect may include the features of any of the first to fourth aspects, wherein the step of measuring the mass to charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of temperature and incubation period is performed using a mass spectrometer.

[0010]

[0011] A seventh aspect may include the features of any of the first to sixth aspects, and may further include determining the heterogeneity of the virus population at each increase in at least one of temperature and incubation period based on the mass resolution of at least one mass peak of interest in each mass spectrum.

[0011]

[0012] An eighth aspect may include the features of any of the first to seventh aspects, and may further include determining that aggregation has occurred if, at each increase in at least one of temperature and incubation period, the respective mass spectrum contains distinguishable particles having a mass greater than that of the highest mass capsid in the virus preparation, and the optimal at least one of the temperature and incubation period is less than the respective temperature and incubation period for the respective mass spectrum at which aggregation occurred.

[0012]

[0013] A ninth aspect may include the features of any of the first to eighth aspects, and may further include treating the other samples of the virus preparation by heating each of the other samples of the virus preparation to the determined optimal temperature for an optimal incubation period, in order to minimize or at least reduce heterogeneity thereof.

[0013]

[0014] A tenth aspect may include the features of any of the first to ninth aspects, wherein the virus preparation is a virus preparation solution, and wherein generating virus ions includes generating virus ions from the virus preparation solution using an electrospray ionization source.

[0014]

[0015] An eleventh aspect may include the features of any of the first to tenth aspects, wherein the step of repeatedly increasing at least one of the temperature and the incubation period comprises controlling a first thermal energy device coupled to the virus preparation to heat the virus preparation.

[0015]

[0016] A twelfth aspect may include the features of any of the first to eleventh aspects, wherein the step of repeatedly increasing at least one of the temperature and the incubation period includes controlling a second thermal energy device positioned to transfer thermal energy to the generated ions to heat the generated ions.

[0016]

[0017] In a thirteenth aspect, a method for reducing heterogeneity of a virus preparation may include sequentially increasing at least one of the temperature and incubation period of the virus preparation at increasing temperatures; generating virus ions from the virus preparation at each increase in the at least one of the temperature and incubation period; measuring the mass-to-charge ratio and charge magnitude of at least some of the virus ions generated at each increase in the at least one of the temperature and incubation period; determining a mass spectrum at each increase in the at least one of the temperature and incubation period based on the respective mass-to-charge ratio and charge magnitude values; and determining, based on the mass spectrum, an optimal temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0017]

[0018] A fourteenth aspect may include the features of the thirteenth aspect, and may further include varying the cooling profile to correspond to a manner in which increased temperatures are decreased after each incubation period, and determining, based on the mass spectrum, an optimum of a temperature and incubation period that, together with the optimum cooling profile, minimizes or at least reduces heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0018]

[0019] A fifteenth aspect may include the features of the thirteenth aspect, and may further include the steps of varying the heating profile to correspond to the manner in which the temperature of at least one of the virus preparation and the generated virus ions is increased, and determining, based on the mass spectrum, an optimal heating profile as well as an optimal temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0019]

[0020] A sixteenth aspect may include the features of the fifteenth aspect, and may further include the step of varying the cooling profile to correspond to the manner in which increased temperatures are decreased after each incubation period, and determining, based on the mass spectrum, an optimal cooling profile as well as an optimal heating profile and an optimal temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of virus capsids in the virus preparation.

[0020]

[0021] A seventeenth aspect can include the features of any of the thirteenth to sixteenth aspects, wherein the step of measuring the mass to charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of temperature and incubation period is performed using a charge detection mass spectrometer.

[0021]

[0022] An eighteenth aspect can include the features of any of the thirteenth to sixteenth aspects, wherein the step of measuring the mass to charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of temperature and incubation period is performed using a mass spectrometer.

[0022]

[0023] A nineteenth aspect may include the features of any of the thirteenth to eighteenth aspects, and may further include determining the heterogeneity of the virus population at each increase in at least one of temperature and incubation period based on the mass resolution of at least one mass peak of interest in each mass spectrum.

[0023]

[0024] A twentieth aspect may include the features of any of the thirteenth to nineteenth aspects, and may further include determining that aggregation has occurred if, at each increase in at least one of temperature and incubation period, the respective mass spectrum contains distinguishable particles having a mass greater than that of the highest mass capsid in the virus preparation, and the optimal at least one of the temperature and incubation period is less than the respective temperature and incubation period for the respective mass spectrum at which aggregation occurred.

[0024]

[0025] A twenty-first aspect may include the features of any of the thirteenth to twentieth aspects, and may further include treating the other samples of the virus preparation by heating each of the other samples of the virus preparation to the determined optimal temperature for an optimal incubation period, in order to minimize or at least reduce heterogeneity thereof.

[0025]

[0026] A twenty-second aspect may include the features of any of the thirteenth to twenty-first aspects, wherein the virus preparation is a virus preparation solution, and wherein generating virus ions includes generating virus ions from the virus preparation solution using an electrospray ionization source.

[0026]

[0027] A 23rd aspect may include the features of any of the 13th to 22nd aspects, wherein the step of continuously increasing at least one of the temperature and the incubation period comprises controlling a first thermal energy device coupled to the virus preparation to heat the virus preparation. [Brief explanation of the drawings]

[0027] [Figure 1]

[0028] FIG. 1 is a simplified diagram of an embodiment of an instrument for repeatedly generating charged particles from a virus preparation and then determining and analyzing the mass of the charged particles, wherein the virus preparation and / or the charged particles are subjected to at least one range of different temperatures, incubation periods, heating profiles, and / or cooling profiles. [Figure 2]

[0029] FIG. 2 is a simplified flow diagram of an embodiment of a process for controlling one or more of the thermal energy sources depicted in FIG. 1 to subject a virus preparation and / or charged particles to at least one range of different temperatures, incubation periods, heating profiles, and / or cooling profiles, then controlling an instrument to generate charged particles from the virus preparation, and determining and analyzing the mass of the charged particles at each combination of temperature, incubation period, heating profile, and / or cooling profile to determine an optimal combination of at least one of temperature, incubation period, heating profile, and / or cooling profile that minimizes or at least reduces heterogeneity of the virus preparation without aggregating remaining virus capsids in the preparation. [Figure 3]

[0030] FIG. 3 is a simplified flow diagram of an embodiment of a process for processing viruses according to a predetermined optimal set of temperatures, incubation periods, heating profiles, and / or cooling profiles using the process illustrated in FIG. 2, with the goal of minimizing or at least reducing heterogeneity of a viral preparation without aggregating any remaining viral capsids in the preparation. [Figure 4]

[0031] FIG. 4A is a mass versus abundance plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation under ambient conditions (25° C.).

[0032] FIG. 4B is a mass versus abundance plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 45° C. over an exemplary incubation period of 10 minutes.

[0033] FIG. 4C is a mass versus abundance plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 50° C. over an exemplary incubation period of 10 minutes.

[0034] FIG. 4D is a mass versus abundance plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 55° C. over an exemplary incubation period of 10 minutes.

[0035] FIG. 4E is a mass versus abundance plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 60° C. over an exemplary incubation period of 10 minutes.

[0036] FIG. 4F is a mass versus abundance plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation ramped to 65° C. over an exemplary incubation period of 10 minutes. [Figure 5]

[0037] FIG. 5A is a mass versus intensity plot illustrating the operation of the process of FIG. 2 for another exemplary virus preparation under ambient conditions (25° C.).

[0038] FIG. 5B is a mass versus intensity plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 55° C. over an exemplary incubation period of 20 minutes.

[0039] FIG. 5C is a mass versus intensity plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 55° C. over an exemplary incubation period of 30 minutes.

[0040] FIG. 5D is a mass versus intensity plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 55° C. over an exemplary incubation period of 40 minutes.

[0041] FIG. 5E is a mass versus intensity plot illustrating the operation of the process of FIG. 2 for an exemplary virus preparation elevated to 55° C. over an exemplary incubation period of 60 minutes. DETAILED DESCRIPTION OF THE INVENTION

[0028]

[0042] For the purposes of promoting an understanding of the principles of the present disclosure, reference will be made to a number of exemplary embodiments illustrated in the accompanying drawings, and specific language will be used to describe the same.

[0043] The present disclosure relates to devices and techniques for repeatedly generating charged particles from a virus preparation and then determining and analyzing the mass of the charged particles, wherein the virus preparation and / or the charged particles are subjected to at least one range of different temperatures, incubation periods, heating profiles, and / or cooling profiles for the purpose of determining an optimal combination of at least one of temperatures, incubation periods, heating profiles, and / or cooling profiles that minimizes or at least reduces heterogeneity of the preparation without aggregating remaining virus capsids in the preparation. The present disclosure also relates to devices and techniques for continuously processing a virus preparation by subjecting the virus preparation to a predetermined optimal combination of temperatures, incubation periods, heating profiles, and / or cooling profiles to produce a processed virus preparation in which heterogeneity of the preparation is minimized or at least reduced without aggregating remaining virus capsids in the preparation. The devices and techniques illustrated in the accompanying drawings and described herein can be used as examples to construct a library of optimal combinations of temperatures, incubation periods, heating profiles, and / or cooling profiles for treating different preparations of viruses, each with the goal of minimizing or at least reducing the heterogeneity of such preparations without aggregating the remaining viral capsids in the preparation. For purposes of this document, the term "incubation period" should be understood to mean the amount of time spent at a particular temperature by a viral preparation and / or by charged particles generated from the viral preparation. The term "aggregation" should be understood to mean the adhesion or attachment of two or more viral capsids or capsid fragments to each other, which typically occurs in a viral preparation at various combinations of elevated temperature and incubation period. For purposes of this document, the terms "ion(s)" and "charged particle(s)" should be understood to be synonymous and, therefore, may be used interchangeably.

[0029]

[0044] 1, there is shown a diagram of an instrument 10 for repeatedly generating charged particles from a virus preparation and then determining and analyzing the mass of the charged particles, wherein the virus preparation and / or the charged particles are subjected to at least one range of different temperatures, incubation periods, heating profiles, and / or cooling profiles. In the illustrated embodiment, the instrument 10 illustratively includes an ion source region 12 having an outlet coupled to the inlet of a mass spectrometer 14.

[0030]

[0045] The ion source region 12 illustratively includes an ion generator 18 configured to generate ions, i.e., charged particles, from the sample 16. In the illustrated embodiment, the ion generator 18 is implemented in the form of a conventional electrospray ionization (ESI) source having a pump 18A connected at a solution inlet to an injection tube 18B and at a solution outlet to a capillary 18C having a capillary outlet located in the ion source region 12 of the instrument 10. The ESI source 18 is operable in a conventional manner to draw solution, e.g., at ambient pressure, through the injection tube 18B into the pump 18A and eject a fine spray or droplets of charged solution particles through the outlet of the capillary 18C into the ion source region 12 of the instrument 10. In alternative embodiments, the ion generator 18 may be any conventional device or apparatus for generating ions from a sample and may be positioned outside or inside the ion source region 12. As one illustrative example of the latter, which should not be considered limiting in any way, structure 25 depicted in FIG. 1 may represent an ion generator 18 in the form of a conventional matrix-assisted laser desorption ionization (MALDI) source or other conventional ion generator configured to generate ions from a sample 16 disposed inside ion source region 12. In some embodiments, structure 25 depicted in FIG. 1 may alternatively or additionally represent an ion injection interface, such as any of the structures disclosed in co-pending International Application No. PCT / US2019 / 035379, filed June 4, 2019, the disclosure of which is incorporated herein by reference in its entirety. In other embodiments, structure 25 may be omitted.

[0031]

[0046] The sample 16 from which ions are generated may illustratively be any virus preparation, such as any mixture or solution of or containing any type of virus; one non-limiting example of these is AAV, as described above. In alternative embodiments, the sample 16 may be any mixture, solution, or other form of biological and / or non-biological components. In the example illustrated in FIG. 1 , the sample 16 is, for example, a virus preparation dissolved, dispersed, or otherwise supported in solution 16A within a container 16B; however, in other embodiments, the sample 16 may not be in solution or part of a solution. In the exemplary embodiment illustrated in FIG. 1 , the container 16B is shown offset downward from the injection tube 18B of the ESI source 18; it will be understood that the container 16B is movable upward in direction D so that the container 16B is in fluid communication with the sample solution 16A during injection.

[0032]

[0047] In the illustrated embodiment, voltage source VS1 is electrically connected to processor 20 via a single path numbered J (J can be any positive integer) and is further electrically connected to ion generator 18 via a single path numbered K (K can also be any positive integer). In some embodiments, voltage source VS1 may be implemented in the form of a single voltage source, while in other embodiments, voltage source VS1 may include any number of separate voltage sources. In some embodiments, voltage source VS1 may be configured or controlled to generate and supply one or more time-invariant (i.e., DC) voltages of selectable magnitudes. Alternatively or additionally, voltage source VS1 may be configured or controlled to generate and supply one or more switchable time-varying voltages, i.e., one or more switchable DC voltages. Alternatively or additionally, voltage source VS1 may be configured or controlled to generate and supply one or more time-varying signals of selectable shape, duty cycle, peak magnitude, and / or frequency.

[0033]

[0048] The processor 20 is illustratively conventional and may include a single processing circuit or multiple processing circuits. The processor 20 illustratively includes or is coupled to a memory 22 having instructions stored therein that, when executed by the processor 20, cause the processor 20 to control the voltage source VS1 to generate one or more outputs for selectively controlling the operation of the ion generator 18. In some embodiments, the processor 20 may be implemented in the form of one or more conventional microprocessors or controllers. In such embodiments, the memory 22 may be implemented in the form of one or more conventional memory units having instructions stored therein in the form of one or more microprocessor-executable instructions or instruction sets. In other embodiments, the processor 20 may alternatively or additionally be implemented in the form of a field programmable gate array (FPGA) or similar circuitry. In such embodiments, the memory 22 may be implemented in the form of programmable logic blocks contained within and / or external to the FPGA, into which instructions can be programmed and stored. In still other embodiments, the processor 20 and / or the memory 22 may be implemented in the form of one or more application-specific integrated circuits (ASICs). Those skilled in the art will recognize other forms in which processor 20 and / or memory 22 may be implemented, and will understand that any such other forms of implementation are contemplated by and intended to fall within the scope of the present disclosure. In some alternative embodiments, voltage source VS1 may itself be programmable to selectively generate one or more constant and / or time-varying output voltages.

[0034]

[0049] In the illustrated embodiment, voltage source VS1 is configured, illustratively in response to control signals generated by processor 20, to generate one or more voltages to cause ion generator 18 to generate ions from sample 16 in a conventional manner. In some embodiments, sample 16 is positioned outside ion source region 12, as shown in FIG. 1 , while in other embodiments, ion source 18 may be positioned within ion source region 12. In the illustrated embodiment, electrospray ionization (ESI) source 18 is configured to generate ions from sample 16 in the form of a fine mist of charged droplets in response to one or more voltages supplied by VS1. As noted above, ESI and MALDI represent only two examples of numerous conventional ion generators, and ion generator 18 can be or include any such conventional device or apparatus for generating ions from a sample, whether the sample is in solution or not.

[0035]

[0050] At least one thermal energy source is configured to thermally selectively energize, i.e., transfer thermal energy, to charged particles within sample 16 and / or ion generator 18 and / or ion source region 12. In the illustrated embodiment, for example, thermal energy source 24 is shown operatively coupled to container 16B carrying solution 16A containing a virus preparation, and in this embodiment, thermal energy source 24 is configured to transfer thermal energy to virus preparation solution 16A via container 16B. Alternatively or additionally, thermal energy source 24' may be operatively coupled to ion generator 18. In some such embodiments, thermal energy source 24' may be coupled to pump 18A and / or injection tubing 18B, and in such embodiments, thermal energy source 24' is configured to transfer thermal energy to virus preparation solution 16A via pump 18A and / or tubing 18B, e.g., prior to ionization of solution 16A. In other such embodiments, a thermal energy source 24' may be coupled to capillary 18C, and in such embodiments, thermal energy source 24' is configured to transfer thermal energy to solution 16A within capillary 18C and / or to the charged particles exiting capillary 18C. Alternatively or additionally, a thermal energy source 24'' may be operatively coupled to ion source region 12 of instrument 10, and in such embodiments, thermal energy source 24'' is configured to transfer thermal energy to the charged particles within ion source region 12, i.e., to the charged particles exiting ion generator 18, and to the charged particles before entering mass spectrometer 14.

[0036]

[0051] In some embodiments, the thermal energy generated by the thermal energy source 24, 24′, 24″ can be in the form of heat transferred from the energy source 24, 24′, 24″ to the sample 16, the ion generator 18, and / or the charged particles; in other embodiments, the thermal energy can be in the form of heat transferred from the sample 16, the ion generator 18, and / or the charged particles to the energy source 24, 24′, 24″, i.e., cooling the sample particles. In some embodiments, the energy source 24, 24′, 24″ can include both heating and cooling capabilities such that the sample temperature can be swept from ambient temperature from warmer to cooler, or from cooler to warmer, or from cooler to cooler, from cooler to less cool, from cool or cool to warm or hot, from warm or hot to cool or cool, from warm to warmer, from warm to less warm, from warm to hot, from hot to warm, etc. Exemplary heat sources 24, 24′, 24″ may include, but are not limited to, conventional solution heaters and heating units, one or more radiation sources such as infrared, laser, microwave, or any at any radiation wavelength, one or more heated gas or other fluid(s), and the like; exemplary cooling sources 24, 24′, 24″ may include, but are not limited to, conventional solution chillers, one or more cooled gas or other fluid(s), and the like. Some examples of thermal energy sources 24″ and their operation for heating charged particles are described in co-pending International Application No. PCT / US2018 / 064005, filed December 5, 2018, the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize other structures and / or techniques for controlling the temperature of virus preparation 16 by heating or cooling before or after generation of charged particles from the virus preparation, and will understand that any such other structures and / or techniques are intended to fall within the scope of the present disclosure.

[0037]

[0052] In some embodiments, illustrated by the example of FIG. 1 , thermal energy sources 24, 24′, 24″ are electrically connected to voltage source VS1, which is configured to generate one or more corresponding voltages in response to one or more control signals generated by processor 20 to control the thermal energy generated by thermal energy sources 24, 24′, 24″. In alternative embodiments, thermal energy sources 24, 24′, 24″ may be configured to selectively generate thermal energy in response to control signals generated by processor 20, and in such embodiments, thermal energy sources 24, 24′, 24″ may be electrically connected to processor 20 directly or via conventional circuitry. In some embodiments including thermal energy source 24 and / or thermal energy source 24′, the voltage / current supplied thereto by voltage source VS1 or thermal energy source 24, 24′ itself may not be controlled by processor 20, but rather may be controlled by a separate conventional control circuit C, shown in dashed line representation in FIG. 1 . In some embodiments, thermal energy source 24, 24′, 24″ may be a conventional, manually controlled thermal energy source, such as a manually controlled heater and / or ice bath; in such embodiments, the operation of thermal energy source 24, 24′, 24″ is not controlled by processor 20 or control circuit C, but is manually controlled, for example, by manually controlling the thermal energy source, manually monitoring the temperature, for example, with a conventional thermometer or temperature sensor, and / or manually monitoring the incubation period with a conventional timer, stopwatch, or similar device. In either case, thermal energy source 24, 24′, 24″ may be implemented in the form of one or more conventional heaters or heating elements and / or one or more conventional coolers or cooling elements.

[0038]

[0053] In embodiments in which the thermal energy source 24, 24', 24'' is controlled by the processor 20 or by the control circuit C, the thermal energy source 24, 24', 24'' responds to one or more voltages generated by the voltage source VS1 and / or one or more control signals generated by the processor 20 or the control circuit C to control the temperature of the sample 16, the temperature of the ion generator 18 and / or the temperature of the charged particles in the ion source region 12, and the incubation period, i.e., the duration for which the thermal energy source 24, 24', 24'' is controlled to any particular temperature.

[0039]

[0054] In some embodiments, thermal energy source 24, 24′, 24″ is configured to respond to one or more voltages generated by voltage source VS1 to achieve a target elevated temperature as quickly as practicable, given the physical limitations of thermal energy source 24, 24′, 24″. In alternative embodiments, thermal energy source 24, 24′, 24″ may be programmed to control or configured to respond to signals generated by processor 20 or control circuit C to achieve a target elevated temperature according to any of a number of different heating profiles. Examples of such heating profiles may include, but are not limited to, a linearly increasing, e.g., ramping, temperature profile, a non-linear or piecewise linearly increasing temperature profile, or a combination thereof. In some such embodiments, the duration of the heating profile, i.e., from the current temperature to the target elevated temperature, may also be controlled by processor 20 or control circuit C.

[0040]

[0055] In some embodiments, the thermal energy source 24, 24′, 24″ is configured to achieve the target reduced temperature as quickly as practicable, given the physical limitations of the thermal energy source 24, 24′, 24″, in response to one or more voltages generated by the voltage source VS1. As an example, the thermal energy source 24, 24′, 24″ can be configured to achieve the target reduced temperature by simply turning off or lowering the thermal energy source 24, 24′, 24″, in which case the target reduced temperature would be achieved over a duration during which the thermal energy source 24, 24′, 24″ and the sample 16, ion generator 18, and / or ion source region 12 together cool to the target reduced temperature. In alternative embodiments, the thermal energy source 24, 24′, 24″ can be programmed to, or configured in response to, control signals generated by the processor 20 or control circuit C to achieve the target reduced temperature according to any of a number of different cooling profiles. Examples of such cooling profiles may include, but are not limited to, a linearly decreasing, e.g., ramped, temperature profile, a non-linear or piecewise linearly decreasing temperature profile, or a combination thereof. In some such embodiments, the duration of the cooling profile, i.e., from the current temperature to the target reduced temperature, may also be controlled by processor 20 or control circuit C.

[0041]

[0056] In some embodiments, the charged particles in the sample 16, ion source 18, and / or ion source region 12 are cooled or actively cooled as just described, such that analysis by the mass spectrometer 14 is performed on the charged particles at or near ambient temperature. In other embodiments, the charged particles in the sample 16, ion source 18, and / or ion source 12 may be cooled to below ambient temperature, such that analysis by the mass spectrometer 14 is performed on charged particles cooled to a temperature below ambient. In still other embodiments, the charged particles in the sample 16, ion source 18, and / or ion source 12 are heated to one or more elevated temperatures for one or more incubation periods as just described, but are not subsequently cooled substantially, such that analysis by the mass spectrometer 14 is performed on charged particles heated to one or more elevated temperatures for one or more incubation periods, respectively.

[0042]

[0057] Mass spectrometer 14 illustratively includes two sections, ion processing region 26 and ion detection region 28, coupled together. Second voltage source VS2 is electrically connected to processor 20 via a single path numbered L (L may be any positive integer) and to ion processing region 26 via a single path numbered M (M may also be any positive integer). In some embodiments, voltage source VS2 may be implemented in the form of a single voltage source, while in other embodiments, voltage source VS2 may include any number of separate voltage sources. In some embodiments, voltage source VS2 may be configured or controlled to generate and supply one or more time-invariant (i.e., DC) voltages of selectable magnitude. Alternatively or additionally, voltage source VS2 may be configured or controlled to generate and supply one or more switchable time-invariant voltages, i.e., one or more switchable DC voltages. Alternatively or additionally, voltage source VS2 may be configured or controllable to generate and supply one or more time-varying signals of selectable shape, duty cycle, peak magnitude, and / or frequency. As one specific example of the latter embodiment, which should not be considered limiting in any way, voltage source VS2 may be configured or controllable to generate and supply one or more time-varying voltages in the form of one or more sinusoidal (or other shaped) voltages in the radio frequency (RF) range.

[0043]

[0058] In some embodiments, mass spectrometer 14 is configured to simultaneously measure both the mass-to-charge ratio and charge magnitude of charged particles generated by ion generator 18, so that processor 20 can then determine ion mass based on these measurements. In such embodiments, ion detection region 28 is electrically connected to the inputs of each of number N charge detection amplifiers CA (N may be any positive integer), the outputs of which are electrically connected to processor 20, as shown in FIG. 1. Each charge detection amplifier(s) CA, illustratively conventional, generates a corresponding charge detection signal at its output in response to the charge induced by the charged particles on one or more respective charge detectors disposed in charge detection region 28 and provides the charge detection signal to processor 20.

[0044]

[0059] In one embodiment, in which the mass analyzer 14 is provided in the form of a mass spectrometer configured to simultaneously measure both the mass-to-charge ratio and charge magnitude of charged particles generated by the ion generator 18, the mass analyzer 14 can be implemented in the form of a charge-detection mass spectrometer (CDMS), in which the ion processing region 26 is or includes a conventional mass analyzer or mass analyzer, and the ion detection region 28 illustratively includes one or more corresponding CDMS charge detectors. In some embodiments, the one or more CDMS charge detectors may be provided in the form of one or more electrostatic linear ion traps (ELITs), and in other embodiments, the one or more CDMS charge detectors may be provided in the form of at least one orbitrap. In some embodiments, the CDMS charge detector(s) may include at least one ELIT and at least one orbitrap. While CDMS is illustratively a single-particle technology operable to measure the mass-to-charge ratio and charge magnitude values ​​of a single ion, some CDMS detectors are designed and / or operable to measure the mass-to-charge ratio and charge magnitude of two or more charged particles at once. Some examples of CDMS instruments and / or techniques that may be implemented in or as mass spectrometer 14 of FIG. 1, as well as some examples of CDMS charge detectors and / or techniques, are disclosed in co-pending International Application Nos. PCT / US2019 / 013251, PCT / US2019 / 013274, PCT / US2019 / 013277, PCT / US2019 / 013278, PCT / US2019 / 013280, PCT / US2019 / 013283, PCT / US2019 / 013284, and PCT / US2019 / 013285, all filed January 11, 2019, the disclosures of which are all incorporated herein by reference in their entireties.

[0045]

[0060] In another embodiment, in which the mass analyzer 14 is provided in the form of a mass analyzer configured to simultaneously measure both the mass-to-charge ratio and the charge magnitude of the charged particles generated by the ion generator 18, the mass analyzer 14 may be implemented in the form of a mass analyzer configured to measure the mass-to-charge ratio of the charged particles and further configured to simultaneously measure the charge magnitude of the charged particles. In such an embodiment, the ion processing region 26 is or includes an ion acceleration region and / or a scanning mass-to-charge ratio filter, and the ion detection region 28 illustratively includes a charge detector array disposed within a field-free drift region or drift tube. In such an embodiment, a conventional ion detector 30, such as a conventional microchannel plate detector or other conventional ion detector, is positioned at the exit end of the drift region or drift tube and is electrically connected to a processor, as shown by the dashed line representation in FIG. 1 . Some examples of such mass analyzers are disclosed in co-pending International Application No. PCT / US2020 / 065301, filed December 16, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0046]

[0061] Regardless of the particular form in which the mass spectrometer 14 is provided, various sections of the instrument 10 are controlled to subatmospheric pressure for its conventional operation. In the illustrated embodiment, for example, a so-called vacuum pump P1 is operatively coupled to the ion source region 12, another vacuum pump P2 is operatively coupled to the ion processing region 26 of the mass spectrometer 14, and yet another vacuum pump P3 is operatively coupled to the ion detection region 28 of the mass spectrometer. In the illustrated embodiment, each of the pumps P1, P2, and P3 is operatively coupled to the processor 20 such that the processor 20 is configured to control the operation of each of the pumps P1, P2, and P3, and thus individually control the pressure in each of the three respective regions 12, 26, and 28. In alternative embodiments, one or more of the pumps P1, P2, and / or P3 may be manually controlled. In still other embodiments, more or fewer pumps may be implemented to control the pressure in more or fewer respective portions of the instrument 10. The pressures in the regions of regions 12, 26, and 28 are illustratively set in a conventional manner to provide a positive gas flow in the direction of region 28.

[0047]

[0062] Apparatus 10 further illustratively includes one or more peripheral devices 32 operatively coupled to processor 20 via a single path numbered P, where P may be any positive integer. Peripheral device(s) 32 may be or include any one or combination of conventional peripheral devices, such as, but not limited to, one or more monitors, keyboards, keypads, point-and-click devices, printers, graphical displays, etc.

[0048]

[0063] 2, a simplified flowchart is shown depicting an exemplary process 100 for controlling thermal energy source(s) 24, 24′, 24″ to subject virus preparation 16 and / or charged virus preparation particles generated by ion generator 18 to at least one range of different temperatures, incubation periods, heating profiles, and / or cooling profiles, and also for controlling instrument 10 to generate charged particles from the virus preparation and determine and analyze the mass of the charged particles at each combination of temperature, incubation period, heating profile, and / or cooling profile to determine an optimal combination of at least one of temperature, incubation period, heating profile, and / or cooling profile that minimizes or at least reduces heterogeneity of the virus preparation without aggregating remaining virus capsids in the preparation. Some of the steps of process 100 are provided by way of example in the form of instructions stored in memory 22 and executable by processor 20 to perform the corresponding functions described below, while others of the steps may be performed manually or by control circuit C shown in FIG. 1.

[0049]

[0064] Process 100 begins at step 102, in which a sample 16 of a virus preparation is prepared or obtained. As discussed above, virus preparation 16 may contain, without limitation, any type of virus or combination of viruses. For purposes of the following description of process 100, virus preparation 16 is illustratively a solution virus preparation, such as the ESI source depicted in FIG. 1 that generates charged particles therefrom in the form of an atomized mist or droplets, as described above. However, it will be understood that in other embodiments, virus preparation 16 may be provided in a non-solution state, and / or the ion generator in other embodiments may be another conventional ion generator, examples of which are described above.

[0050]

[0065] Process 100 proceeds from step 102 to step 104, in which processor 20 is operable to follow execution of corresponding instructions stored in memory 22 to control ion generator 18 to generate charged particles from virus preparation 16, which are directed by instrument 10 through ion source region 12 to mass spectrometer 14, for example, via a pressure differential between atmospheric pressure in ESI source 18 and a vacuum in ion source region 12, and / or via a pressure differential between the vacuum in ion source region 12 and a lower vacuum in mass analyzer 14, and / or via an injection interface in embodiments including interface 25. Processor 20 is further operable in step 104 to control mass analyzer 14 to measure the mass-to-charge ratio and charge magnitude of the charged particles generated as described above, and then calculate the masses of the charged particles based on the measured mass-to-charge ratio and charge magnitude to generate a mass spectrum of charged particle masses. In step 104, the virus preparation is illustratively at ambient temperature, e.g., 25°C, and has not yet been subjected to elevated temperature treatment, and the measurements made by instrument 10 in step 104 are also at ambient temperature. In alternative embodiments, virus preparation 16 and / or measurements made by instrument 10 may be at higher or lower temperatures than ambient.

[0051]

[0066] An example of a mass spectrum 300 generated in step 104 is shown in FIG. 4A. The exemplary mass spectrum 300 is represented in FIG. 4A as a plot of abundance versus mass of a virus preparation solution containing AAV8 with an EF1a-GFP genome and having a broad mass peak at approximately 4.6 MDa. The temperature of the virus preparation 16 was 25° C., and the mass spectrum 300 was measured by the instrument 10, also at 25° C. In some alternative embodiments, the mass spectrum 300 may take the form of measured ion intensity versus mass; in other alternative embodiments, the mass spectrum 300 may be represented in the form of particle charge versus particle mass (i.e., a scatter plot).

[0052]

[0067] After step 104, process 100 proceeds to step 106, where counter numbers, e.g., M, N, P, Q, and R, are illustratively set to starting values, such as 1. Thereafter, in step 108, the virus preparation 16, the ion generator 18, and / or the charged particles residing within the ion source region 12 are illustratively heated to an elevated temperature T(M) (i.e., T1 is the elevated temperature in the first run of step 108) using a heating profile P (e.g., heating profile 1 in the first run of step 108) over an incubation period N (e.g., incubation period 1 in the first run of step 108). The temperature change(s), i.e., temperature step size(s), between the measurement taken at ambient conditions, e.g., 25°C, in step 104 and the temperature T(1), and between each T(M) in each run of step 108, may have any integer or non-integer value and may or may not have the same value in each run of step 108. In the first execution of step 1, T(1) is illustratively higher than the temperature condition of step 104. In subsequent executions of step 108, T(M) may or may not change relative to the previous execution of step 108, and any change in T(M) in any such subsequent executions of step 108 may or may not be uniform or constant. The virus preparation 16, the ion generator 18, and / or the charged particles residing within the ion source region 12 may be heated in step 108 using any one or combination of the various devices, apparatus, and / or techniques described above with respect to FIG. 1 . In some embodiments, for example, the processor 20 may be operable to execute instructions stored in the memory 22 to cause the processor 20 to control the voltage source V1 to control the thermal energy source 24 to heat the virus preparation 16 using a heating profile P for an incubation period N corresponding to the temperature T(M).Alternatively or additionally, processor 20 may be operable to execute instructions stored in memory 22 to cause processor 20 to control voltage source V1 to control thermal energy source 24′ to heat ion generator 18 using heating profile P to a temperature T(M) for a corresponding incubation period N in a manner that heats virus preparation 16 contained in any portion thereof, and / or to cause processor 20 to control voltage source V1 to control thermal energy source 24″ to heat charged particles emitted by ion source 18 into ion source region 12 using heating profile P to a temperature T(M) for a corresponding incubation period N. Alternatively, control circuit C may be programmed to control any one or combination of thermal energy sources 24, 24′, 24″ instead of or in addition to their control by processor 20. In yet other embodiments, the temperature of the virus preparation 16 and / or the ion generator 18 and / or the ion source region 12 may be manually controlled over an incubation period corresponding to a temperature T(M) using a heating profile P.

[0053]

[0068] The incubation period(s), i.e., the duration spent by the virus preparation 16, the ion generator 18, and / or the charged particles residing within the ion source region 12 at the set temperature T(M) in each execution of step 108, may have any one or combination of days, hours, minutes, seconds, and / or fractions of a second, and the incubation period in any execution of step 108 may or may not have the same duration as the incubation step in any other execution of step 108. The heating profile(s), i.e., the duration and / or manner in which the temperature(s) of the virus preparation 16, the ion generator 18, the charged particles exiting the ion generator 18 and / or the charged particles residing within the ion source region 12 are increased in step 108 to a temperature higher than in step 104 or to a temperature higher than in a previous run of step 108, can be or have any desired heating profile, some non-limiting examples of which are described above, and the heating profile used in any increase in temperature T(M) in any run of step 108 may or may not be the same as that used in any other run of step 108.

[0054]

[0069] In some embodiments of process 100, the virus preparation 16, the ion generator 18, and / or the charged particles resident within the ion source region 12 are cooled to a temperature lower than the temperature at step 108 following step 108 and before being processed by the apparatus 10. In such embodiments, process 100 illustratively includes step 110, to which process 100 proceeds after performance of step 108, in which the virus preparation 16, the ion generator 18, and / or the charged particles resident within the ion source region 12 are cooled to a reduced temperature, T(R), using a cooling profile Q, e.g., heating profile 1 during the first performance of step 110. The cooling profile(s), i.e., the duration and / or manner in which the temperature(s) of the virus preparation 16, the ion generator 18, the charged particles exiting the ion generator 18, and / or the charged particles resident in the ion source region 12 are reduced in step 110 to a temperature lower than that in step 108, can be or have any desired cooling profile, some non-limiting examples of which are described above, and the cooling profile used in any reduction in temperature T(Q) in any execution of step 110 may or may not be the same as that used in any other execution of step 110. The virus preparation 16, the ion generator 18, and / or the charged particles resident in the ion source region 12 may be cooled in step 110 using any one or combination of the various devices, apparatus, and / or techniques described above with respect to FIG.

[0055]

[0070] In some embodiments, the virus preparation 16, the ion generator 18, and / or the charged particles resident in the ion source region 12 are cooled to ambient temperature(s), e.g., 25° C., after each execution of step 108 in which the virus preparation 16, the ion generator 18, and / or the charged particles resident in the ion source region 12 are heated to an elevated temperature above ambient temperature, so that measurements performed by the mass spectrometer 14 in either case are performed on the charged particles at ambient temperature, e.g., 25° C. In one exemplary such embodiment, each execution of step 108 is performed by heating only the virus preparation 16 to the elevated temperature T(M) for an incubation period N using a heating profile P, and then subsequently cooling the virus preparation 16 to ambient temperature in step 110, so that the virus preparation is unaffected by the instrument 10 until the heating, incubation, and cooling steps are complete. In an alternative embodiment, step 110 may be omitted such that the charged particles resulting from heating the virus preparation 16, the ion generator 18, and / or the charged particles resident within the ion source region 12 to T(M) for the corresponding incubation period at each run step 108 are measured by the instrument 10 at or near the same temperature(s) T(M).

[0056]

[0071] Following step 110, in embodiments that include step 110 and that otherwise follow step 108, process 100 proceeds to step 112, where processor 20 is again operative pursuant to execution of corresponding instructions stored in memory 22 to control ion generator 18 to generate charged particles from virus preparation 16, which are directed by instrument 10 through ion source region 12 to mass spectrometer 14, and control mass analyzer 14 to measure the mass-to-charge ratios and charge magnitudes of the generated charged particles as described above, and then calculate the masses of the charged particles based on the measured mass-to-charge ratios and charge magnitudes to generate an updated mass spectrum of charged particle masses. In some embodiments, as described above, virus preparation 16 is illustratively at ambient temperature, e.g., 25°C, and measurements made by instrument 10 in step 112 are also made at ambient temperature; however, in alternative embodiments, virus preparation 16 and / or measurements made by instrument 10 in step 112 may be at temperatures higher or lower than ambient temperature, as also described above.

[0057]

[0072] Following step 112, process 100 proceeds to steps 114 and 116, in which the updated mass spectrum determined during the most recent execution of step 112 is compared to the most recent previously determined mass spectrum, e.g., the mass spectrum determined during step 104 during the first execution of step 114 and otherwise the mass spectrum determined during a previous execution of step 114, to determine whether the updated mass spectrum indicates an improvement in mass peak resolution without viral capsid aggregation. In some embodiments, steps 114 and 116 are performed by processor 20; in other embodiments, either or both of steps 114 and 116 may be performed manually, i.e., by visually comparing the updated mass spectrum with the previous mass spectrum. In either case, the mass peak width may be determined in a conventional manner, via processor 20, or visually.

[0058]

[0073] Aggregation can also be determined visually or via processor 20. For example, when two or more viral capsids or capsid fragments adhere or attach to one another during aggregation, which generally occurs at various combinations of sufficiently high temperature and incubation period, the adhered or attached capsids generally result in charged particles having higher mass and higher charge than non-aggregated capsids. Thus, the onset of aggregation can be detected either visually or automatically by processor 20 by determining whether an updated mass spectrum shows increased mass and / or charge values.

[0059]

[0074] In either case, in step 116, if the comparison made in step 114 indicates that the updated mass spectrum exhibits an improvement in mass peak resolution without aggregation, process 100 proceeds to step 118, where one or more of the counters M, N, P, Q, and / or R are advanced and / or reset before looping back to step 108. As detailed above, one or more of the temperature of the virus preparation 16, the temperature of one or more components of the ion generator 18, the charged particle temperature in the ion source region 12, the incubation period, the heating profile, and the cooling profile may or may not be changed during each execution of step 118. Two different examples are described below with respect to Figures 4A-4F and 5A-5E.

[0060]

[0075] In step 116, if the comparison performed in step 114 indicates that the updated mass spectrum does not exhibit an improvement in mass peak resolution or exhibits a detectable amount of aggregation, the process proceeds to step 120, where the variable values ​​that produced the most recent previous mass spectrum are recorded and stored in memory 22 as an optimal combination of, for example, temperature, incubation period, heating profile, and in some embodiments, cooling profile, i.e., conditions for processing a virus preparation or the like, with the objective of minimizing or at least reducing heterogeneity of the preparation without aggregating remaining viral capsids in the preparation. It will be understood that other combinations of temperature, incubation period, heating profile, and in some embodiments, cooling profile, i.e., conditions for processing a virus preparation that also minimizes or at least reduces heterogeneity of the preparation without aggregating remaining viral capsids in the preparation, are possible, and that in some embodiments, cooling profile, conditions resulting from execution of process 100 using other values ​​of one or more of the variables may also be recorded.

[0061]

[0076] As briefly discussed above, process 100 can be used to build a library of optimal combinations of temperatures, incubation periods, heating profiles, and / or cooling profiles, each for processing the same preparation of virus, different preparations of virus, and / or preparations of different viruses, with the goal of minimizing or at least reducing heterogeneity of such preparations without aggregating remaining viral capsids in the preparations. For example, following recording of at least one optimal combination of temperatures, incubation periods, heating profiles, and / or cooling profiles obtained from performing process 100 as just described, another process can be performed to apply the optimal combination of conditions to a similar, yet unprocessed viral preparation. Referring now to FIG. 3, a simplified flow diagram of one example of such a process 200 is shown. Process 200 illustratively begins at step 202, in which a viral preparation is prepared or obtained. The viral preparation can be prepared or obtained in any form, e.g., a mixture, a solution, a bulk form, etc., and can contain, without limitation, any type of virus or combination of viruses. Thereafter, in step 204, a previously recorded optimal combination of temperature and incubation period, and in some embodiments, heating profile and / or cooling profile, is obtained. In some embodiments, more than one optimal combination may have been previously recorded, and in such embodiments, one of such multiple optimal combinations may be selected manually or automatically. Thereafter, in step 206, the virus preparation is heated for an incubation period corresponding to the selected optimal temperature. In some embodiments, the selected optimal combination may include an optimal heating profile, and in such embodiments, the virus preparation may be heated to the optimal temperature using the optimal heating profile in step 206.In some embodiments, the selected optimal combination may alternatively or additionally include an optimal cooling profile, in which the virus preparation may be heated to an optimal temperature for an optimal incubation period in step 206, followed by cooling the virus preparation using the optimal cooling profile. In either case, following step 206, the processed virus preparation has minimized, or at least reduced, heterogeneity without causing any remaining viral capsids to aggregate. [Example]

[0062]

[0077] 4A-4F, examples of steps 102-118 of process 100 illustrated in FIG. 2 are shown. As discussed above, FIG. 4A depicts an example mass spectrum 300 generated in step 104 of process 100 in the form of an abundance versus mass plot of a previously unprocessed (by process 100) virus preparation solution 16 containing AAV8 with an EF1a-GFP genome. The temperature of virus preparation 16 was 25° C., and mass spectrum 300 was similarly measured at 25° C. by instrument 10. As illustrated in the example of FIG. 4A, mass spectrum 300 has a broad mass peak at approximately 4.6 MDa.

[0063]

[0078] FIG. 4B depicts another mass spectrum 302 obtained from performance of step 108 of process 100 in which the temperature of virus preparation 16 was increased by controlling a conventional heating coil 24 coupled to the virus preparation to raise the temperature of virus preparation 16 to 45°C as rapidly as possible, and then the temperature of virus preparation 16 was maintained at 45°C for a 15-minute incubation period. In the illustrated example, process 100 included step 110 in which, after the incubation period, virus preparation 16 was removed from the heating coil, cooled on ice for 1 minute, and then allowed to warm naturally to 25°C. After cooling to 25°C, step 112 was performed on the cooled virus preparation 16 using instrument 10 similarly operating at 25°C. Comparing mass spectrum 302 to mass spectrum 300 in step 114, it is clear from FIGS. 4A and 4B that mass spectrum 302 exhibits an improvement in mass peak resolution over that of mass spectrum 300. Furthermore, there does not appear to be any aggregation in mass spectrum 302 because the resulting mass spectrum 302 does not appear to exhibit any peaks higher in mass than that of the only mass peak depicted in mass spectrum 300. Therefore, step 116 proceeds to step 118 where, in this case, only the temperature value is changed by increasing it by 5°C.

[0064]

[0079] The just-described process steps 108-118 are repeated four additional times, exposing virus preparation 16 to 50°C, 55°C, 60°C, and 65°C, respectively, for 15-minute incubation periods. The resulting mass spectra 304, 306, 308, and 310, shown in Figures 4C-4F, respectively, each exhibit an improvement in mass peak resolution over that of a previously determined mass spectrum with no discernible aggregation. The example shown in Figures 4A-4F did not continue beyond 65°C, and therefore, the onset of aggregation was not observed in this example. Therefore, it is not possible to discern from Figures 4A-4F whether any additional improvement in mass peak resolution can be realized by continuing process 100. Similarly, it is not possible to discern from Figures 4A-4F whether the 15-minute incubation period for exemplary virus preparation 16 at 65°C represents an optimal combination of temperature and incubation period that minimizes heterogeneity of virus preparation 16 without aggregating remaining viral capsids. However, from Figures 4A-4F, it can be concluded that an incubation period for exemplary virus preparation 16 of 15 minutes at 65°C substantially reduces the heterogeneity of virus preparation 16 without causing any remaining viral capsids to aggregate. [Example]

[0065]

[0080] 5A-5E, another example of process 100 shown in FIG. 2 is shown, including step 120. In the illustrated example, FIG. 5A depicts an example mass spectrum 400 generated in step 104 of process 100 in the form of a plot of relative ion intensity versus mass of a previously unprocessed (by process 100) simulated virus preparation 16, representative of a virus preparation that may contain, for example, AAV. The temperature of simulated virus preparation 16 was 25° C., and the simulated measurements performed by instrument 10 to generate mass spectrum 400 were also at 25° C. As illustrated by the example of FIG. 5A, mass spectrum 400 has several peaks, each corresponding to a different content of viral capsid. For example, mass peak 402 at approximately 3.8 MDa is due to empty capsids, i.e., those containing no genome or partial genome; mass peaks at approximately 4.3 MDa and 4.6 MDa are due to partial capsids, i.e., those containing a partial genome or partial genomes; mass peak 406 at approximately 5 MDa is due to complete capsids, i.e., those each containing a single genome; and mass peak 408 at approximately 5.2 MDa is due to overpackaged capsids, i.e., those containing a genome of interest and another portion or complete genome of interest.

[0066]

[0081] 5B depicts another mass spectrum 410 obtained from performance of step 108 of process 100, in which the temperature of simulated virus preparation 16 was increased stepwise from 25° C. to 55° C. over a simulated incubation period of 20 minutes. In the illustrated example, process 100 included step 110, in which, following completion of the incubation period, the temperature of simulated virus preparation 16 was decreased stepwise from 55° C. back to 25° C. for performance of step 112 (in which simulated measurements by instrument 10 were performed on simulated, cooled virus preparation 16 with instrument 10 operating at 25° C.). Comparing mass spectrum 410 with mass spectrum 400 in step 114, it is apparent from FIGS. 5A and 5B that mass spectrum 410 exhibits an improvement in mass peak resolution for each capsid type over that of mass spectrum 400. Furthermore, there does not appear to be any aggregation in mass spectrum 410, as the resulting mass spectrum 410 does not appear to exhibit any peaks of higher mass than those attributed to the overpackaged capsid depicted in mass spectrum 400. While the mass peaks 402 and 406 of the empty and full capsids, respectively, appear to be of higher mass resolution, with stable or increased signal intensities, the mass peaks 404 and 408 of the partial and overpackaged capsids, respectively, appear to have decreased signal intensities compared to mass spectrum 400. In either case, step 116 proceeds to step 118, where, in this example, only the incubation period is altered by increasing it by 10 minutes. The temperature increase remains the same at 55°C.

[0067]

[0082] The just-described process steps 108-118 were repeated three more times, exposing the simulated virus preparation 16 to 55°C for three gradually increasing incubation periods of 30, 40, and 60 minutes, respectively. The resulting mass spectra 420 and 430, shown in Figures 5C and 5D, each exhibit an improvement in mass peak resolution for the empty capsid 402 and the full capsid 406, respectively, over those of the previously determined mass spectra with no discernible aggregation. As Figures 5A-5D also demonstrate, partial and overpackaged capsids begin to degrade and continue to degrade under increased incubation durations at elevated temperatures, as indicated by the decrease in intensity under such conditions and their eventual disappearance in Figure 5D of the partial and overpackaged capsid mass peaks 404 and 408, respectively, indicating that such capsids are not stable under elevated temperatures and their respective incubation periods. However, it should be understood that such instability of partial capsids and overpackaged capsids under the conditions depicted in Figures 5A-5D may be representative of the particular exemplary virus preparation 16 used, but is not necessarily representative of other types of virus preparations, and that in other types of virus preparations, one or any combination of capsid types may exhibit such instability while remaining capsid types remain stable.

[0068]

[0083] As further shown in Figure 5E, the resulting mass spectrum 440 similarly exhibits an improvement in mass peak resolution of empty capsid 402 and complete capsid 406 over that of the previously determined mass spectrum 430. However, mass spectrum 440 also exhibits high-mass components in the 6-7 MDa range, i.e., components with masses greater than that of the original highest mass peak 408 attributed to overpackaged capsids, which is indicative of at least some aggregation of remaining empty capsids and / or complete viral capsids. Additionally, lower mass peaks 444, 446, and 488, e.g., between 0.2 and 2 MDa, are also observed in spectrum 440 depicted in Figure 5E. Peak 444 is attributed to single-stranded DNA resulting from degradation of some of the capsids, and peak 446 is attributed to double-stranded DNA resulting from the merging of some of the single-stranded DNA. Peak 448 is attributed to proteins associated with the genome of the degraded partial capsid and / or overpackaged capsid.

[0069]

[0084] In the exemplary implementation of process 100 depicted in Figures 5A-5E, mass resolution of the peak of interest improves with each incrementally increasing incubation period at a typical virus preparation temperature of 55°C, although the onset of aggregation appears to occur between 40 and 60 minutes of incubation time. Thus, in this exemplary implementation of process 100, the optimal combination of variables that minimizes or at least reduces heterogeneity of the virus preparation without aggregation is a 40-minute incubation period at temperature = 55°C. If a heating profile is to be included in the optimal combination of variables, the heating profile in this particular example is a step change, which in a practical application would correspond to controlling thermal energy source 24 to increase the temperature of the virus preparation from 25°C to 55°C as quickly as possible. If a cooling profile is to be included in the optimal combination of variables, the cooling profile in this particular example is similarly a step change, which in a practical application would correspond to cooling the virus preparation in, for example, an ice bath or other rapid cooling environment. In either case, the optimal combination of variables in this example can then be used in process 300 depicted in FIG. 3 to heat-treat similar virus preparations with the goal of minimizing or at least reducing heterogeneity without causing any remaining capsids to aggregate.

[0070]

[0085] While the present disclosure has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered illustrative and not restrictive in nature, it being understood that only exemplary embodiments thereof have been shown and described, and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

Claims

1. 1. A method for reducing heterogeneity in a virus preparation, excluding virus preparations in which heterogeneity is increased by increasing at least one of virus temperature and incubation period at one or more increased virus temperatures, comprising: generating virus ions from the virus preparation; repeatedly increasing at least one of the temperature and incubation period at the increased temperature of at least one of the virus preparation and the generated virus ions; measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of the temperature and the incubation period; determining a mass spectrum at each increase in at least one of the temperature and the incubation period based on respective mass-to-charge ratio and charge magnitude values; determining, based on the mass spectrum, the optimum temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation; A method comprising:

2. Varying the cooling profile to correspond to the manner in which the increased temperature is decreased after each incubation period; Based on the mass spectra, determining an optimal cooling profile along with an optimal temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation. Further comprising: The method of claim 1.

3. Varying a heating profile to correspond to the manner in which the temperature of at least one of the virus preparation and the generated virus ions is increased; determining an optimal heating profile, along with an optimal one for the temperature and the incubation period, that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation, based on the mass spectrum; Further comprising: The method of claim 1.

4. Varying the cooling profile to correspond to the manner in which the increased temperature is decreased after each incubation period; determining an optimal cooling profile, along with the optimal temperature and incubation period, that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation based on the mass spectrum; Further comprising: The method of claim 3.

5. measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of the temperature and the incubation period using a charge-detection mass spectrometer; The method according to any one of claims 1 to 4.

6. measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of the temperature and the incubation period using a mass spectrometer; The method according to any one of claims 1 to 4.

7. determining the heterogeneity of the virus population at each increment of at least one of said temperature and said incubation period based on the mass resolution of at least one mass peak of interest in each mass spectrum; Further comprising: The method according to any one of claims 1 to 6.

8. determining that aggregation has occurred if, at each increase in at least one of the temperature and the incubation period, the respective mass spectra contain distinguishable particles having a mass greater than that of the highest mass capsid in the virus preparation. Further comprising: at least one of the optimum temperatures and incubation periods is less than the respective temperatures and incubation periods of the respective mass spectra at which aggregation occurred; The method according to any one of claims 1 to 7.

9. treating the other samples of said virus preparation by heating each of said other samples of said virus preparation to the determined optimal temperature for an optimal incubation period in order to minimize or at least reduce heterogeneity thereof; Further comprising: The method according to any one of claims 1 to 8.

10. the virus preparation is a virus preparation solution; generating the virus ions includes generating the virus ions from the virus preparation solution using an electrospray ionization source; The method according to any one of claims 1 to 9.

11. repeatedly increasing at least one of the temperature and the incubation period comprises controlling a first thermal energy device coupled to the virus preparation to heat the virus preparation. The method according to any one of claims 1 to 10.

12. repeatedly increasing at least one of the temperature and the incubation period includes controlling a second thermal energy device positioned to transfer thermal energy to the generated ions to heat the generated ions. The method according to any one of claims 1 to 11.

13. 1. A method for reducing heterogeneity in a virus preparation, excluding virus preparations in which heterogeneity is increased by increasing at least one of virus temperature and incubation period at one or more increased virus temperatures, comprising: sequentially increasing at least one of temperature and incubation period at increasing temperatures of the virus preparation; generating virus ions from the virus preparation at each increase in at least one of the temperature and the incubation period; measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of the temperature and the incubation period; determining a mass spectrum at each increase in at least one of the temperature and the incubation period based on respective mass-to-charge ratio and charge magnitude values; determining, based on the mass spectrum, the optimum temperature and incubation period that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation; A method comprising:

14. Varying the cooling profile to correspond to the manner in which the increased temperature is decreased after each incubation period; determining an optimal cooling profile, along with an optimal one for the temperature and the incubation period, that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation, based on the mass spectrum; Further comprising: The method of claim 13.

15. Varying a heating profile to correspond to the manner in which the temperature of at least one of the virus preparation and the generated virus ions is increased; determining an optimal heating profile, along with an optimal one for the temperature and incubation period, that together minimizes or at least reduces heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation based on the mass spectrum; Further comprising: The method of claim 13.

16. Varying the cooling profile to correspond to the manner in which the increased temperature is decreased after each incubation period; determining, based on the mass spectrum, an optimal heating profile and an optimal cooling profile, together with the optimal temperature and incubation period, that together minimize or at least reduce heterogeneity of the virus preparation without causing aggregation of viral capsids in the virus preparation; Further comprising:

16. The method of claim 15.

17. measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of the temperature and the incubation period is performed using a charge-detection mass spectrometer; The method according to any one of claims 13 to 16.

18. measuring the mass-to-charge ratio and charge magnitude of at least some of the generated virus ions at each increase in at least one of the temperature and the incubation period using a mass spectrometer; The method according to any one of claims 13 to 16.

19. determining the heterogeneity of the virus population at each increase in at least one of the temperature and the incubation period based on the mass resolution of at least one mass peak of interest in each mass spectrum; Further comprising: The method according to any one of claims 13 to 18.

20. determining that aggregation has occurred if, at each increase in at least one of the temperature and the incubation period, the respective mass spectra contain distinguishable particles having a mass greater than that of the highest mass capsid in the virus preparation. Further comprising: at least one of the optimum temperatures and incubation periods is less than the respective temperatures and incubation periods of the respective mass spectra at which aggregation occurred; The method according to any one of claims 13 to 19.

21. treating the other samples of said virus preparation by heating each of said other samples of said virus preparation to the determined optimal temperature for an optimal incubation period in order to minimize or at least reduce heterogeneity thereof; Further comprising: The method according to any one of claims 13 to 20.

22. the virus preparation is a virus preparation solution; generating the virus ions includes generating the virus ions from the virus preparation solution using an electrospray ionization source; The method according to any one of claims 13 to 21.

23. sequentially increasing at least one of the temperature and the incubation period comprises controlling a first thermal energy device coupled to the virus preparation to heat the virus preparation. The method according to any one of claims 13 to 22.

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