Ion separator

Abstract

A method of separating ions comprising: rotating a housing about an axis such that gas therein is rotated around the axis; and then transmitting ions into the gas such that the ions are driven around the axis by the gas and thus experience a centrifugal force that separates the ions.

Description

[0001] ION SEPARATOR

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority from and the benefit of United Kingdom patent application No. 2404163.4, which was filed on 22 March 2024. The entire contents of this application are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates generally to mass spectrometers and method of mass spectrometry in which ions are separated.

[0006] BACKGROUND

[0007] There is increasing interest in the use of viral capsids to deliver a DNA cargo, such as in vaccines and gene therapy applications. Current research focusses on the capsids of Adeno-Associated Virus (AAV). AAVs have a molecular weight of about 3.7 MDa and can encapsulate a DNA cargo having a molecular weight of up to about 1.5 MDa. Future therapies will need to deliver a DNA cargo having a molecular weight that is larger than this and therefore methods of analysis that are suitable for larger capsids are of interest.

[0008] An important aspect of quality assurance or quality control in capsid-based vaccine and gene therapy applications is the quantification of the particles that are empty, the particles that contain a full DNA cargo, and the particles incorporating a truncated DNA cargo (which are known as partially filled capsids). It is known to use Analytical UltraCentrifugation to separate empty, partially filled and full capsids. However, this process is relatively slow and requires large and complex equipment to perform it.

[0009] SUMMARY

[0010] From a first aspect the present invention provides a method of separating ions comprising: rotating a housing about an axis such that gas therein is rotated around the axis; and then transmitting ions into the gas such that the ions are driven around the axis by the gas and thus experience a centrifugal force that separates the ions.

[0011] The centrifugal force causes ions having different masses to be separated from each other in a direction that is radially outward from the axis of rotation. More specifically, the centrifugal force urges the ions through the gas in the housing and so the ions experience a drag force caused by the gas such that ions having different masses are caused to be separated from each other.

[0012] It has been found that rotating the housing that encloses a chamber of gas is a particularly good way to cause the gas to rotate around the axis so as to cause the centrifugal separation of the ions. For example, this technique creates a gas flow around the axis that is substantially uniform throughout the housing, which may be beneficial for ion separation.

[0013] There may be substantially no gas flow within the housing in a direction parallel to the axis of rotation.

[0014] The centrifugal force separates the ions in a radial direction that is orthogonal to the axis of rotation, and may cause the ions to travel in the radial direction until they pass out of the housing and / or strike an ion detector.

[0015] The housing may be toroidal and / or a gas chamber inside the housing that contains the rotating gas may be toroidal.

[0016] The housing and / or gas chamber may have a substantially rectangular crosssection in the plane that is defined by the axis of rotation and an axis orthogonal to the axis of rotation.

[0017] Preferably, the rectangular cross-section is elongated in the radial direction that extends orthogonally to the axis of rotation.

[0018] The ions may be transmitted into the housing and the gas through one or more ion entrance opening located on a radially inner side of the housing that faces the axis of rotation.

[0019] The one or more ion entrance opening may comprise one or more elongated slit that extends partway along a radially inner wall of the housing, in a direction orthogonal to the axis of rotation; or the one or more ion entrance opening may be an elongated slit that extends fully along a radially inner wall of the housing, in a direction orthogonal to the axis of rotation.

[0020] The method may comprise: transmitting the ions into the housing and the gas through one or more ion entrance opening in a wall of the housing; providing one or more ion detector radially outwards of the one or more ion entrance opening; and detecting ions that have been separated in the housing, or ions derived therefrom, with the one or more ion detector. For example, the separated ions may be fragmented or reacted so as to produce fragment or product ions that are detected at the one or more detector.

[0021] The method may comprise determining the mass of any given ion that is detected by the ion detector from the duration of time between that ion entering the rotating gas and that ion, or an ion derived therefrom, being detected by the ion detector.

[0022] For example, the step of determining the mass of any given ion may comprise: measuring, estimating, or otherwise obtaining the mobility of the ion through the gas; determining the ratio of the mass of the ion to the mobility of the ion in the radial direction through the gas in the housing, based on the duration of time between that ion entering the rotating gas and that ion, or an ion derived therefrom, being detected by the ion detector; and calculating the mass of the ion from said ratio and said mobility.

[0023] For instance, if the diameter of the ionised particle is known, this may be used, along with said ratio, to calculate the mass since diameter is related to mobility.

[0024] An ion gate may be provided in, or upstream of, the one or more ion entrance opening, and the time that the ion enters the rotating gas may be determined from the time that the ion gate is opened. The ion gate has at least one electrode and a voltage supply that is controlled so as to maintain the ion gate closed such that ions cannot pass through the ion entrance opening, then to open the ion gate and allow ions to enter the housing as an ion packet, and to then close the ion gate again so as to prevent ions from entering the housing.

[0025] The mass of any given ion may be determined from the duration of time between the ion gate being opened and the time that the ion, or an ion derived therefrom, is detected by the ion detector.

[0026] The ions may be ionised capsid particles, e.g. capsid particles of the same type but that may encapsulate different DNA cargos.

[0027] Accordingly, the ions may be ionised capsid particles, wherein at least some of the capsid particles encapsulate a DNA cargo, and wherein the method comprises determining whether capsid particles detected at the detector contain no DNA cargo, a full DNA cargo, or a partially full DNA cargo based on the calculated mass for the ion.

[0028] The capsid particles may be viral capsid particles, such as capsid particles of an Adeno Virus, capsid particles of an Adeno Associated Virus, or capsid particles of a Lentivirus. Alternatively, the capsid particles may be of other types, such as liposome capsid particles.

[0029] The capsid particles may be of the same type, e.g. from the same virus or liposome, but they may encapsulate different mass DNA cargos. The mass of the capsid particle shell that is used to encapsulate the DNA cargo may be known, as the type of capsid particle may have been selected. This information, along with the determined mass of the ionised capsid particle, may be used to determine the DNA cargo inside the detected capsid particle. The mass of the full DNA cargo may also be known. As such, the method may determine if the detected ionised capsid particles has no DNA cargo, a full DNA cargo, or a partially full DNA cargo based on the determined mass.

[0030] The method may comprise determining the proportion of the detected capsid particles that have no DNA cargo, and / or a full DNA cargo, and / or a partially full DNA cargo.

[0031] The one or more ion detector may be located inside the housing so as to rotate therewith.

[0032] The ion detector may have an ion detecting surface that extends, in the circumferential direction, along substantially the entire length of the radially outer wall of the housing. Alternatively, the ion detecting surface may extend, in the circumferential direction, along only a portion of the length of the radially outer wall. Multiple such ion detecting surfaces may be provided on the radially outer wall that are spaced apart in the circumferential direction.

[0033] The one or more ion detector may be a charge sensitive detector, such as a Faraday plate, for detecting ions separated by the separator. The ion detector may detect the charge of the ion.

[0034] The housing may be a substantially closed housing such that the gas in the housing is substantially confined by the housing. For example, the housing may be sealed, except that it may have one or more ion entrance openings that are arranged such that ions may enter into the housing. However, it is contemplated that the housing may also have one or more ion exit opening located on a radially outer side of the housing.

[0035] The housing may comprise an ion entrance opening and an ion detector that are arranged on the housing such that when the housing is rotated, only ions having a preselected ratio of mass to mobility through the gas are able to strike the detector and ions having other ratios of mass to mobility do not strike the detector.

[0036] Ions may be continuously supplied through the ion entrance opening and the method may determine that ions having said pre-selected ratio of mass to mobility are present when ions are detected at the detector and determines that ions having said preselected ratio of mass to mobility are not present when ions are not detected at the detector.

[0037] The ion separator may have multiple such ion detectors. For example, the separator housing may comprise an ion entrance opening, a first ion detector and a second ion detector, wherein the ion entrance opening and ion detectors are arranged such that when the housing is rotated only ions having a first pre-selected range of mass to mobility ratios are able to strike the first ion detector and ions having other mass to mobility ratios do not strike the first detector, and only ions having a second, different pre-selected range of mass to mobility ratios are able to strike the second ion detector and ions having other mass to mobility ratios do not strike the second detector.

[0038] Ions may be continuously supplied through the ion entrance opening and the method may determine that ions having said first pre-selected mass to mobility ratio are present when ions are detected at the first ion detector and determines that ions having said first pre-selected mass to mobility ratio are not present when ions are not detected at the first ion detector. Similarly, the method may determine that ions having said second pre-selected mass to mobility ratio are present when ions are detected at the second ion detector and determines that ions having said second pre-selected mass to mobility ratio are not present when ions are not detected at the second ion detector.

[0039] As described above, the ions may be ionised capsid particles, e.g. capsid particles of the same type but that may encapsulate different DNA cargos. These embodiments may be used to determine whether capsid particles contain no DNA cargo, a full DNA cargo, or a partially full DNA cargo.

[0040] For example, the first ion detector may be arranged such that when the housing is rotated only ionised capsid particles having a mass to mobility ratio corresponding to particles containing a full DNA cargo are able to strike the first ion detector, whereas ions having other mass to mobility ratios do not strike the first detector. Similarly, the second ion detector may be arranged such that when the housing is rotated only ionised capsid particles having a mass to mobility ratio corresponding to particles containing no DNA cargo are able to strike the second ion detector, whereas ions having other mass to mobility ratios do not strike the second detector. Additionally, or alternatively, a third ion detector may be arranged such that when the housing is rotated only ionised capsid particles having a range of mass to mobility ratios corresponding to particles containing a partially full DNA cargo are able to strike the third ion detector, whereas ions having other mass to mobility ratios do not strike the third detector.

[0041] The method may comprise determining the proportion of the detected capsid particles that have no DNA cargo, and / or a full DNA cargo, and / or a partially full DNA cargo.

[0042] Ions are separating in the rotating housing and may exit the housing through one or more ion exit opening located on a radially outer side of the housing that faces away from the axis of rotation; wherein the ions then pass to one or more ion detector that is located outside the housing.

[0043] In these embodiments, the one or more ion detectors may be arranged such that they are static and do not rotate with the rotating housing. However, it is contemplated that they could be arranged to rotate with the housing, e.g. at the same angular rate.

[0044] The one or more ion exit opening may comprise one or more elongated slit that extends partway along a radially outer wall of the housing in a direction orthogonal to the axis of rotation; or the one or more ion entrance opening may be an elongated slit that extends fully along a radially outer wall of the housing in a direction orthogonal to the axis of rotation.

[0045] Preferably, the gas in the housing is maintained at a pressure of at least 1x104Pa, at least 5x104Pa, or at least 1x105Pa during separation of the ions. This avoids the need for a vacuum system and has been found to be acceptable for the separation of large mass ions, such as ionised capsid particles.

[0046] Accordingly, the gas within the housing may be substantially at atmospheric pressure during separation of the ions; and / or the housing may not be evacuated by a vacuum system that sucks gas out of the housing. By substantially at atmospheric pressure it is meant that the gas within the housing is at atmospheric pressure when the housing is not rotating, but that the pressure of the gas may increase due to rotation of the housing.

[0047] However, it is contemplated that the housing could be evacuated such that the gas pressure is less than atmospheric pressure when the housing is rotating and ions are being separated. For example, the gas in the housing may be maintained at a pressure of < 5x104Pa or < 1x104Pa during rotation of the housing and separation of the ions.

[0048] The housing may be rotated around the axis at > 5,000 rpm, > 10,000 rpm, > 15,000 rpm, > 20,000 rpm, > 25,000 rpm, or > 30,000 rpm.

[0049] The housing may have a maximum dimension, e.g. in the radial direction, of < 100 mm, < 90 mm, < 80 mm, < 70 mm, < 60 mm, or < 50 mm.

[0050] The method comprises generating ions with an ion source, such as an electrospray ion source. The ion source is arranged such that ions are provided to the one or more ion entrance openings. The ion source may provide ions to, or generate ions in, the region that is surrounded by the housing. For example, the ion source may direct ions along the axis of rotation into the region that is surrounded by the housing. The axis of rotation may be substantially vertical. In such embodiments, the ion source may direct ions downwards. For example, the ion source may be an electrospray ion source that generates an electrospray plume along the axis of rotation.

[0051] A liquid or gas chromatography device may be used to separate an analytical sample and supply it to an ion source that generates the ions. These ions, or ions derived therefrom, may be supplied to the ion separator device described herein for separation and detection.

[0052] The method may comprise providing a liquid chromatography device that is arranged to rotate around the rotational axis with the housing such that a centrifugal force is provided within the liquid chromatography device that drives an analytical sample through the liquid chromatography device so that different components in the sample elute from the liquid chromatography device at different times, and ionising the eluting components so as to form ions, and supplying these ions, or ions derived therefrom into said housing to be separated.

[0053] For example, the liquid chromatography device may comprise a chromatography column that is arranged with its longitudinal axis such that the sample entrance to the column is arranged radially inwards of the sample exit from the column. The column may be arranged with its longitudinal axis extending in the radial direction.

[0054] It will be appreciated that the methods described herein are methods of mass spectrometry.

[0055] From a second aspect the present invention provides a method of separating ions comprising: rotating a housing about an axis such that gas therein is rotated around the axis; transmitting ionised capsid particles into the gas through one or more ion entrance opening in a wall of the housing such that the ionised capsid particles are driven around the axis by the gas and thus experience a centrifugal force that separates the ionised capsid particles; providing one or more ion detector radially outwards of the one or more ion entrance opening, and detecting ionised capsid particles that have been separated, or ions derived therefrom, with the one or more ion detector; determining the mass of any given ionised capsid particle that is detected by the one or more ion detector from the duration of time between that ionised capsid particle entering the rotating gas and that ionised capsid particle, or an ion derived therefrom, being detected by the one or more ion detector; and determining if the detected ionised capsid particles has no DNA cargo, a full DNA cargo, or a partially full DNA cargo based on the determined mass.

[0056] The capsid particles may be of the same type, e.g. from the same virus or liposome, but they may encapsulate different mass DNA cargos. The mass of the capsid particle shell that is used to encapsulate the DNA cargo may be known, as the type of capsid particle may have been selected. This information, along with the determined mass of the ionised capsid particle, may be used to determine the DNA cargo inside the detected capsid particle. The mass of the full DNA cargo may also be known. As such, the method may determine if the detected ionised capsid particles has no DNA cargo, a full DNA cargo, or a partially full DNA cargo based on the determined mass. The method may determine the proportion of the detected capsid particles that have no DNA cargo, and / or a full DNA cargo, and / or a partially full DNA cargo.

[0057] The gas within the housing maybe substantially at atmospheric pressure during separation of the ionised capsid particles; and / or wherein the housing is not evacuated by a vacuum system that sucks gas out of the housing.

[0058] The method according to the second aspect of the invention may have any of the features described in relation to the first aspect of the invention.

[0059] The first aspect of the present invention also provides an ion separating apparatus set up and configured to perform the methods described herein.

[0060] Accordingly, the present invention provides an ion separator apparatus comprising: an ion source; and a housing arranged to receive ions from the ion source, or ions derived therefrom; wherein the housing contains a gas and is rotatable about an axis such that the gas therein is rotated around the axis and ions received in the housing are driven around the axis by the gas and thus experience a centrifugal force that separates the ions.

[0061] The apparatus may comprise a motor configured to rotate the housing around the axis.

[0062] The ion separator apparatus may have any of the features described above in relation to the method of the first and second aspects of the present invention.

[0063] The ion separator apparatus may be arranged and configured to perform any of the methods described herein.

[0064] For example, the housing may be arranged such that when it is rotated around the axis the centrifugal force separates the ions in a radial direction that is orthogonal to the axis of rotation, and causes the ions to travel in the radial direction until they pass out of the housing and / or strike an ion detector.

[0065] The housing may be toroidal and / or a gas chamber inside the housing that contains the gas may be toroidal. The housing and / or gas chamber may have a substantially rectangular cross-section in the plane that is defined by the axis of rotation and an axis orthogonal to the axis of rotation. Preferably, the rectangular cross-section is elongated in the radial direction that extends orthogonally to the axis of rotation.

[0066] The housing may have one or more ion entrance opening located on a radially inner side of the housing that faces the axis of rotation, for receiving the ions into the housing. The one or more ion entrance opening may comprise one or more elongated slit that extends partway along a radially inner wall of the housing, in a direction orthogonal to the axis of rotation; or the one or more ion entrance opening may be an elongated slit that extends fully along a radially inner wall of the housing, in a direction orthogonal to the axis of rotation.

[0067] The apparatus may comprise one or more ion detector located radially outwards of the one or more ion entrance opening such that ions that have been separated by the ion separator, or ions derived therefrom, are detected by the ion detector.

[0068] The apparatus may be configured to determine the mass of any given ion that is detected by the ion detector from the duration of time between that ion entering the rotating gas and that ion, or an ion derived therefrom, being detected by the ion detector. For example, at least one of the one or more ion entrance openings may be an ion gate that is configured to open and close so as to admit a packet of ions into the housing. The ion gate has at least one electrode and a voltage supply that is controlled so as to maintain the ion gate closed such that ions cannot pass through the ion entrance opening, then to open the ion gate and allow ions to enter the housing as an ion packet, and to then close the ion gate again so as to prevent ions from entering the housing. The apparatus may automatically determine the mass of any given ion from the duration of time between the ion gate being opened and the time that the ion, or an ion derived therefrom, is detected by the ion detector.

[0069] The apparatus may include a supply of an analytical sample that comprises capsid particle, such that the ion source ionises the capsid particles. The ions may therefore be ionised capsid particles, e.g. capsid particles of the same type but that may encapsulate different DNA cargos. The apparatus may be configured to automatically determine whether capsid particles detected at the detector contain no DNA cargo, a full DNA cargo, or a partially full DNA cargo based on the calculated mass for the ion. The apparatus may be configured to automatically determine the proportion of the detected capsid particles that have no DNA cargo, and / or a full DNA cargo, and / or a partially full DNA cargo.

[0070] The capsid particles may be viral capsid particles, such as Adeno Virus capsid particles, Adeno Associated Virus capsid particles, or Lentivirus capsid particles. Alternatively, the capsid particles may be of other types, such as liposome capsid particles.

[0071] The one or more ion detector may be located inside the housing so as to rotate therewith. The ion detector may have an ion detecting surface that extends, in the circumferential direction, along substantially the entire length of the radially outer wall of the housing. Alternatively, the ion detecting surface may extend, in the circumferential direction, along only a portion of the length of the radially outer wall. Multiple such ion detecting surfaces may be provided on the radially outer wall that are spaced apart in the circumferential direction. The one or more ion detector may be a charge sensitive detector, such as a Faraday plate, for detecting ions separated by the separator. The ion detector may detect the charge of the ion.

[0072] The housing may be a substantially closed housing such that the gas in the housing is substantially confined by the housing. For example, the housing may be sealed, except that it may have one or more ion entrance openings that are arranged such that ions may enter into the housing. However, it is contemplated that the housing may also have one or more ion exit opening located on a radially outer side of the housing.

[0073] The housing may comprise an ion entrance opening and an ion detector that are arranged on the housing such that when the housing is rotated, only ions having a preselected mass to mobility ratio are able to strike the detector and ions having other mass to mobility ratios do not strike the detector. The ion source and apparatus may be configured to continuously supplied ion through the ion entrance opening and the apparatus may be configured to determine that ions having said pre-selected mass to mobility ratio are present when ions are detected at the detector and determine that ions having said preselected mass to mobility ratio are not present when ions are not detected at the detector. The ion separator may have multiple such ion detectors. For example, the separator housing may comprise an ion entrance opening, a first ion detector and a second ion detector, wherein the ion entrance opening and ion detectors are arranged such that when the housing is rotated only ions having a first pre-selected range of mass to mobility ratios are able to strike the first ion detector and ions having other mass to mobility ratios do not strike the first detector, and only ions having a second, different pre-selected range of mass to mobility ratios are able to strike the second ion detector and ions having other mass to mobility ratios do not strike the second detector.

[0074] The apparatus may be configured such that ions are continuously supplied through the ion entrance opening and the apparatus may determine that ions having said first preselected mass to mobility ratio are present when ions are detected at the first ion detector and determines that ions having said first pre-selected mass to mobility ratio are not present when ions are not detected at the first ion detector. Similarly, the apparatus may determine that ions having said second pre-selected mass to mobility ratio are present when ions are detected at the second ion detector and determines that ions having said second pre-selected mass to mobility ratio are not present when ions are not detected at the second ion detector.

[0075] Although the ion detector(s) have been described as being in the housing, the ion detector(s) may be outside the housing. For example, the apparatus may be configured such that ions that are separated in the rotating housing exit the housing through one or more ion exit opening located on a radially outer side of the housing that faces away from the axis of rotation, and the ions then pass to one or more ion detector that is located outside the housing. In these embodiments, the one or more ion detectors may be arranged such that they are static and do not rotate with the rotating housing. However, it is contemplated that they could be arranged to rotate with the housing, e.g. at the same angular rate.

[0076] The one or more ion exit opening may comprise one or more elongated slit that extends partway along a radially outer wall of the housing in a direction orthogonal to the axis of rotation; or the one or more ion entrance opening may be an elongated slit that extends fully along a radially outer wall of the housing in a direction orthogonal to the axis of rotation.

[0077] Preferably, the apparatus is configured to maintain the gas in the housing at a pressure of at least 1x104Pa, at least 5x104Pa, or at least 1x105Pa during separation of the ions. This avoids the need for a vacuum system and has been found to be acceptable for the separation of large mass ions, such as ionised capsid particles. Accordingly, the apparatus may be configured to maintain the gas in the housing substantially at atmospheric pressure during separation of the ions (i.e. when the housing is rotated), y substantially at atmospheric pressure it is meant that the gas within the housing is at atmospheric pressure when the housing is not rotating, but that the pressure of the gas may increase due to rotation of the housing. Additionally, or alternatively, the housing may not be evacuated by a vacuum system that sucks gas out of the housing. The apparatus may be configured to rotate the housing around the axis at > 5,000 rpm, > 10,000 rpm, > 15,000 rpm, > 20,000 rpm, > 25,000 rpm, or > 30,000 rpm.

[0078] The housing may have a maximum dimension, e.g. in the radial direction, of < 100 mm, < 90 mm, < 80 mm, < 70 mm, < 60 mm, or < 50 mm.

[0079] The axis of rotation may be substantially vertical. In such embodiments, the ion source may direct ions downwards. For example, the ion source may be an electrospray ion source that generates an electrospray plume along the axis of rotation.

[0080] The apparatus may comprise a liquid or gas chromatography device configured to separate an analytical sample and supply it to the ion source. These ions, or ions derived therefrom, are supplied to the rotating housing described herein for separation and detection. Accordingly, the apparatus may comprise a liquid chromatography device that is arranged to rotate around the rotational axis with the housing such that a centrifugal force is provided within the liquid chromatography device for driving an analytical sample through the liquid chromatography device so that different components in the sample elute from the liquid chromatography device at different times. The ion source may be arranged and configured to ionise the eluting components so as to form ions, and supply these ions, or ions derived therefrom, into said housing to be separated.

[0081] For example, the liquid chromatography device may comprise a chromatography column that is arranged with its longitudinal axis such that the sample entrance to the column is arranged radially inwards of the sample exit from the column. The column may be arranged with its longitudinal axis extending in the radial direction.

[0082] BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:

[0084] Figs. 1A-1C show different views of an ion separator according to an embodiment of the present invention;

[0085] Fig. 2A shows a simulation of the gas velocity distribution in the ion separator housing when it is rotating, and Fig. 2B shows a simulation of the gas pressure distribution in the ion separator housing when it is rotating;

[0086] Fig. 3 shows plots of the tangential velocity of the gas in the rotating separator housing as a function of height inside the housing for different radial positions within the housing;

[0087] Fig. 4 shows the inverse of the slip factor plotted over a range of Knudsen numbers in order to highlight the relative influence of viscous and molecular flow effects on a selection of relevant ionised particles;

[0088] Figs. 5A and 5B show the diffusion limited arrival time distributions calculated for full and empty capsid particles of Adenovirus and Adeno-Associated virus, respectively;

[0089] Fig. 6 shows simulations of the detection times of various ionised particles when separated according to an embodiment of the present invention; and Fig. 7 shows another embodiment of the present invention for filtering the ions that reach the detector(s).

[0090] DETAILED DESCRIPTION

[0091] Embodiments of the present invention relate to a gas-phase centrifugal ion separator. The ion separator is used to separate ions using a centrifugal force on the ions. The separator may be used to separate capsids, such as viral capsids. As is well known, capsids are used to encapsulate DNA cargos, e.g. for delivering vaccines and for gene therapy applications. Capsid particles of the same type will have substantially the same diameter and surface properties, but will have different masses if they encapsulate different DNA cargos. It is desired to separate capsid particles of the same type that have different masses, e.g. to determine the proportion of the capsids that encapsulate a full or partially full DNA cargo, or which encapsulate no DNA cargo.

[0092] Figs. 1A-1C show different views of an ion separator according to an embodiment of the present invention. Fig. 1 A shows a perspective view that illustrates only half of the ion separator housing, Fig. 1 B shows a plan view of the ion separator, and Fig. 1C shows a cross-sectional view though the ion separator.

[0093] The ion separator comprises a toroidal housing 2 that contains a gas. The housing surrounds a central axis 4 and is rotatable around that axis. As shown in Fig. 1 C, an ion source 6 is provided for introducing ions into one or more ion entrance openings 8 in a wall of the housing 2. The one or more ion entrance openings 8 are illustrated as being in the radially inner wall 10 of the housing, facing the axis of rotation 4, although they may alternatively be located at other positions on the housing walls.

[0094] The housing 2 may be rotationally symmetric around the rotation axis 4. The housing preferably has a substantially rectangular cross-section in the plane that is defined by the rotation axis 4 and an axis that extends radially from the rotation axis. Preferably, the rectangular cross-section is elongated in the radial direction away from the axis. The housing therefore has a radially inner wall 10 and a radially outer wall 12 that are arranged concentrically around the rotation axis 4 such that they are parallel to each other and spaced apart in the radial direction. The housing also has two side walls 14,16 that extend in the radial direction between the radially inner and outer walls so that the four walls form an enclosed chamber. However, it is contemplated that the chamber may, less preferably, be formed from walls such that it has a different cross-sectional shape, such as an oval or circular cross-section for example.

[0095] The housing 2 may be a substantially closed housing such that the gas in the housing is substantially confined. For example, the housing may be sealed, except that it may have one or more ion entrance openings 8 that are arranged such that ions from the ion source 6 may enter into the chamber enclosed by the housing 2. For instance, the housing may be sealed other than having one or more ion entrance openings 8 on its radially inner side that faces the axis of rotation. As shown in the depicted embodiment, the housing may have a slit 8 that extends through its radially inner wall 10 so as to allow ions to pass through the wall and into the chamber in the housing 2. The housing may have a single slit 8 that extends, in the circumferential direction of the housing (i.e. in the circumferential direction around the axis of rotation 4), either partway along or fully around the radially inner wall 10. For instance, the slit may extend around the entire circumference of the inner wall 10 so as to divide the inner wall into two separate sections that are separated by the slit 8. Alternatively, multiple spaced apart slits may be provided that each extend around a portion of the circumference of the inner wall 10.

[0096] Referring to Fig. 1 C, the ion source 6 is arranged such that ions 3 are provided to the one or more ion entrance openings 8. The ion source may provide ions to, or generate ions in, the region that is surrounded by the housing 2. For example, the ion source may direct ions along the axis of rotation 4 into the region that is surrounded by the housing. The axis of rotation may be substantially vertical and the ion source may direct ions downwards. The ion source may be an electrospray ion source, e.g. that generates an electrospray plume along the axis of rotation, although it is contemplated that the ion source may be a different type of ion source.

[0097] Each of the one or more ion entrance openings 8 may be configured as an ion gate that is controlled to selectively allow ions to enter the housing when the ion gate is open and to prevent ions from entering the housing when the ion gate is closed. As such, the ion gate is controlled such that ions enter the housing as packets of ions.

[0098] The ion separator also comprises one or more ion detector 18 for detecting ions separated by the separator. The one or more ion detector may be arranged inside the chamber on the radially outer wall 12 so as to detect ions 3. The ion detector may have an ion detecting surface that extends, in the circumferential direction, along substantially the entire length of the radially outer wall 12. Alternatively, the ion detecting surface may extend, in the circumferential direction, along only a portion of the length of the radially outer wall. Multiple such ion detecting surfaces may be provided on the radially outer wall that are spaced apart in the circumferential direction. The one or more ion detector may be a charge sensitive detector, such as a Faraday plate, for detecting ions separated by the separator.

[0099] During operation, the housing 2 is rotated around the axis of rotation 4, which causes the gas inside the housing to move such that it also rotates around the axis of rotation. After a period of time, the angular velocity of the gas around the axis of rotation will be similar to that of the rotating housing that encloses it. Ions 3 from the ion source 6 are directed through the ion entrance opening(s) 8 in the housing and pass into the inside of the housing, and into the rotating volume of gas. The ions therefore acquire an angular velocity corresponding to that of the rotating gas and so move around the rotation axis 4 whilst also experiencing a centrifugal force in the radially outward direction. Accordingly, after the ions 3 enter the housing 2 they spiral outwards towards the radially outer wall 12 of the housing, at which point they strike the ion detector 18. Exemplary spiral ion trajectories 20,21 for different ions are shown in Fig. 1 B.

[0100] It will be appreciated that the centrifugal force that any given ion experiences in the radially outward direction is dependent on its mass, whereas the drag force in the opposite direction that the ion will experience is dependent on its size (or more generally, on its mobility through the gas). When the ion separator is used to separate ionised capsid particles that are of the same type but have different DNA cargos, the drag force will be substantially the same for all of these capsid particles since they have substantially the same diameter and surface properties, whereas different centrifugal forces will be exerted on capsid particles having different DNA cargos since they have different masses. Accordingly, full, partially full and empty capsid particles of the same type will travel radially outward within the housing at different rates and hence will become separated from each other and arrive at the ion detector(s) 18 at different times. Fig. 1B shows an example of the spiral ion trajectory for a full capsid particle 20 and an example of the spiral ion trajectory of an empty capsid particle 21 , when the two particles have entered the ion entrance opening 8 at the same time and location. It can be seen that the full capsid particle strikes the ion detector on the radially outer wall before the empty capsid particle does. More generally, ions of different mass-to-diameter ratio (or mass-to-mobility ratio) will have different flight times from the ion entrance opening(s) to the ion detector(s) on the radially outer side of the separator chamber. For example, for ions having the same diameter / mobility, the heavier that the ion is, the shorter the time it will take to travel from the ion entrance opening(s) to the ion detector(s) on the radially outer side of the separator chamber.

[0101] The ion separator may determine the mass to diameter ratio (or mass to mobility ratio) of any given ion based on the duration of time between it entering the ion separator and the time that it is detected by the detector. For example, in embodiments that have an ion gate at the ion entrance opening, the ion separator may determine the mass to diameter ratio (or mass to mobility ratio) of an ion based on the duration of time between the ion gate being opened and the time of detection of the ion at the ion detector. For the case of capsid analysis, the diameter (or mobility) of the capsids may be known since the capsids have been selected to carry the DNA cargo. This may be used, along with the mass to diameter ratio (or mass to mobility ratio) that has been determined, to calculate the mass of each detected capsid particle ion, including the mass of the DNA cargo it is carrying. The mass of the capsid shell itself may also be known, and this may be subtracted in order to determine the mass of the DNA cargo carried by the detected capsid particle.

[0102] Fig. 2A shows the results of a simulation for simulating the velocity of the gas inside the rotating housing 2 as a function of position in the radial direction from the axis of rotation 4 to the radially outer wall 12 of the separator housing. Fig. 2A illustrates the velocity distribution over the area shown by box 24 in Fig. 1 A, i.e. including only half of the cross-section of the housing 2. The different intensities of shading at different radial positions in Fig. 2A illustrate the different tangential velocities (in m / s) of the gas at those different radial positions. In this simulation the gas was at ambient temperature and pressure, and the separation chamber was rotated at 32,000 rpm. The radially inner wall 10 of the separator chamber was located at 35 mm from the rotational axis 4, the radially outer wall 12 of the separator chamber was located at 55 mm from the rotational axis 4, and the height of the separator chamber (in the direction orthogonal to the radial direction) was 6 mm. It can be seen from Fig. 2A that the velocity of the gas inside the housing is slightly lower close to the radially inner wall 10 and slightly higher close to the radially outer wall 12, relative to the radially central region of the housing 2. The gas in the region between the radially inner wall 10 of the housing and the axis of rotation 4 has a lower velocity than the lowest gas velocity in the housing 2.

[0103] Fig. 2B shows the results of a simulation for simulating the gas pressure of the gas in the rotating housing 2 as a function of position in the radial direction from the axis of rotation 4 to the radially outer wall 12 of the separator housing. The parameters for the simulation were the same as those described in relation to Fig. 2A. The different intensities of shading at different radial positions in Fig. 2B illustrate the different air pressures, in Pa, of the gas at those different radial positions. It can be seen that the gas pressure increases as a function of radial position within the rotating housing 2.

[0104] Distortions in the gas flow within the separator chamber may be undesirable as they may affect the separation of the ions within the chamber. Gas flow distortions due to the edge flow effects near the walls of the separation chamber were simulated, and are discussed in relation to Fig. 3.

[0105] Fig. 3 shows four plots of the tangential velocity of the gas in the separator chamber (in m / s) as a function of height inside the chamber (in metres) for four different respective radial positions within the separator chamber. The uppermost plot represents the gas velocity at a radial position of 54 mm from the rotational axis 4, i.e. just inside of the radially outer wall 12. The second plot down represents the gas velocity at a radial position of 45 mm from the rotational axis, i.e. half way between the radially inner wall 10 and radially outer wall 12. The third plot down to intercept the velocity axis (y-axis) represents the gas velocity at a radial position of 35.4 mm from the rotational axis 4, i.e. just inside the housing proximate the radially inner wall 10. The fourth plot down to intercept the velocity axis (y- axis) represents the gas velocity at a radial position of 34.95 mm from the rotational axis 4, i.e. at the radially inner wall 10. It is apparent from Fig. 3 that there are some gas flow distortions very close to the walls of the separator chamber, and also due to the opening 8 in the radially inner wall 10 as shown by the velocity profiles at 34.95 mm and 35.4 mm. However, the gas flow distortion due to the entrance opening was found to occur primarily over the first ~5% of the distance from the radially inner wall 10 to the radially outer wall 12. Also, approximately 90% of the volume of the separator chamber was found to be free from such edge flow distortions.

[0106] As described above, when the ions are injected into the rotating gas the ions will attain the angular velocity of gas and will experience a centrifugal force Fc:

[0107] Fc=ma)2r where m is the mass of the ion, co is the angular velocity of the ion, and r is the radial position of the ion. The motion of an ion in the radially outward direction will be opposed by a drag force FD that is caused by the gas molecules in the separator chamber. Depending on the ratio of the mean free path of an ion within the gas to the size of the ion (i.e. the Knudsen Number, Kn), this drag force may be dominated by either viscous flow around the ion (i.e. by Stokes law) or by molecular collisions (i.e. by Mason-Schamp law). Millikan observed deviations from Stokes law and provided an empirical “slip” correction factor that allows one to estimate drag force in a transition flow regime [R. A. Millikan; Phys. Rev. 22, 1 , 1923], The drag force in a transition flow regime may therefore be estimated as:

[0108] FD= 3n7 Dv('\+(2A / D)(A+Be~cD''2'))~' where q is the viscosity of the gas in the separator chamber, D is the diameter of the ion, v is the velocity of the ion through the gas, A is the mean free path of the ion through the gas, and A, B and c are dimensionless parameters.

[0109] The term on the left is due to Stokes law, whereas the term on the right corresponds to a “slip factor” that is defined using the mean free path of gas A and the dimensionless parameters A, B and c.

[0110] Fig. 4 shows the inverse of the slip factor plotted over a range of Knudsen numbers in order to highlight the relative influence of viscous and molecular flow effects on a selection of relevant ionised particles. Particle 30 is an Adeno Associated Virus having a diameter of 24 nm, particle 32 is an Adeno Virus having a diameter of 80 nm, particle 34 is a Lentivirus having a diameter of 100 nm, and particle 36 is a liposome having a diameter of 200 nm. The graph depicts data for the separation of particles in air that is at ambient pressure and temperature. Locations in the graph that are progressively further to the left indicate a progressively stronger viscous flow regime, whereas locations in the graph that are progressively further to the right indicate a progressively stronger molecular flow regime.

[0111] At steady state, the centrifugal force Fc balances the drag force FD. Therefore, the above equations for Fc and FD can be used to determine the velocity of an ion through the gas in the radial direction (at any given radial position) as:

[0112] An effect analogous to slip is anticipated to influence the diffusional spread of particles such as ions. The diffusion coefficient for viscous flow (Diffuse) and the diffusion coefficient for molecular flow (Diffmoi) may be described using Stokes-Einstein and Einstein- Smoluchowski equations, respectively: where KB is the Boltzmann constant, T is temperature, K is ion mobility in the gas, q is the charge of the ion, P is gas pressure, and M is the mass of gas.

[0113] These equations for the diffusion coefficients can be used to estimate the separation timescales as well as diffusion-limited peak widths for the relevant capsid systems, as shown in Figs. 5A-5B.

[0114] Fig. 5A shows the diffusion limited arrival time distributions calculated for full cargo and empty capsid particles of Adenovirus. Both the full and empty particles had a diameter of 80 nm, whereas the full particle had a mass of 160 MDa and the empty particle had a mass of 125 MDa. The peaks shown by solid lines correspond to the arrival time distributions calculated based on the molecular diffusion coefficient and the peaks shown by dashed lines correspond to the arrival time distributions calculated based on the viscous diffusion coefficient. Fig. 5B shows the diffusion limited arrival time distributions calculated for full cargo and empty capsid particles of Adeno-Associated virus. Both the full and empty particles had a diameter 23.5 nm, whereas the full particle had a mass of 5.0 MDa and the empty particle had a mass of 3.8 MDa. The peaks shown by solid lines correspond to the arrival time distributions calculated based on the molecular diffusion coefficient and the peaks shown by dashed lines correspond to the arrival time distributions calculated based on the viscous diffusion coefficient. The peaks modelled do not account for the pressure gradient and flow distortions in the separator chamber that have been described above.

[0115] Fig. 6 shows SIMION ion trajectory simulations of the detection times of various ionised particles when separated according to an embodiment of the present invention. The model was based on an ion separator as described above, except that the inner radial wall 10 was at a radius of 40 mm from the axis of rotation 4, the radially outer wall 12 (i.e. detector 18) was located at a radius of 65 mm, and the gas velocity distribution was considered to be constant. The simulation was based on molecular flow assumptions and assumed a constant pressure of 1.013x105Pa. Particles having a diameter of 23.5 nm and masses of 3.5 MDa, 4 MDa, 4.5 MDa, 5 MDa, and 5.5 MDa were modelled. These masses were selected as being illustrative of empty, partially filled and full AAV species. Fig. 6 shows the intensity of the ions detected as a function of time and it can be seen that the different mass ions are detected in intensity peaks that occur at different times, and which are resolved from each other in time. Fig. 6 illustrates that the techniques disclosed herein are able to separate capsid particles that differ in mass by relatively small amounts. Larger vector / capsid particles could be separated more easily using the techniques described herein, i.e. by even larger amounts (e.g. Adenovirus, Lentivirus and Liposome particles).

[0116] Fig. 7 shows an embodiment wherein the radially inner wall 10 of the separator housing 2 only has a single ion entrance opening 8 that extends over only a relatively small portion of the circumference around the radially inner wall. The separation chamber also has one or more discrete ion detector 18a, 18b provided on the radially inner side of the radially outer wall 12. In the same manner as described above, ions enter the separator housing 2 through the entrance opening 8 and are caused to spiral radially outwards towards the radially outer wall 12. Ions having different mass to diameter ratios (or mass to mobility ratios) will have different travel times to the radially outer wall 12. As the separator housing is rotating about the rotational axis 4, ions having different mass to diameter ratios (or mass to mobility ratios) will generally reach the radially outer wall 12 at different positions along that wall in the circumferential direction. The operational parameters of the ion separator may be selected such that ions having a selected mass to diameter ratio (or mass to mobility ratio) strike the detector 18a or 18b and ions having other mass to diameter ratios (or other mass to mobility ratios) do not strike the detector. For example, any one or more of the rotational speed of the separator housing, the pressure of the gas in the housing, and the type of gas in the housing, may be selected such that only ions having a selected mass to diameter ratio, or range of mass to diameter ratios, strike the detector 18a and ions having other mass to diameter ratios do not strike the detector 18a. As such, the ion separator device may be used to detect whether or not ions having a pre-selected mass to diameter ratio (or pre-selected mass to mobility ratio) are present in the ions 3 being supplied to the ion separator. In other words, if ions are detected by the detector 18a then the ion separator device automatically determines that ions having the pre-selected mass to diameter ratio (or pre-selected mass to mobility ratio) are present, whereas if ions are not detected by the detector 18a then the ion separator device automatically determines that ions having the pre-selected mass to diameter ratio (or pre-selected mass to mobility ratio) are not present.

[0117] As mentioned above, and shown in Fig. 7, multiple separate ion detectors 18a, 18b may be arranged circumferentially around the radially outer wall. The operational parameters of the ion separator may be selected such that ions having a first pre-selected mass to diameter ratio (or mass to mobility ratio) strike a first of the detectors 18a and ions having a second, different pre-selected mass to diameter ratio (or mass to mobility ratio) strike a second different one of the detectors 18b. For example, as shown in Fig. 7 ions having a first pre-selected mass to diameter ratio have an ion trajectory as shown by the dashed spiral line and strike a first of the detectors 18a, whereas ions having a second, different pre-selected mass to diameter ratio have an ion trajectory as shown by the solid spiral line and strike a second of the detectors 18b. It will be appreciated that third or further ion detectors may be provided for detecting third or further pre-selected mass to diameter ratio (or mass to mobility ratio) ions respectively. It is also contemplated that a continuous positionally sensitive ion detecting surface may extend circumferentially around the radially outer wall and that the detector may be calibrated such that the mass to diameter ratio (or mass to mobility ratio) of any given ion that is detected may be determined according to the position on the detecting surface that it was detected.

[0118] These embodiments enable ions to be supplied substantially continually into the ion entrance opening 8, whilst still determining the mass to diameter ratios (or mass to mobility ratios) of the ions that are present. In other words, it is not required to gate ions into the separator housing 2 in order to determine their mass to diameter ratios (or mass to mobility ratios) from their transit times to the detector(s).

[0119] The separator housing 2 described herein is preferably not evacuated by a vacuum system that sucks gas out of the housing. Rather, the chamber is the housing is substantially at atmospheric pressure, and is only raised above atmospheric pressure due to the rotation of the housing. As there are no vacuum requirements, these embodiments provide a low cost, ultra-compact mass analyser. For example, the overall form factor of the ion separator housing may be similar to a common PC hard disk drive (e.g. having a dimension of 3.5 inch). Thus, it is particularly suitable for integration into, for example, a liquid chromatography system or as a stand-alone device used in point-of-need applications and process monitoring. Also, the ion separator has a relatively low power consumption and so may be operated using a battery and may be portable, such as being a hand-held portable device.

[0120] Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.

[0121] For example, although a single ion entrance opening 8 has been described in various embodiments, it is contemplated that multiple discrete ion entrance openings may be provided around the radially inner wall 10 in the embodiments described herein. Additionally, or alternatively, although the ion entrance opening(s) have been described as being located in the radially inner wall 10, they may alternatively be located in the side walls 14,16, e.g. proximate to the radially inner wall 10.

[0122] Each ion entrance opening 8 may comprise an ion gate having at least one electrode and voltage supply that is controlled so as to switch between blocking ions from entering the opening and allowing ions to pass into the entrance opening. The ion gate may comprise spaced apart electrodes that the ions pass between. When the ion gate is closed a voltage may be applied to at least one of these electrodes such that the ions are directed onto one of the electrodes. When the ion gate is open a different voltage, or ground, may be applied to at least one of these electrodes such that the ions are able to pass between the electrodes and into the separator housing.

[0123] It is contemplated that one or more operational parameter of the ion separators described herein may be varied during the separation of ions. The ion separator may be configured determine the mass to diameter ratio (or mass to mobility ratio) of any given ion that strikes the detector 18 based on its time of detection and the value of the operational parameter(s) at the time of detection. For example, any one or more of the rotational speed of the separator housing 2, the pressure of the gas in the housing, and the type of gas in the housing, may be varied with time and the ion separator may be configured determine the mass to diameter ratio (or mass to mobility ratio) of any given ion that strikes the detector 18 based on its time of detection and the value of these operational parameters at the time of detection.

[0124] The relatively fast separation timescale of the separator device enables it to be coupled with slower upstream chromatography techniques. For example, a liquid or gas chromatography device may separate an analytical sample. The separated sample may be ionized by an ion source 6 and the resulting ions, or ions derived therefrom, may be supplied to the ion separator device described herein for separation and detection. Where a liquid chromatography device is provided, it may be arranged to extend in the radial direction and to rotate around the rotational axis 4 with the separator housing 2 such that a centrifugal force is provided within the liquid chromatography device that drives an analytical sample through the liquid chromatography device so that different components in the sample elute from the liquid chromatography device at different times. For example, the liquid chromatography device may comprise a chromatography column that is arranged with its longitudinal axis such that the sample entrance to the column is arranged radially inwards of the sample exit from the column. The column may be arranged with its longitudinal axis extending in the radial direction.

[0125] A mobility separating device (other than the separator that uses the rotating gas described above) may be provided upstream of the detector 18 so as to separate ions primarily according to their mobility (e.g. according to their diameters), e.g. and substantially not according to their mass. Ions having different mobilities elute from the mobility separating device at different times and then pass to the detector 18. As such, the mobility / diameter of any given ion that is detected is determined from its time of detection at the detector. The mass to diameter ratio, or mass to mobility ratio, is also determined, as has been described above. The instrument is therefore able to calculate the mass of the detected ion based on the determined mass to diameter ratio, or mass to mobility ratio, and the determined diameter or mobility. The mobility separating device may be provided between the ion source 6 and the rotating ion separator housing 2. Alternatively, the mobility separating device may be incorporated into the rotating housing 2. For example, a mobility separator device may be provided in a radially inner portion of the housing such that when ions enter the housing they enter the mobility separating device.

[0126] An ion separation or filter device that separates or filters the ions temporally or spatially may be provided upstream of the rotating separator housing 2, such as an ion mass separator or filter, or a mass-to-charge ratio separator or filter. This may be used to reduce contamination passing into the rotating separator housing 2.

[0127] In order to improve the duty cycle of the analysis by the rotating ion separator described herein, an ion accumulation region such as an ion trap may be provided. The ion accumulation region may be provided upstream of the ion entrance opening(s) 8, or may be provided inside the rotating separator housing 2 adjacent to the ion entrance opening(s) 8. In use, ions are transmitted into the ion accumulation region and may intermittently be released such that they are subsequently separated in the rotating separator housing.

[0128] A single ion source 6 may be provided so as to introduce ions into the opening(s) 8 in the separator housing 2. For example, the ion source may be an electrospray ion source or another type of ion source. Alternatively, a plurality of ion sources may be arranged so as to introduce ions into the ion entrance opening(s) in the rotating separator housing. Different ones of the ion sources may be arranged at different positions, in the circumferential direction, along the radially inner wall 10 of the separator housing and / or at different radial positions from the rotational axis 4. The plurality of ion sources may all be the same type of ion source, such as an electrospray ion source or another type of ions source, or they may be different types of ions sources. Alternatively, or additionally, the plurality of ion sources may ionise analyte from the same sample supply or from different sample supplies.

[0129] An electric field may be provided in the separator housings 2 described herein in order to urge the ions in the radially inward or outward direction. This may be used to control the separation timescale over which the ions are separated or to control the resolution of the separation. For example, the magnitude or direction of the electric field may be varied in order to vary the separation timescale or separation resolution.

[0130] AC and / or DC voltage supplies may be provided that supply AC and / or DC voltages to electrodes that arranged within the separator housing 2 disclosed herein. These voltages and electrodes may be arranged and configured so as to confine the ions within the separator housing. For example, the electrodes may be provided on the side walls 14,16 of the separator housing and the voltages may be applied to the electrodes so as to repel the ions away from the side walls.

[0131] Alternatively, the electrodes may be provided on the side walls 14,16 of the separator housing and the voltages may be applied to the electrodes so that the separator housing acts as a field asymmetric ion mobility spectrometer (FAIMS). In other words, an oscillating voltage may be applied which causes the ions to oscillate between the two side walls but to have an overall motion between the side walls that is towards a first of the side walls, whereas a DC voltage difference is applied between the side walls so as to urge the ions in the opposite direction towards the second of the side walls. These voltages may be controlled such that only ions having a particular (FAIMS) ion mobility range reach the ion detector 18. The voltages may be varied with time such that ions having different (FAIMS) ion mobilities reach the ion detector at different respective times. The (FAIMS) ion mobility of a given ion that is detected may be determined based on the time of its detection and the voltages that were applied to the side walls at that time.

[0132] The ion detectors 18 described herein may be electrical ion detectors that detect the impact of an ion thereon due to the electrical signal that is generated by the ion hitting the detector surface. Alternatively, the ion detector may be an optical ion detector that optically detects when an ion has reached the radially outer wall 12 of the separator housing 2. Multiple detector types may be used in tandem.

[0133] The rotating separator housing 2 has been described as containing one or more ion detector 18 and / or various other electrical components such as electrodes. In order to provide electrical communication to and / or from these components, an electrically conductive brush, or another type of electrical contact, may be arranged so as to extend between the housing and a non-rotating electrical component outside of the housing when the housing is rotating. Electrical signals can therefore be transmitted between the housing and the non-rotating electrical component via the conductive brush or other type of electrical contact. Alternatively, or additionally, the electrical signals may be transmitted to and / or from the housing wirelessly, such as by inductive and / or capacitive coupling. Battery driven circuitry may be located on the rotating separator housing so as to transmit electrical signals wirelessly from the electrical housing. A magnet may be rotated relative to the separator housing so as to transfer electrical signals to an inductor on the separator housing and / or a magnet on the separator housing may rotate relative to an inductor that is not on the housing so as to transfer electrical signals from the housing.

[0134] The separator housing 2 may be configured such that ions strike the detector(s) 18 inside the housing and do not exit the housing. The separator housing may therefore have no exit openings for the ions to exit whilst the separator housing is rotating. For example, the separator housing may have no openings in the radially outer wall 12.

[0135] Alternatively, the separator housing 2 may have one or more opening in a radially outer portion of the separator housing, such as through the radially outer wall 12. The opening(s) may be arranged such that gas flows out of the separator housing through the opening(s) when the housing is rotating. For example, an array or ion entrance openings may be provided towards or on the radially inner portion or wall of the housing and an array or exit openings may be provided towards or on the radially outer portion or wall of the housing. Such a gas flow may be used to spatially focus the ions. Alternatively, one or more ion exit apertures may be arranged through the radially outer wall 12 of the housing such that, when the housing is rotating, ions are separated by mass to diameter ratio (or mass to mobility ratio) in the housing and then the separated ions pass out of the housing through the ion exit opening(s) at different times. One or more ion detector 18 may be arranged outside of the rotating housing for detecting the ions that pass through the ion exit opening(s). Accordingly, the ion detector(s) need not rotate with, or be arranged within, the housing 2. The ion detector(s) may have any of the features that have been described above in relation to the embodiments that have the ion detectors in the housing. For example, an ion detecting surface may extend substantially continuously around the entire perimeter of the separator housing 2. The detecting surface may or may not be a positional detecting surface. Alternatively, multiple different ion detectors may be provided spaced circumferentially around the perimeter of the separator housing.

[0136] Different gas conditions may be provided in the separator housing 2 at different times, so as to perform different analyses on the ions. For example, gases having different viscosity coefficients and masses may be used to probe surface and porosity properties of the analyte ions. The use of heavy gases may be used to reduce the diffusion coefficient in the molecular flow regime.

[0137] Alternatively, the gas provided into the separator housing 2 may include a gas, and / or reagent ions, that reacts with the analyte ions so as to fragment the analyte ions, cause a conformational change in the analyte ions, or produce adduct ions from the analyte ions. For example, a charge transfer reagent may be introduced into the housing to react with the analyte ions. The resulting ions are then detected by the detector(s).

[0138] Other techniques may be used to fragment the ions within the separator housing, e.g. in order to perform MS / MS experiments. For example, the ions within the rotating housing may be fragmented by photo-activation, such as by providing a laser within the rotating housing or by directing a laser beam into the housing. One or more laser entrance aperture or window may be provided in the housing such that a laser beam can be directed through the aperture(s) orwindow(s) and into the housing. The aperture or window may be arranged in one of the side walls of the housing and may be annular, with an axis of symmetry around the axis of rotation, such that the laser can pass into the housing whilst the housing rotates and without the laser having to move. Alternatively, one or more arcshaped windows or apertures may be provided for the same purpose.

[0139] It is contemplated that the laser may be activated so as to only fragment ions having a pre-selected mass to diameter ratio (or mass to mobility ratio). For example, in embodiments in which the ion entrance opening 8 is an ion gate, the laser may be pulsed at a time that is synchronised with the opening of the ion gate, and delayed relative thereto, such that the ions having the pre-selected ratio are passing through the region where the laser is incident and are fragmented. Alternatively, or additionally, an array of laser entrance apertures or windows may be provided at different radial positions around the housing such that ions having different pre-selected mass to diameter ratios (or mass to mobility ratios) are fragmented by the laser.

[0140] Alternative techniques of fragmenting the ions are contemplated, such as collision induced dissociation (e.g. surface induced dissociation) or electron-based dissociation such as electron impact dissociation, electron capture dissociation or electron transfer dissociation.

[0141] In the embodiments in which the ion detector(s) is arranged outside of the separator housing, the ions may be fragmented or activated after leaving the separator housing and prior to reaching the detector(s).

[0142] In the embodiments described above, the separator housing encloses a toroidal chamber that extends continuously around entire circumference of housing 2. In other words, the gas and ions are free to move around the entire circumference of the toroidal region inside the separator housing. However, it is also contemplated that one or more wall may be arranged inside the separator housing such that the interior space is divided into multiple compartments. For example, one or more wall may be arranged so as to divide the interior of the housing circumferentially into multiple different compartments, e.g. into two or more sectors. The one or more wall may extend substantially in the radial direction from the rotational axis 4 and may be substantial straight or curved in the plane orthogonal to the rotational axis.

[0143] The wall(s) may be arranged inside the housing such that gas and ions inside any given compartment may be substantially unable to exit that compartment when the housing is rotating. As such, each compartment may include an ion detector as described above.

[0144] Alternatively, one or more wall may be arranged inside the separator housing so as to divide the interior of the housing radially into multiple different compartments, e.g. a radially inner compartment and a radially outer compartment. Ions may travel between these compartments by passing through an aperture in the wall between them.

[0145] Different types of gas, a mixture of gases having different gas compositions, or different gas pressures may be provided in the different respective compartments described herein.

[0146] Multiple separator housings of the types described herein may be stacked one on top of the other in a direction along the rotational axis 4, such that the multiple separator housings are rotated together about the rotational axis. The different separator housings may have the same configuration or they may have different configurations, such as by having different numbers or layouts of compartments therein. Ions from the same ion source 6 may be supplied to the entrance opening(s) 8 in each of the separator housings 2, or ions from different ions sources may be supplied to different, respective, separator housings.

[0147] Different types of gas, a mixture of gases having different gas compositions, or different gas pressures may be provided in the different separator housings.

Claims

171343-02v1Claims:

1. A method of separating ions comprising: rotating a housing about an axis such that gas therein is rotated around the axis; and then transmitting ions into the gas such that the ions are driven around the axis by the gas and thus experience a centrifugal force that separates the ions.

2. The method of claim 1, wherein the centrifugal force separates the ions in a radial direction that is orthogonal to the axis of rotation, and causes the ions to travel in the radial direction until they pass out of the housing and / or strike an ion detector.

3. The method of claim 1 or 2, wherein the housing is toroidal and / or a gas chamber inside the housing that contains the rotating gas is toroidal.

4. The method of claim 3, wherein the housing and / or gas chamber has a substantially rectangular cross-section in the plane that is defined by the axis of rotation and an axis orthogonal to the axis of rotation.

5. The method of any preceding claim, wherein the ions are transmitted into the housing and the gas through one or more ion entrance opening located on a radially inner side of the housing that faces the axis of rotation.

6. The method of claim 5, wherein the one or more ion entrance opening comprises one or more elongated slit that extends part way along a radially inner wall of the housing, in a direction orthogonal to the axis of rotation; or wherein the one or more ion entrance opening is an elongated slit that extends fully along a radially inner wall of the housing, in a direction orthogonal to the axis of rotation.

7. The method of any preceding claim, comprising: transmitting the ions into the housing and the gas through one or more ion entrance opening in a wall of the housing; providing one or more ion detector radially outwards of the one or more ion entrance opening; and detecting ions that have been separated in the housing, or ions derived therefrom, with the one or more ion detector.

8. The method of claim 7, comprising determining the mass of any given ion that is detected by the ion detector from the duration of time between that ion entering the rotating gas and that ion, or an ion derived therefrom, being detected by the ion detector.

9. The method of claim 8, wherein an ion gate is provided in, or upstream of, the one or more ion entrance opening, and the time that the ion enters the rotating gas is determined from the time that the ion gate is opened.

10. The method of claim 8 or 9, wherein the ions are ionised capsid particles, wherein at least some of the capsid particles encapsulate a DNA cargo, and wherein the method comprises determining whether capsid particles detected at the detector contain no DNA cargo, a full DNA cargo, or a partially full DNA cargo based on the calculated mass for the ion.

11. The method of claim 10, comprising determining the proportion of the detected capsid particles that have no DNA cargo, and / or a full DNA cargo, and / or a partially full DNA cargo.

12. The method of any one of claims 7-11, wherein the one or more ion detector is located inside the housing so as to rotate therewith.

13. The method of any one of claims 7-12, wherein the housing comprises an ion entrance opening and an ion detector that are arranged on the housing such that when the housing is rotated, only ions having a pre-selected ratio of mass to mobility through the gas are able to strike the detector and ions having other ratios of mass to mobility do not strike the detector.

14. The method of claim 13, wherein ions are continuously supplied through the ion entrance opening and the method determines that ions having said pre-selected ratio of mass to mobility are present when ions are detected at the detector and determines that ions having said pre-selected ratio of mass to mobility are not present when ions are not detected at the detector.

15. The method of any one of claims 7-11, wherein ions are separating in the rotating housing and exit the housing through one or more ion exit opening located on a radially outer side of the housing that faces away from the axis of rotation; and wherein the ions then pass to one or more ion detector that is located outside the housing.

16. The method of any preceding claim, wherein the gas within the housing is substantially at atmospheric pressure during separation of the ions; and / or wherein the housing is not evacuated by a vacuum system that sucks gas out of the housing.

17. The method of any preceding claim, comprising providing a liquid chromatography device that is arranged to rotate around the rotational axis with the housing such that a centrifugal force is provided within the liquid chromatography device that drives an analytical sample through the liquid chromatography device so that different components inthe sample elute from the liquid chromatography device at different times, and ionising the eluting components so as to form ions, and supplying these ions, or ions derived therefrom into said housing to be separated.

18. A method of separating ions comprising: rotating a housing about an axis such that gas therein is rotated around the axis; transmitting ionised capsid particles into the gas through one or more ion entrance opening in a wall of the housing such that the ionised capsid particles are driven around the axis by the gas and thus experience a centrifugal force that separates the ionised capsid particles; providing one or more ion detector radially outwards of the one or more ion entrance opening, and detecting ionised capsid particles that have been separated, or ions derived therefrom, with the one or more ion detector; determining the mass of any given ionised capsid particle that is detected by the one or more ion detector from the duration of time between that ionised capsid particle entering the rotating gas and that ionised capsid particle, or an ion derived therefrom, being detected by the one or more ion detector; and determining if the detected ionised capsid particles has no DNA cargo, a full DNA cargo, or a partially full DNA cargo based on the determined mass.

19. The method of claim 18, wherein the gas within the housing is substantially at atmospheric pressure during separation of the ionised capsid particles; and / or wherein the housing is not evacuated by a vacuum system that sucks gas out of the housing.

20. An ion separator apparatus comprising: an ion source; and a housing arranged to receive ions from the ion source, or ions derived therefrom; wherein the housing contains a gas and is rotatable about an axis such that the gas therein is rotated around the axis and ions received in the housing are driven around the axis by the gas and thus experience a centrifugal force that separates the ions.

21. The ion separator apparatus of claim 20, arranged and configured to perform the method of any one of claims 1-19.

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