Method and apparatus for analysis of therapeutic payloads carried by nanoparticle delivery vehicles

Abstract

There is described a method of determining at least one property of a therapeutic payload and / or nanoparticle delivery vehicles, the therapeutic payload and nanoparticle delivery vehicles forming a diffusely scattering liquid suspension sample, the method comprising: locating the sample within an integrating cavity; directing probe light to a first port of the integrating cavity; receiving a portion of the probe light from a second port of the integrating cavity following scattering from the sample; measuring one or more spectral features of the probe light received at the second port; and determining the at least one quantitative property of the therapeutic payload and / or nanoparticle delivery vehicles in the liquid suspension from the measured one or more spectral features of the probe light received at the second port. Apparatus is also described.

Description

[0001] Method and Apparatus for Analysis of Therapeutic Payloads Carried by Nanoparticle Delivery Vehicles

[0002] Technical Field

[0003] The present disclosure relates to methods and apparatus for determining a property, such as the loading, of therapeutic payloads in nanoparticle delivery vehicles. Examples of such nanoparticle delivery vehicles include, but are not limited to, lipid-based delivery vehicles. In particular, the apparatus and methods relate to analysis of diffusely scattering suspensions comprising the nanoparticle delivery vehicles. As well as analysis of the therapeutic payloads, the nanoparticle delivery vehicles may also be analysed.

[0004] Background

[0005] Lipid nanoparticle (LNP) drug products comprise a drug payload that is carried by a nanoparticle delivery vehicle comprised of lipids. The drug is a pharmaceutical drug that may be carried into the body by the LNP. Recent advances in this and similar technologies mean that vaccines, such as the mRNA Covid vaccine, may also be carried into the body in this way. During development and manufacturing it is necessary to quantify the amount of payload present in the LNPs.

[0006] Quantification of RNA, DNA, proteins, etc. is conventionally performed by UV- Visible spectroscopic analysis where the RNA, DNA, protein, etc. is held in a buffer solution. RNA will typically absorb light at UV wavelengths. Such UV-Visible spectroscopic analysis is common but careful dilution is often required such that the sample is provided to the instrument within the dynamic range of the instrument. Thermo Fisher Scientific’s NanoDrop Microvolume Spectrophotometers and C-Tech’s SoloVPE CTech™ SoloVPE® System are developments of such instruments where the standard UV-Visible approach is adapted to avoid needing to dilute the sample to the dynamic range of the instrument.

[0007] Conventional UV-Visible spectroscopy and these adapted instruments cannot be applied to measure RNA, DNA and proteins carried or encapsulated by LNPs because the lipid nanoparticles scatter strongly at UV wavelengths. This is at least partly due to their size which may be around 100nm. Classically scattering varies with wavelength, λ, in proportion to λ-4. Hence, scattering may also dominate over absorption at other wavelengths for other nanoparticles. Figure 1 is a schematic diagram and schematic graph of conventional UV-Visible spectroscopy where UV or visible light is directed at a sample such as a sample in a cuvette. The light that is transmitted through the sample is analysed, an extinction spectrum is calculated, and absorption bands identified. The wavelength and intensity or magnitude of the absorption bands allows the type and quantity of species such as RNA, DNA, proteins etc. to be determined. However, as shown in figure 2 for LNP particles the UV-Visible spectroscopic light is heavily scattered so the spectrum is dominated by the scattering. This results in it not being possible to determine absorption values for the payload of the LNP.

[0008] In more detail, figure 3 shows the spectrum obtained with conventional UV-Visible spectroscopy applied to LNPs mixed with RNA at two different concentrations. RNA would conventionally be quantified based on the absorbance at 260nm (known as A260). The Beer-Lambert law states that absorption is proportional to concentration. In figure 3 the top curve was taken for a sample containing twice the concentration of RNA as the lower curve, but the difference in absorbance is not a factor of 2 because the extinction spectrum is dominated by scattering from the LNPs. Hence, UV-Visible spectroscopy cannot be used for reliable analysis of RNA, DNA and protein payloads carried by LNP nanoparticles.

[0009] Such encapsulated RNA is conventionally quantified using a fluorescence assay, typically RiboGreen, which is time-consuming and prone to systematic and operator errors.

[0010] Accordingly, there is a need for an improved technique for measuring RNA and other payloads carried by LNPs.

[0011] Summary of the Invention

[0012] The present invention provides a method of determining at least one property of a therapeutic payload and / or nanoparticle delivery vehicles, the therapeutic payload and nanoparticle delivery vehicles forming a diffusely scattering liquid suspension sample, the method comprising: locating the sample within an integrating cavity; directing probe light to a first port of the integrating cavity; receiving a portion of the probe light from a second port of the integrating cavity following scattering from the sample; measuring one or more spectral features of the probe light received at the second port; and determining the at least one property of the therapeutic payload and / or nanoparticle delivery vehicles in the liquid suspension from the measured one or more spectral features of the probe light received at the second port. The inventors have realised that the method described herein can be used to measure complex drug or vaccine delivery vehicles when the delivery vehicle is highly scattering. For example, as mentioned above it is common to use standard UV-Visible spectroscopy to analyse and quantify RNA. However, as discussed above the nanoparticle delivery vehicles can be highly scattering and prevent accurate measurement of RNA or other payload. The method described herein corrects for the scattering by using the integrating cavity to allow the effect of scattering to be largely eliminated. Hence, this technique may be known as scatter-corrected absorption (SCA). The present technique is unique in that species that are highly scattering at UV-visible wavelengths may surround or encapsulate the payload that it is desired to measure. Although in other cases the payload may only partly be surrounded by the nanoparticle delivery vehicle, or be in suspension outside of the vehicle. In development stages the payload and the delivery vehicle may be held in the suspension together but without the payload having been loaded into the vehicle. Hence, because the delivery vehicle is highly scattering and may also take up more volume in suspension than the payload it is generally very difficult to make a UV- visible spectroscopy measurement of the payload. The use of the integrating sphere and measurement techniques of the present invention overcome the scattering difficulties and an accurate measurement of the payload becomes possible. The technique also allows measurement of the absorption of the highly scattering delivery vehicle. Hence, the technique may be applied to measure the payload and / or delivery vehicle, and whether or not the payload is loaded into or onto the delivery vehicle, but where the presence of the delivery vehicle and its scattering makes conventional UV-visible spectroscopy inaccurate. The scatter-corrected absorption technique is independent of particle size or distribution and can be used to quantify payload. The method is particularly applicable to particles in the size range 10-150nm and especially 60-150nm. For example, for LNPs which are often in the 60-80nm range, Adeno-associated viruses (AAVs) which may be in the range 20- 25nm or larger, lentiviruses or exosomes, which might be up 150nm, and antibody-drug conjugates (ADCs) which are particularly variable in size. The method is applicable to diffusely scattering or turbid samples and does not require the use of fluorescent dyes.

[0013] By nanoparticle delivery vehicle we mean a nano-sized transport unit that can carry therapeutics which form a payload on or in the delivery vehicle, although the measurement technique also measures the payload when in a suspension with the delivery vehicle but not loaded to the vehicle. In general, encapsulation or loading efficiency of payloads to NPVs is high or the buffer which may contain unloaded payloads is exchanged after manufacture. Hence, the encapsulation or loading efficiency is generally known and the payload inside the NPV can be readily quantified from the measurements of this invention. The delivery vehicle may be lipid based such as a lipid nanoparticle and may carry drugs or vaccines. As we set out herein other types of nanoparticle delivery vehicle and payload are possible.

[0014] The method of the present invention does not rely on knowing particle size, particle size distribution (Pdi) or refractive index of the material, in order to estimate the scattering and / or scattering-corrected-absorption. Furthermore, as mentioned above the method allows for characterising NPVs themselves, e.g., by composition or concentration.

[0015] The method allows the determination of a property of the payload and / or the NPV. The property may be a quantitative property, such as the amount or concentration of payload and / or NPV in the sample, or may be a qualitative property such as the type of payload, such as the type of RNA carried, or the type of NPV.

[0016] The step of determining the at least one property of the therapeutic payload in the liquid suspension may be performed at the apparatus comprising the light source or may be performed at a different location, such as remotely.

[0017] The step of determining the at least one property of the therapeutic payload and / or NPV in the liquid suspension may comprise determining one or more absorbance spectral features of the therapeutic payload and / or NPV from the measured one or more spectral features of the probe light. The one or more absorbance spectral features may be a peak or a trough which is detected in the received probe light and is indicative of absorbance at a particular wavelength or range of wavelengths. The wavelength of the peak or trough may be a characteristic of the therapeutic payload and / or NPV and is therefore indicative of the presence of the payload and / or NPV.

[0018] The step of determining one or more absorbance spectral features of the therapeutic payload and / or NPV may comprise applying a calibration transform to the one or more measured spectral features of the probe light to determine the absorbance spectral features of the therapeutic payload and / or NPV. The calibration transform may transform the measured light or spectrum from a raw measurement that comprises, for example, features of the instrument, to a spectrum or value that is indicative of the presence, or not, or the therapeutic payload.

[0019] The method may comprise performing linear decomposition to determine the absorbance or quantity of LNPs.

[0020] The calibration transform may be based on a calibration of the integrating sphere and / or an absorption coefficient of the therapeutic payload at the wavelength of the probe light. We refer here to absorption coefficient but this is used interchangeably with the term extinction coefficient since we are only concerned with absorption.

[0021] The at least one property of the therapeutic payload and / or NPV may be a measure of, or may be related to, the quantity or concentration of the payload and / or NPV in the sample.

[0022] The at least one property of the therapeutic payload and / or NPV maybe the absorbance spectrum of the payload and / or NPV. The method may further comprise determining from the absorbance spectrum of the payload and / or NPV one or more characteristics relating to the payload and / or NPV and identifying the payload and / or NPV from the characteristics.

[0023] The probe light may enter the integrating cavity through the first port in a first axial direction and may be received at the second port in a second axial direction. The second axial direction may be offset from the first axial direction such that probe light received at the second port is scattered from the integrating cavity and / or sample. By offset we may mean that the probe light cannot be transmitted directly, such as without scattering, from the first port to the second port.

[0024] The method may further comprise directing probe light between third and fourth ports of the integrating cavity. The fourth port may be arranged opposing a third port such that the fourth port receives probe light transmitted, such as transmitted directly without scattering, from the third port, and measuring the transmitted light and determining an extinction value for probe light received at the fourth port. An extinction value may be a measure of the remaining light arriving at the fourth port, after any scattering and absorption.

[0025] The method does not require the sample to be moved between making an absorption measurement and making an extinction measurement.

[0026] The method may further comprise quantifying an amount of scattering of the suspension sample based on the extinction value and determined absorbance spectral features. A measure of scattering may be used to determine the amount of concentration of the scattering nanoparticle delivery vehicles.

[0027] The third port may be the same as the first port such that the fourth port receives probe light transmitted directly, such as without scattering, from the first port.

[0028] The fourth port may be the same as the second port such that the second port receives probe light transmitted directly, such as without scattering, from the third port.

[0029] The probe light may include one or more wavelengths in the UV-visible spectrum, and may extend into the near infrared (NIR), such as including light covering a range 240nm to 750nm, 240 to 650nm, at around 260 nm, one or more discrete wavelengths or lines between 240nm and 320nm, or the probe light may scan a range of wavelengths between 240nm to 750nm, 240nm to 650nm or between 240nm and 320nm, such as by tuning the probe light source output wavelength. For example, RNA tends to absorb around 260nm but some payloads absorb at different wavelengths. The commercial liposome cancer therapy encapsulating doxorubicin (actually as nanocrystals) absorbs in the red such as up to around 600nm, for example. Polymeric nanoparticles absorb at longer wavelengths and metal nanoparticles scatter a lot and absorb in the visible. Gold NPs with a drug bound to the outside results in absorption bands that are otherwise masked by the scatter in the visible. Silver nanoparticles absorb at up to 750nm. Nevertheless, the majority of xNA molecules absorb in the 240 to 320nm range.

[0030] The nanoparticle delivery vehicle carrying a therapeutic payload may comprise one or more of: a lipid-based nanoparticle carrying RNA, antibody-drug conjugates, viral vectors such as lentiviruses and AAVs, liposomes, exosomes, polymeric nanoparticles, liposomebased systems such as lipoplexes, metal nanoparticles, SPIONs, polymeric micelles, dendrimers, nanoemulsions, silica particles and nanogels.

[0031] The nanoparticle delivery vehicles may comprise a lipid-based nanoparticle and the payload may comprise RNA such as mRNA, SiRNA, ssRNA.

[0032] The payload may comprises one or more of: oligonucleotides, DNA, pharmaceuticals, vaccines, oncological treatments and gene editing treatments. The payload may additionally or alternatively comprise small molecule drugs. Small molecule drugs are standard pharma products made by chemistry methods generally. Encapsulating small molecule drugs has numerous potential benefits, especially if combined with the other payloads mentioned. For example, they may extend the release profile or minimise collateral damage of, e.g., highly toxic cancer drugs. An example small molecule drug is doxorubicin.

[0033] The method may comprise analysing the therapeutic payload and the NPV to determine a quantity ratio of the NPV to payload.

[0034] The present invention may provide a nanoparticle vehicle analysis or measurement / characterization method comprising: obtaining / receiving at least one or a plurality of scatter-corrected absorbance measurements / data or scatter-corrected absorbance spectrum measurements / data for a liquid sample or solution containing a plurality of nanoparticle vehicles, the method comprising the method of any preceding claim, and wherein the liquid sample or solution comprises the diffusely scattering liquid suspension sample, and wherein the step of obtaining / receiving at least one or a plurality of scatter-corrected absorbance measurements / data or scatter-corrected absorbance spectrum measurements / data comprises measuring one or more spectral features of the probe light. A property of the therapeutic payload may be a quantitative property such as an absorbance spectrum.

[0035] The step of determining at least one property of the therapeutic payload in the liquid suspension may comprise using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using an extinction coefficient of the payload of the nanoparticle vehicles at an absorption wavelength of interest, a quantity of a loading of the payload of the nanoparticle vehicles of the liquid sample or solution.

[0036] The step of determining at least one property of the therapeutic payload in the liquid suspension may comprise using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using an extinction coefficient of the payload and an extinction coefficient of the carrier component of the nanoparticle vehicles at an absorption wavelength of interest, a quantity ratio of the payload to carrier component / fraction of the nanoparticle vehicles of the liquid sample or solution.

[0037] The step of determining at least one property of the therapeutic payload in the liquid suspension may comprise using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using extinction coefficients of constituent carrier elements of the carrier component / fraction of the nanoparticle vehicle, a quantity ratio of the constituent carrier elements of the carrier component / fraction of the nanoparticle vehicles.

[0038] The step of determining at least one property of the therapeutic payload in the liquid suspension may comprise using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data and a measured or determined nanoparticle vehicle size to determine a nanoparticle vehicle concentration of the nanoparticle vehicles in the liquid sample or solution.

[0039] The step of determining at least one property of the therapeutic payload in the liquid suspension may comprise using at least one scattering spectrum or scattering spectrum data and measured or determined nanoparticle vehicle size to determine a nanoparticle vehicle concentration of the nanoparticle vehicles in the liquid sample or solution.

[0040] The present invention provides an apparatus for determining at least one property of a therapeutic payload and / or nanoparticle delivery vehicles, the therapeutic payload and nanoparticle delivery vehicles forming a diffusely scattering liquid suspension, the apparatus comprising: an integrating cavity configured to receive a sample of the liquid suspension into a sample space; a light source arranged to direct probe light through a first port into the integrating cavity; a detector for measuring probe light received at a second port of the integrating cavity, the second port arranged such that the light received is following scattering from the sample in the sample space. The apparatus may optionally comprise an analyser configured to determine the at least one property of the therapeutic payload and / or nanoparticle delivery vehicles in the liquid suspension from the one or more spectral features of the probe light received at the second port. The therapeutic payload may be carried or encapsulated by the NPV in the liquid suspension.

[0041] The property determined by the analyser may be a quantitative property, such as the amount or concentration of payload and / or nanoparticle delivery vehicles in the sample, or may be a qualitative property such as the type of payload, the type RNA carried, and / or the type of nanoparticle delivery vehicles.

[0042] The analyser may be further configured to determine one or more absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles from the measured one or more spectral features of the probe light, and the at least one property of the therapeutic payload and / or nanoparticle delivery vehicles is determined from the one or more absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles. The one or more absorbance spectral features may be a peak or a trough which is detected in the received probe light and is indicative of absorbance at a particular wavelength or range of wavelengths. The wavelength of the peak or trough may be a characteristic of the therapeutic payload and / or nanoparticle delivery vehicles and is therefore indicative of the presence of the payload and / or nanoparticle delivery vehicles.

[0043] The analyser may be configured to determine the absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles by applying a calibration transform to the one or more measured spectral features of the probe light to determine the absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles.

[0044] The calibration transform may be based on a calibration of the integrating sphere and / or an absorption coefficient of the therapeutic payload and / or nanoparticle delivery vehicles at the wavelength of the probe light.

[0045] The light source may be configured to direct probe light to enter the integrating cavity through the first port in a first axial direction and the second port is arranged to receive probe light scattered from the integrating sphere and / or sample at the second port in a second axial direction, wherein the second axial direction is offset from the first axial direction.

[0046] The integrating sphere may further comprise third and fourth ports. The fourth port may be arranged opposing a third port to receive probe light transmitted directly, such as without scattering, from the third port. The analyser may be further configured to determine an extinction value for probe light received at the fourth port. The analyser may be further configured to quantify an amount of scattering of the sample of suspension based on the extinction value and the determined absorbance spectral features.

[0047] The third port may be the same as the first port such that the fourth port is configured to receive probe light transmitted directly, such as without scattering at least some of the light, from the first port.

[0048] The fourth port may be the same as the second port such that the second port is configured to receive probe light transmitted directly, such as without scattering at least some of the light, from the third port.

[0049] The probe light may include one or more wavelengths in the UV-visible spectrum and may extend into the near infrared (NIR), such as including light covering a range 240nm to 750nm, 240 to 650nm, at around 260 nm, at one or more discrete wavelengths or lines between 240nm and 320nm, or to scan or tune a range across of wavelengths between 240nm to 750nm, 240nm to 650nm or between 240nm and 320nm.

[0050] The analyser may be configured to determine at least one quantitative property of the nanoparticle delivery vehicle carrying a therapeutic payload, and wherein the nanoparticle delivery vehicle carrying a therapeutic payload may comprise one or more of: a lipid-based nanoparticle carrying RNA, antibody-drug conjugates, viral vectors such as lentiviruses and AAVs, liposomes, exosomes, polymeric nanoparticles, liposome-based systems such as lipoplexes, metal nanoparticles, SPIONs, polymeric micelles, dendrimers, nanoemulsions, silica particles and nanogels.

[0051] The analyser may be configured to determine at least one quantitative property of the nanoparticle delivery vehicle carrying a therapeutic payload, and wherein the nanoparticle delivery vehicle may be a lipid-based nanoparticle and the payload comprises RNA such as mRNA, SiRNA, ssRNA.

[0052] The analyser may be configured to determine at least one quantitative property of the nanoparticle delivery vehicle carrying a therapeutic payload, and wherein the payload may comprise one or more of: oligonucleotides, DNA, pharmaceuticals, vaccines, oncological treatments and gene editing treatments.

[0053] The present invention further provides a nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device, such as comprising the apparatus as set out herein, wherein the integrating cavity may comprise a reflective inner wall or walls, the integrating cavity may be configured to receive a cuvette, the cuvette being configured to contain a liquid sample or solution including at least one or a plurality of nanoparticle vehicles, the liquid sample or solution comprising the diffusely scattering liquid sample; wherein the integrating cavity may comprise at least one light inlet port and at least one light outlet port, the or each light inlet port being configured to receive light from the light source and the or each light outlet port being configured to deliver light to a spectrometer, wherein the spectrometer may comprises the detector; wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is optionally a lipid-based drug delivery vehicle analyzer or a lipid-based drug delivery vehicle measurement / characterization device, and is configured to operate in a diffusely reflecting mode in which light from the light source follows a light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the at least one or the plurality of nanoparticle vehicles contained in the liquid sample or solution.

[0054] The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device may be for or may be configured for: determining a payload quantity or payload concentration of the nanoparticle vehicles, and / or determining a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or determining a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or determining a nanoparticle vehicle concentration in a solution or formulation. By nanoparticle vehicle we may mean the nanoparticle delivery vehicle.

[0055] The nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device may be a lipid-based nanoparticle analyzer or a lipid- based nanoparticle measurement / characterization device for or configured for determining a lipid-based nanoparticle concentration in a solution or in a formulation, or for or configured for determining a lipid-based drug delivery vehicle concentration in a solution or in a formulation.

[0056] The lipid-based nanoparticle vehicle may comprise or consist of a (solid) lipid nanoparticle and / or a liposome.

[0057] The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device may further include a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided. The light path adjuster may be configured such that when the light path adjuster is in a first configuration, the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is in a transmission mode in which light from the light source follows a first light path from the or one of the light inlet port(s) to the liquid sample such that the light from the light source irradiates the liquid sample directly before the light transmitted by the sample is transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample. The light path adjuster may be further configured such that when the light path adjuster is in a second configuration, the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is in the diffusely reflecting mode in which light from the light source follows a second light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid or liquid matrix contained in the liquid sample.

[0058] The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device may further comprise a controller, or a controller configured to control the light path adjuster to selectively adjust the path of light through the apparatus.

[0059] The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device may be a (solid) lipid nanoparticle drug delivery vehicle drug load quantity analyzer or (solid) lipid nanoparticle drug delivery vehicle drug load quantity measurement / characterization device; or a (solid) lipid nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio analyzer or (solid) lipid nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio measurement / characterization device; or a (solid) lipid nanoparticle drug delivery vehicle lipidic component ratio analyzer or (solid) lipid nanoparticle drug delivery vehicle lipidic component ratio measurement / characterization device; or a liposome drug delivery vehicle analyzer or measurement / characterization device; or a liposome nanoparticle drug delivery vehicle drug load quantity analyzer or liposome nanoparticle drug delivery vehicle drug load quantity measurement / characterization device; or a liposome nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio analyzer or a liposome nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio measurement / characterization device; or a liposome nanoparticle drug delivery vehicle lipidic component ratio analyzer or liposome nanoparticle drug delivery vehicle lipidic component ratio measurement / characterization device.

[0060] The present invention further provides a method of determining at least one property of nanoparticle delivery vehicles or a therapeutic payload carried by nanoparticle delivery vehicles, the nanoparticle delivery vehicles forming a diffusely scattering liquid suspension sample, the method comprising: locating the sample within an integrating cavity; directing probe light to a first port of the integrating cavity; receiving a portion of the probe light from a second port of the integrating cavity following scattering from the sample; measuring one or more spectral features of the probe light received at the second port; and determining the at least one property of the nanoparticle delivery vehicles or the therapeutic payload in the liquid suspension from the measured one or more spectral features of the probe light received at the second port.

[0061] The present invention may further comprises a computer program configured to perform methods of analysis described herein and performed by the analyser or controller. The present in invention further provides a computer readable medium having stored thereon the computer program, that is, instructions which when operated by a computer cause the computer to run analyser steps set out herein. Hence, the analyser described herein may comprises a computer having a processor, memory and user interface.

[0062] The present disclosure relates to nanoparticle vehicles or nanoparticle delivery vehicles configured to transport or deliver a payload or cargo.

[0063] Such payload delivery transport may occur, for example, in a medium such as the human or animal body.

[0064] The nanoparticle vehicle or nanoparticle delivery vehicle includes a carrier component and a payload component. The payload is held or carried by the nanoparticle vehicle or nanoparticle delivery vehicle and can eventually be released from the nanoparticle vehicle or nanoparticle delivery vehicle, for example, when certain release conditions are satisfied.

[0065] The present disclosure relates to characterization of nanoparticle vehicles or nanoparticle delivery vehicles.

[0066] The present disclosure relates, for example, to a nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device.

[0067] The present disclosure relates, for example, to a nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device, for example, for: - determining a payload quantity or payload concentration of the nanoparticle vehicles, and / or

[0068] - determining a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or

[0069] - determining a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or determining a nanoparticle vehicle concentration in a solution or formulation.

[0070] The present disclosure also relates, for example, to a nanoparticle vehicle analysis method or measurement / characterization method.

[0071] The present disclosure relates to a nanoparticle vehicle analysis method or measurement / characterization method, for example, for:

[0072] - determining a payload quantity or payload concentration of the nanoparticle vehicles, and / or

[0073] - determining a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or

[0074] - determining a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or determining a nanoparticle vehicle concentration in a solution or formulation.

[0075] Brief Summary of the Drawings

[0076] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings of which:

[0077] Figure 1 is a schematic diagram and schematic graph of conventional UV-Visible spectroscopy;

[0078] Figure 2 is a schematic diagram and schematic graph of conventional UV-Visible spectroscopy as applied to a highly scattering LNP;

[0079] Figure 3 is a spectrum obtained with conventional UV-Visible spectroscopy applied to LNPs carrying RNA at two different concentrations;

[0080] Figure 4 is a schematic diagram of apparatus according to an embodiment of the present invention;

[0081] Figure 5 is a schematic diagram of apparatus according to an alternative embodiment of the present invention;

[0082] Figures 6a and 6b are graphs of a liquid suspension of RNA and lipid nanoparticles measured using conventional UV-visible spectroscopy and the technique of the present invention; Figures 7a and 7b are graphs comparing extinction and absorption measurements for varying amounts of LNP and RNA;

[0083] Figure 8 is a graph showing a linear relationship between RNA concentration and absorption at 260nm;

[0084] Figures 9a and 9b are graphs showing further comparisons of conventional UV- visible spectroscopy with that of the present invention, including a comparison showing the extent to which scattering dominates in conventional spectroscopy;

[0085] Figure 10 is a graph showing a comparison between raw measurements, calibrated measurements and a reference;

[0086] Figure 11 is a table comparing results produced according to the present invention with a known assay technique; and

[0087] Figures 12a and 12b are graphs respectively comparing extinction measurements of three samples with absorption measurements of the three samples.

[0088] Detailed Description

[0089] Figure 4 is a schematic diagram of apparatus 100 according to the present invention. The apparatus comprises an integrating cavity 110 which has walls that are coated with a diffusely reflecting material. The integrating cavity may be a sphere but many other shapes are possible. The integrating cavity has a first port 130 and a second port 140. A light source 150 is provided that emits in the desired UV-Visible wavelength band. As we will discuss below this may be a limited range of wavelengths, such as at 260nm alone, a wider range of wavelengths, or may comprises a scanning / tuning wavelength source. The light source 150 is configured to direct probe light into the integrating cavity 110 at first port 130. The probe light is preferably directed such that it diffusely scatters at the surface of the integrating cavity. The apparatus further comprises a sample space 120 which may be a cuvette. The sample space is configured to be able to hold a liquid suspension sample in the integrating cavity. In the present invention the sample is diffusely scattering such that probe light impinges on the sample. If the wavelength matches an absorption band the sample will absorb some of the problem. Much of the probe light will also be scattered by the sample but the integrating cavity will return this sample scattered light back to the sample for further passes and possibilities for absorption by the sample. The second port is offset from first port such that light cannot travel a straight line path from first port to second port but has to undergo scattering from the integrating cavity and / or the sample. The probe light arriving at the first port 130 may travel along a straight line such that it travels along a first axial direction. Probe light being received at second port 140 may be received in a second axial direction along which the light passes to the detector. The first and second axial direction are offset from one another. For example, they are not directly at opposing positions on opposite sides of the integrating cavity. The integrating cavity may have a centre line and at least one of the first and second ports is offset form the centreline. The sample space or holder may be on the centreline.

[0090] Light exits the integrating sphere at second port 140 and is directed to a detector 160. The detector may form part of a spectrometer, may be configured with a filter to scan a detection wavelength band. The apparatus further comprises an analyser 170 which receives signals from the detector and determines a quantitative or qualitative property of the sample. The signals from the detector may be representative of one or more spectral features of the probe light received at the second port. For example, if a single wavelength such as 260nm is used the signal may be a representation of the amount of light received. Alternatively, the signal may form a spectrum of the amount of light received across a range of wavelengths. Based on the received signal the analyser determines a property of the sample. The property may be a quantitative property of the sample, such as an amount of RNA in the sample. Alternatively, the output may be a spectrum of the absorption of the sample across a range of wavelengths such as 240nm and 320nm. Other wavelength ranges may alternatively be used.

[0091] The analyser may determine one or more spectral features of the sample based on the one or more spectral features of the probe light received at the detector at the second port. For example, the analyser may store calibration data or a calibration transform. The calibration data may comprise a calibration of the integrating sphere and / or a calibration relating to the relative absorbance of the sample species at 260nm. For example, it is common to use a standard absorption coefficient for RNA, e.g. 0.025 (μg / ml)-1x cm'1, but accuracy can be improved by determining the absorption coefficient of a specific RNA formulation. Other calibration information may also be included. Various possibilities exist for the size of the sample holder or cuvette, for example, a 10mm cuvette containing 1 ml of sample or a 1 mm cuvette containing 100μl of sample may be used.

[0092] The arrangement of figure 4 is provided to measure the absorption of diffusely scattering samples, which cannot be measured by conventional UV-visible spectroscopy.

[0093] Figure 5 shows an alternative embodiment which comprises additional ports, namely third port 130’ and fourth port 140’. Third port 130’ may be configured to receive probe light from light source 150 or from a secondary light source but preferably substantially the same as light source 150. Fourth port 140’ may be configured to receive probe light from the integrating sphere that has passed directly from third port 130 and through the sample. Probe light from fourth port may be collected by detector 160 or by a secondary detector which is substantially the same as the detector 160. The arrangement of third port 130’ and fourth port 140’ allows an extinction value to be measured for light passing directly through the sample. Such a measurement is substantially the same as the extinction measurement made by conventional UV-visible spectrometers. In the present case, for samples that are diffusely scattering such as LNPs containing RNA much of the probe light will be scattered and will dominate over the probe light absorbed by the RNA. Nevertheless, as we will describe below the inclusion the additional ports allows a scattering measurement to be made to confirm the functionality of the apparatus with diffusely scattering samples.

[0094] Although the embodiment of figure 5 includes four ports, in other embodiments only three ports may be included. For example, the second port 140 may be omitted such that the straight through extinction measurement, as discussed in relation to figure 5, may be measured by directing probe light from third port 130’ to fourth port 140’. The absorption measurement may be made by directing probe light to first port 130 and collecting light at fourth port 140’. In this way the two ports used for the absorption measurement are still offset such that light scattered around the integrating sphere is collected. In a further example, the first port 130 may be omitted. Again the straight through extinction measurement, as discussed in relation to figure 5, may be measured by directing probe light from third port 130’ to fourth port 140’. The absorption measurement may be made by directing probe light to third port 130’ and collecting light at second port 140. Again the two ports used for the absorption measurement are still offset such that light scattered around the integrating sphere is collected. In the arrangement of figure 5 and the related arrangement the sample is not moved between the extinction measurement and the absorption measurement.

[0095] For conventional UV-Visible spectrometers and according to the Beer-Lamber law the absorbance A of a sample is given by: where Io is the input light intensity to the sample, I is the light intensity after passing through the sample, ε is the molar attenuating coefficient or absorptivity of the species, 1 is the optical path length through the sample and c is the concentration of the absorbing species. Hence, provided the attenuating coefficient for the particular species and the path length are known, the concentration of the species in the sample can be determined.

[0096] For the integrating cavity approach of the present invention, the Beer-Lambert law can still be considered to apply. However, an effective path length that can be used in the above equation needs to be determined by a calibration procedure. This procedure requires measurement of known concentrations of species at the wavelength(s) of interest and applying a curve fit to obtain an effective path length as a function of wavelength for the apparatus. The wavelength variation of the effective path length takes into account that the integrating cavity walls may have different reflectivity as the wavelength changes. Once the calibration has determined an effective path length the apparatus may be used to measure diffusely scattering samples.

[0097] Figures 6a and 6b show two graphs of a liquid suspension of RNA and lipid nanoparticles. In figure 6a the measurement is an extinction measurement made using conventional UV-visible spectroscopy. The vertical axis shows the optical density, i.e. a measure of the light lost in passing through the sample, and the horizontal axis shows how this varies with wavelength between 240nm and 320nm. The graph of figure 6a has two curves. The top curve is for empty LNPs (no RNA inside) in a buffer solution comprising 20μg / ml RNA and the bottom curve is for empty LNPs (no RNA inside) in a buffer solution comprising 10μg / ml RNA. In other words the sample for the top curve has twice as much RNA and the sample for the bottom curve. The conventional wavelength for measuring RNA concentration is 260nm. At this wavelength we would expect to see the sample for the top curve having twice the absorbance of the bottom curve due to the different concentrations. However, as can be seen in figure 6a the ODs are respectively 0.52 and 0.77. This is a result of the heavy scattering caused by the LNPs.

[0098] Figure 6b uses the apparatus and method of the present invention. The same two sample compositions are examined. At the 260 nm wavelength the respective OD values are 0.27 and 0.54, which is a doubling in OD in line with a doubling in concentration. In figure 6b the dotted lines are for further samples without the LNPs, which show that all of the RNA in the mixed samples has been correctly measured. Using the method of the present invention the effect of scattering has been eliminated. Hence, these measurements in figure 6b can be considered to be related to a scattered-corrected absorption (SCA). For the conventional technique the extinction measures absorbance and scattering together and they cannot be separated. Hence, for conventional UV-visible spectroscopy extinction can be considered to be given by:

[0099] Extinction = Absorbance + Scattering

[0100] Figures 7a and 7b show further comparison between conventional UV-Visible spectroscopy and the apparatus and methods of the present invention. As previously mentioned, most lipids do not absorb at 260nm, which is the typical RNA absorption peak, so if the scattering by the LNP can be eliminated then we can measure directly the amount or concentration of the RNA from the absorption peak. For figures 7a and 7b data is shown for the following samples:

[0101] A. 10μg / ml RNA concentration with empty (unloaded) LNPs (100%);

[0102] B. 10μg / ml RNA concentration with empty (unloaded) LNPs (50% by mass compared to A);

[0103] C. 10μg / ml RNA concentration without empty (unloaded) LNPs (0%);

[0104] D. 5μg / ml RNA concentration with empty (unloaded) LNPs (100% by mass compared to A and B); and

[0105] E. 5μg / ml RNA concentration with empty (unloaded) LNPs (200% by mass compared to A and B).

[0106] Hence, the samples have two amounts of RNA. Samples A-C have the most RNA at 10μg / ml concentration. Samples D and E have 50% of the RNA of samples A-C, namely 5μg / ml concentration.

[0107] Figure 7a is a similar graph to that of figure 6a, but figure 7a shows the extinction values measured using conventional UV-visible spectroscopy. Samples A, B and C are marked on figure 7a with “x”. These samples have the same amounts of RNA but differing amounts of unloaded LNPs. The conventionally measured OD values are different. This is a result of the differing amounts of scattering caused by the differing amounts of LNPs. In figure 7b, which has OD values measured according to the present invention these three samples have substantially the same absorption at around 0.27 to 0.28 at 260nm. The same can be seen for samples D and E, which are indicated in the graphs by triangles. Comparing A-B-C with D-E it can be seen that the samples having twice the amount of RNA have twice the absorption. Thus the amount of absorption varies linearly with the amount of RNA. In general based on these samples it has been found that samples measured according to the present invention and with the same amounts of RNA have the same OD measures to within <3%.

[0108] Figure 8 compares the nominal concentration of RNA to the absorption at 260nm for nine samples having various amounts of RNA and LNPs measured. The fit is highly linear showing that absorption can be taken as proportional to the amount of RNA independent of the amount of LNP present and the resultant scattering.

[0109] Figure 9a is a graph showing a further comparison of conventional UV-visible spectroscopy with that of the present invention. This graph shows the extent to which scattering dominates in conventional spectroscopy. In conventional spectroscopy the extinction is measured which is the top curve (B) in the figure. As mentioned, it is known that light scatters as a function of particle diameter to the sixth power and as a function of wavelength to the -4thpower. The amount of scattering can be inferred from a fit to the extinction measurement. A fit may be made using an equation of the form: and the scattering is inferred assuming no scattering occurs between 340 and 370nm and the extinction E, absorption A and scattering S are related by:

[0110] Using these equations the scattering is estimated and shown as the top dashed line C in the figure, and the absorption can also be estimated and is shown as bottom dashed line E. By the present invention absorption is measured (line A, indicated as SCA spectrum). As can be seen by the lower lines the estimated absorption from conventional UV-visible spectroscopy does not match that measured by the technique and has a significant error, such as > 25%.

[0111] Figure 9b shows similar information as figure 9a, except that the figure also shows a dotted line which is a reference measurement of RNA without LNPs present using a conventional absorption / extinction method. It can be seen in figure 9b that calculating the absorption from the scattering difference (III) underestimates the actual absorption of the RNA (IV), whereas comparing to figure 9a as it can be seen that the SCA measurement is a good estimate of the RNA. In practice extrapolating the amount of scattering using the conventional method described abode does not take into account the fact that scatter is affected when absorption is present. Hence, conventional methods include inherent inaccuracies whereas the SCA method of the present invention provides a more accurate measurement.

[0112] Figure 10 is a graph showing the calibration of the method according to the present invention. The curves on the graph are for 20μg / ml RNA in buffer. The dashed line at the top of the graphs shows the measured (raw) data, which after applying a calibration transform as discussed herein produces the solid curve in the figure. Also shown in the figure is a measure of the same sample measured using the conventional UV-visible extinction method. No LNPs or other scattering species are present in the measurements of figure 10. A very good match can be seen between the calibrated measurements according to the present invention and the reference measurement.

[0113] For some LNPs the lipid may absorb at the 260nm measurement wavelength. Measurements of absorption can be made of empty LNPs and RNA loaded LNPs using the method of the present invention. . Since absorption is additive, by subtracting the absorbance of the empty LNP from the loaded measurements (taking into account any concentration differences etc.), the absorbance for the RNA can be found. Hence, quantification of the LNPs and RNA can be determined and a loading ratio of the LNPs can be found.

[0114] The technique of the present invention has also been compared to other conventional techniques such as the RiboGreen fluorescent assay technique. A comparison of results is shown in the table in figure 11 . The results of figure 11 are for two different LNP formulations (SM102-LNP and a proprietary absorbing LNP) with two different RNAs (RNA1 and RNA2). The result for 260nm absorbing LNP is determined by linear decomposition as discussed on the preceding page. The results are all for a nominal concentration of 400μg / ml of RNA. The results from the present invention are comparable to those of the assay technique. The results were obtained for the present technique using a common standard extinction coefficient of 0.025 (μg / ml)-1x cm'1but accuracy can be improved by using or determining the extinction coefficient for the actual RNA of interest.

[0115] The scatter-corrected absorption method as applied to nanoparticle delivery vehicles carrying payloads according to the present invention differs from analysis of other turbid or diffusely scattering samples. In many scattering samples the scattering samples absorb light in the liquid phase with scattering from solids or semi-solids held in the liquid such as in suspension or colloid. However, in the nanoparticle delivery vehicle application, the delivery vehicles themselves typically absorb little in the liquid. The RNA absorbs whether in the liquid or in the delivery vehicle. Typically formulations tend towards 100% encapsulation efficiency so the amount of RNA measured by the SCA is a good indicator of the actual RNA or other payload content. We have described the technique, which we have referred to above as scatter corrected absorption (SCA), with reference to lipid nanoparticles (LNPs) carrying RNA. The technique is also applicable to a number of other nanomedicine examples. For example, other nanoparticles delivery vehicles carrying other therapeutic payloads may be measured, such as the following delivery vehicles:

[0116] • antibody-drug conjugates,

[0117] • viral vectors such as lentiviruses and AAVs,

[0118] • liposomes,

[0119] • exosomes,

[0120] • polymeric nanoparticles,

[0121] • liposome-based systems such as lipoplexes,

[0122] • metal nanoparticles,

[0123] • SPIONs,

[0124] • polymeric micelles,

[0125] • dendrimers,

[0126] • nanoemulsions, and

[0127] • nanogels.

[0128] We have discussed that the payload may comprise RNA. This may, for example, be mRNA, SiRNA, ssRNA. The payload may alternatively be one or more of:

[0129] • oligonucleotides,

[0130] • DNA,

[0131] • pharmaceuticals,

[0132] • vaccines,

[0133] • oncological treatments, and

[0134] • gene editing treatments.

[0135] As additional information we now provide further information regarding the background and related to the invention.

[0136] Nanoparticle vehicles or nanoparticle delivery vehicles may carry or transport payloads of various compositions, for example, comprising a drug, biologies or bioactive compounds. The payload may, for example, comprise a therapeutic agent, for example an oligonucleotide such as mRNA, siRNA, DNA, or the payload may comprise an active agricultural agent or ingredient, or an active pharmaceutical agent or ingredient. Nanoparticle delivery vehicles or systems are engineered technologies using nanoparticles for the targeted delivery and / or controlled release of a payload.

[0137] For example, nanoparticle drug delivery vehicles or systems are widely known for the targeted delivery and / or controlled release of therapeutic agents. One example of such nanoparticle delivery vehicles or systems is a lipid-based delivery vehicle. Lipid-based delivery vehicles include lipid nanoparticles such as (solid) lipid nanoparticles and liposomes.

[0138] Lipid nanoparticles are nanoparticles composed of lipids and have been developed as vehicles for small-molecule delivery. Lipid nanoparticles were first approved in 2018 as a drug delivery vehicle for the siRNS drug Patisiran, sold under the brand name Onpattro, and are now a key component of COVID-19 mRNA vaccines.

[0139] A lipid nanoparticle can be, for example, typically substantially spherical and can have an average diameter between 10 and 1000 nanometres, and typically in the range 60nm to 150nm or 80nm to 150nm.

[0140] Solid lipid nanoparticles may possess, for example, a solid lipid core matrix that can solubilize lipophilic molecules. The lipid core can be stabilized by surfactants, for example, an emulsifier. The lipid components may include one or more of, for example, triglycerides, diglycerides, monoglycerides, fatty acids, steroids or waxes.

[0141] Biological membrane lipids such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol) may be used as stabilizers.

[0142] The drug payload may, for example, be embedded in the interior of the solid lipid nanoparticle.

[0143] The lipid nanoparticles used in COVID-19 mRNA vaccines are generally made up of four types of lipids: an ionizable cationic lipid (whose positive charge binds to negatively charged mRNA), a PEGylated lipid (for stability), a phospholipid (for structure), and cholesterol (for structure).

[0144] Lipidic fractions and compositional make-up of the lipid nanoparticle vary by formulation and are typically include 3 to 4 components, e.g., cholesterol, a stabilising phospholipid, a PEGylated lipid, etc.

[0145] A liposome can be considered to be a ‘hollow’ lipid nanoparticles which may, for example, have a phospholipid bilayer and an interior composed of aqueous substance. The drug payload may, for example, be embedded in the interior of this lipid nanoparticle.

[0146] A liposome may, for example, comprise a substantially spherical vesicle composed of synthetic or natural phospholipids that self-assemble. The vesicle can have an aqueous core (surrounded by a hydrophobic membrane) that can be loaded with a wide variety of hydrophobic or hydrophilic molecules, for example, for therapeutic purposes.

[0147] Particle size, particle concentration, payload concentration and payload-to-particle loading are crucial parameters to measure in the development and production of nanoparticle vehicles (NPVs) or nanoparticle vehicle systems, for applications in therapeutics, agriculture, skincare and beyond. A nanoparticle vehicle (nanoparticle delivery vehicle) or NPV system comprises (i) a particle or carrier component and (ii) a payload component.

[0148] The particle or carrier component may be made of or comprise various materials, including but not limited to:

[0149] • Lipidic formulations or a Lipidic composition

[0150] • Polymer formulations or a Polymer composition

[0151] • Liposome formulations or a Liposome composition

[0152] • Other nanoparticle formulations or nanoparticle composition

[0153] The payload fraction or component may be made of or comprise:

[0154] • mRNA

[0155] • siRNA

[0156] • DNA

[0157] • Other therapeutic payloads

[0158] • Active agricultural ingredients

[0159] • Active pharmaceutical ingredients

[0160] Practical, accurate and affordable analytical techniques are needed to measure these characteristics. Optical methods, including UV-Vis spectroscopy, are used to measure these parameters.

[0161] In a standard UV-Vis instrument, when samples scatter light, the transmitted intensity of light as a function of wavelength will be influenced by both the scattering and the absorbance of a sample. This produces the wavelength dependent “extinction” spectrum of the sample, where extinction = scattering + absorbance. Normally the absorbance spectrum is what is of interest when using UV-Vis spectroscopy, because it can be used to quantify the concentration of analytes, via the Beer-Lambert law. When samples scatter, light scattering will interfere with obtaining a true absorption spectrum of the sample, yielding the Beer-Lambert law invalid. As a result, concentration estimates will be incorrect.

[0162] Nanoparticle vehicles (NPVs) are typically in the size range of 60nm or 80 nm - 150 nm diameter. Due to their size and refractive index, NPVs scatter light significantly in the UV-region - where payloads and NPV particle fractions absorb light. Therefore, attempting to use regular UV-Vis spectroscopy techniques to optically measure the components of a NPV system (e.g. NPV concentration, payload / NPV concentrations, payload loading) is challenging, due to the interference by light scattering in obtaining a useful absorption spectrum.

[0163] A method of subtracting / removing the scattering contribution from the extinction measurement is necessary to obtain accurate measurements. Such methods include:

[0164] A. Background subtraction: Approximating sample scattering by measuring the UV-Vis spectrum in a region where samples do not absorb light and removing the measured value as a “flat background” from the measured extinction spectrum

[0165] B. Approximate-model subtraction: The wavelength dependent light scattering spectrum of the sample can be approximated by a mathematical model (e.g.

[0166] Rayleigh scattering) and to produce a theoretical scattering spectrum, which can be removed as a background from the measured extinction spectrum.

[0167] C. Measurement of sample scattering: An orthogonal or separate measurement of the sample’s light scattering spectrum can be performed, which can then be subtracted from the measured extinction spectrum.

[0168] However these approaches will have significant drawbacks in certain scenarios, particularly when:

[0169] 1 . The NPV scattering spectrum is significantly stronger than the absorption spectrum of the components. In this instance, the absorbance spectrum will be almost indistinguishable over the large scattering background, and background subtraction methods will be insufficient in isolating an accurate estimate of the absorbance (analogous to fluorescence interference in Raman spectroscopy).

[0170] 2. The NPV components and the payload / payload components have overlapping absorbance spectra. In this instance, background subtraction will be insufficient in deconvoluting the relative contributions to absorbance by each NPV component.

[0171] Other techniques for characterizing NPV components include:

[0172] • Fluorescent assays (Ribogreen assay): Requires a number of sample preparation steps and at least 10 minutes per sample measurement.

[0173] • Field Flow Fractionation Multi-Angle Light Scattering (FFF-MALS): Only measures particle size, not concentration and is very complicated. • Dynamic Light Scattering (DLS): Only measures particle size, not concentration. Can only measure particles above a certain size threshold, and has a strong bias towards detecting large particles in samples.

[0174] • Particle Tracking Analysis (PTA): Can measure particle size and concentration, but can only measure particles above a certain size threshold. Samples require significant dilution to be measured.

[0175] A goal of the present invention and disclosure is to provide an alternative device and an alternative method for characterizing nanoparticle vehicles or nanoparticle vehicle systems, or to provide a device and a method for characterizing nanoparticle vehicles or nanoparticle vehicle systems that addresses the above-mentioned inconveniences of known devices and methods.

[0176] A goal of the present disclosure is to provide a nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device.

[0177] A goal of the present disclosure is to provide a nanoparticle vehicle analyzer or a nanoparticle measurement / characterization device for determining, for instance, a payload quantity or payload concentration of the nanoparticle vehicles, and / or a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or a nanoparticle vehicle concentration in a solution or formulation.

[0178] A goal of the present disclosure is provide to a nanoparticle vehicle analysis method or measurement / characterization method.

[0179] A goal of the present disclosure is provide to a nanoparticle vehicle analysis method or measurement / characterization method for determining, for example, a payload quantity or payload concentration of the nanoparticle vehicles, and / or a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or a nanoparticle vehicle concentration in a solution or formulation.

[0180] Embodiments of the present invention provide a nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device including: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette or sample holder within the integrating cavity, the cuvette or sample holder being configured to contain a liquid sample or solution including at least one or a plurality of nanoparticle vehicles; wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the or each light inlet port being configured to receive light from at least one light source and the or each light outlet port being configured to deliver light to a detector or spectrometer; wherein the lipid-based drug delivery vehicle analyzer or the lipid-based drug delivery vehicle measurement / characterization device is configured to operate in a diffusely reflecting mode in which light from the light source follows a light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the detector or spectrometer for wavelength analysis of the light to provide a property such as an absorbance spectrum of the at least one or the plurality of nanoparticle vehicles contained in the liquid sample or solution.

[0181] In a particular embodiment, the nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device is a lipid-based nanoparticle vehicle analyzer or a lipid-based nanoparticle vehicle measurement / characterization device. The lipid-based nanoparticle vehicle may comprise or consist of, for example, a (solid) lipid nanoparticle and / or a liposome.

[0182] Embodiments of the present invention concern a nanoparticle vehicle analysis or measurement / characterization method comprising: providing the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device, or providing an apparatus configured to measure or determine at least a scatter-corrected absorbance measurement or a scatter-corrected absorbance spectrum; and / or obtaining / receiving at least one or a plurality of scatter-corrected absorbance measurements / data or scatter-corrected absorbance spectrum measurements / data for a liquid sample or solution containing a plurality of nanoparticle vehicles.

[0183] In a particular embodiment, the nanoparticle vehicle analysis or measurement / characterization method is a lipid-based nanoparticle vehicle analysis or measurement / characterization method. The lipid-based nanoparticle vehicle may comprise or consist of, for example, a (solid) lipid nanoparticle and / or a liposome. The invention provides an alternative device and an alternative method to known devices and methods. The invention provides a device and a method that addresses the previously-mentioned inconveniences of known devices and methods.

[0184] The invention furthermore overcomes the shortcomings of traditional UV-Vis based methods by employing an integrating-cavity based spectrophotometer to perform the absorbance measurements. The use of an integrating cavity based apparatus allows for the elimination of the effects of scattering on the measured absorbance spectrum, thereby producing a “pure absorbance” spectrum, which more accurately represents the characteristics of the nanoparticle vehicles.

[0185] The invention assures both a priori scatter-correction and absolute quantification using, for example, Beer-Lambert-like quantification.

[0186] The device and method of the present invention assures a unique scatter-correction permitting an accurate compositional measurement. Payload concentrations are determined independently of particle size / scatter.

[0187] The device and method of the present invention removes the unwanted scatter contribution and assures accurate or more accurate measurements of concentrations or characterization measurements of nanoparticle vehicles, for example, drug delivery nanoparticle vehicles.

[0188] The device and method of the present invention advantageously assures a quick and accurate measure of a payload (for example, drug) concentration, where only a simple dilution is required for the measurements. The device and method of the present invention advantageously assures a quick and accurate measure of the payload-to-carrier (for example, drug-to-lipid) concentration ratio.

[0189] Integrating cavities have never been used or suggested before as a way to characterize nanoparticle vehicles or lipid-based drug delivery vehicles.

[0190] The nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device of the present invention may for example be, or comprise or consist of the spectrometer apparatus disclosed in international patent application WO2018070882, the entire contents of which are fully incorporated herein by reference, and also included as an Annex of this application. This apparatus is commercially available from MARAMA LABS sold as their CloudSpecTM apparatus.

[0191] In some embodiments, the nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device may differ in a number of aspects from the apparatus described in WO2018070882. For example, the analyzer / device described herein may not include the input light path adjuster 13 and / or the output light path adjuster 13B. Additionally, the integrating cavity may only include an input port P2 and an output port P4 arranged for absorbance measurements.

[0192] The use of an integrating sphere to measure the absorbance of a scattering sample physically / optically eliminates the contribution of light scattering to absorbance measurements, and therefore:

[0193] • is much more effective in obtaining a true-absorbance measurement compared to background-subtraction methods.

[0194] • does not require any measurement or calculation of the scattering spectrum (which in other techniques is required, in order to subtract a suitable scattering spectrum) to obtain the scatter-corrected absorbance. Particularly, it does not rely on particle size, particle size distribution (Pdi) or refractive index of the material.

[0195] This approach opens up a number of valuable applications over existing techniques, given below.

[0196] A. Fast Payload Measurement

[0197] CloudSpec removes the interferences from light scattering in absorbance measurements of NPVs through the use of an integrating sphere, and thus can measure accurate concentrations of the components of interest. The benefits of CloudSpec are:

[0198] • Measure payload concentration quickly - only simple dilution of the sample required

[0199] • Measure payload-to-carrier (for example, payload-to-lipid) concentration ratio

[0200] • Measure significantly lower payload concentrations in scattering samples than with background-subtraction methods

[0201] • Ability to characterise NPVs themselves, e.g., by composition or concentration.

[0202] Most NPVs scatter in the UV and not much in the visible, making scatter-correction difficult or impossible.

[0203] The CloudSpec apparatus, as described in WO2018070882, can produce two spectra of a liquid sample that can be measured simultaneously; the first is the “extinction spectrum”, where the sample is measured in a transmission geometry and the reduction in light intensity is a result of sample absorbance and scattering. This is the equivalent spectrum produced by a traditional UV-Vis spectrophotometer. The second is the “absorption spectrum”, where the sample is measured in an integrating-cavity geometry and the reduction in light intensity is a result of sample absorbance only.

[0204] Exemplary measurements using the CloudSpec apparatus were performed on liquid samples comprising nanoparticle vehicles contained in a liquid. The resulting measurements manifestly confirm the ability of the CloudSpec apparatus and / or an integrating-cavity arrangement to provide a true and useful absorption spectrum of the nanoparticle vehicle sample, that is, a scatter-corrected absorption that can be exploited to characterize multiple features of the nanoparticle vehicles.

[0205] Some results of the exemplary measurements of extinction and absorption are shown in the Figures 12(a) and 12(b) that were performed on nanoparticle vehicles. The nanoparticle vehicles included a particle / carrier component composed of a (solid) lipid nanoparticle and a payload component that was either (i) absent, (ii) composed of mRNA, or (iii) composed of siRNA.

[0206] The lipid nanoparticles used in the measurements typically comprise 3 to 4 constituent carrier elements, e.g., cholesterol, a stabilizing phospholipid, a PEGylated lipid. The nanoparticle vehicle average size / diameter (measured by DLS) for the above- mentioned lipid nanoparticle vehicles was respectively (i) 56.4 nm, (ii) 96.9 nm, and (iii) 76 nm.

[0207] The liquid sample was provided in the previously mentioned cuvette that is received within the integrating cavity.

[0208] The samples were diluted (20x) in tris buffer and measured against a distilled water blank. No stirring was performed during measurement.

[0209] As can be seen in figure 12(b), a strong absorption characteristic feature is obtained around 260nm corresponding to the payload absorption (mRNA OR siRNA). Nanoparticle vehicles having an absent payload contribute negligibly to absorption at this characteristic wavelength permitting the measured absorption at this wavelength to be exploited to characterize, for example, the payload concentration.

[0210] It is manifestly logical that this approach and principle can be expanded and applied to characterize various features of the nanoparticle vehicles.

[0211] The measured extinction results of Figure 12(a) confirm that the previously mentioned correction approach, of subtracting / removing the scattering contribution of the nanoparticle vehicles having no payload from the extinction measurements of the nanoparticle vehicles having a payload, to try to obtain accurate measurements will not provide an adequate correction, and not permit to obtain the accurately measured absorption values presented in Figure 12(b).

[0212] As shown in figure 12(a), scatter masks lipid and RNA absorption, so much so that by OD600 there is no signal. Figure 12(b) shows that obtaining the true lipid and RNA absorption enables absolute and relative concentration measurement. The approach and principle of the present disclosure can characterize various features of the nanoparticle vehicles such as the previously mentioned Payload Loading in NPVs.

[0213] As mentioned, measuring payload load in NPVs is difficult and is often done by inconvenient and resource-intensive techniques, such as FFF-MALS and HPLC. UV / Vis spectroscopy could, in principle, be used to quantify payload in the NPVs, e.g., by using the A260 peak, but particle scattering causes artefacts that vary in magnitude between NPV systems.

[0214] NPVs both absorb in the UV and scatter light due to factors such as refractive index, particle size, particle size distribution and composition, which creates a significant error in using optical absorption to quantify components. Since these factors vary between formulations and manufacturing processes it is inconvenient or impossible to build a simple correction that does not rely on other input data, e.g., particle size or concentration. The CloudSpec enables both a priori scatter-correction and absolute quantification using Beer- Lambert-like quantification.

[0215] NPVs vary by formulation and are usually 3 to 4 constituent carrier elements, e.g., cholesterol, a stabilising phospholipid, a PEGylated lipid, etc.

[0216] The device and method of the present disclosure can advantageously be used, for example, as follows:

[0217] 1 . Using scatter-corrected absorption to quantify the loading of payload in an NPV formulation, using the extinction coefficient of the payload

[0218] 2. Using scatter-corrected absorption to quantify the loading of payload and / or carrier (e.g. lipidic) fraction in an NPV formulation using the extinction coefficients of the payload and the carrier (lipidic) fractions (either combined or separately)

[0219] 3. Using scatter-corrected absorption to quantify the relative carrier (e.g. lipidic) component fractions using the extinction coefficients of the individual carrier (lipidic) constituent elements

[0220] 4. All of the above in relation to various lipidic-based vehicle payloads, e.g., liposomes

[0221] A further exemplary and valuable application over existing techniques is:

[0222] B. Fast Particle Concentration measurement

[0223] NPVs tend to absorb in the UV, but they also scatter light significantly. CloudSpec removes the scatter, enabling Beer-Lambert concentration determination. The benefits of CloudSpec are: • Measurement of particle concentration by mass, using scatter-corrected absorption.

[0224] • Measurement of particle concentration (using externally measured size information).

[0225] • Use of extinction (scatter) to approximate particle size parameters.

[0226] In other words, the approach and principle of the present disclosure can characterize the feature of the nanoparticle vehicles that is Particle Concentration of NPVs.

[0227] Measuring concentration of NPVs in solution is difficult because the NPVs are small and discrete within the buffer solution, and so extinction coefficient varies by particle number and size. UV / Vis spectroscopy could, in principle, be used to measure particle concentration in the NPVs, e.g., by using the absorption of the payload or carrier (lipidic) fractions, but particle scattering causes artefacts that vary in magnitude between NPV systems and the particle size may or may not be possible to approximate using scatter- corrected absorption SCA.

[0228] NPVs both absorb in the UV and scatter light due to factors such as refractive index, particle size, particle size distribution and composition, which creates a significant error in using optical absorption for quantification. Since these factors vary between formulations and manufacturing processes it is inconvenient or impossible to measure particle concentration. The CloudSpec enables both a priori scatter-correction and absolute quantification using Beer-Lambert-like quantification.

[0229] Particle concentration can be measured using SCA if the particle size is known, e.g., from DLS measurements.

[0230] The scatter spectrum and Mie theory may be used to approximate the size, negating the need for other measurements (such as DLS).

[0231] The device and method of the present disclosure can thus advantageously be used, for example, as follows:

[0232] 1 . Using scatter-corrected absorption to quantify the particle concentration of NPVs in a formulation using the extinction coefficient of the payload and the particle size measured by a technique such as DLS, PTA, etc.

[0233] 2. Using the scattering spectrum to quantify the particle concentration of NPVs in a formulation using the calculated scattering spectrum (from Mie Theory) and the particle size measured by a technique such as DLS, PTA etc

[0234] The above and other objects, features and advantages of the present invention and the manner of realizing them will become more apparent, and the invention itself will best be understood from a study of the following description. The above general description and the detailed description given below, serve to explain the features of the invention of the present disclosure. Exemplary and non-limiting embodiments of the invention of the present disclosure are now provided.

[0235] According to embodiments, an exemplary nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device of the present disclosure may comprise, be based on, or consist of the apparatus described in international patent application WO2018070882, the entire contents of which are fully incorporated herein by reference, and also included as Annex of this application.

[0236] The nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device is, for example: a nanoparticle vehicle payload quantity or payload concentration analyzer or measurement / characterization device, and / or a nanoparticle vehicle payload-to-carrier content / concentration ratio analyzer or measurement / characterization device, and / or a nanoparticle vehicle ratio or fractional carrier component content analyzer or measurement / characterization device, and / or a nanoparticle vehicle solution concentration analyzer or measurement / characterization device.

[0237] The nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device includes, for example,: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette configured to contain a liquid sample within the integrating cavity, wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the or each light inlet port being configured to receive light from at least one light source and the or each light outlet port being configured to deliver light to a spectrometer; wherein the nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device is configured to operate in a diffusely reflecting mode in which light from the light source follows a light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample. Particular embodiments of the nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device are described in international patent application WO2018070882, and also recited in the claims.

[0238] In a particular embodiment, the nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device is, for example, a lipid-based nanoparticle vehicle analyzer or a lipid-based nanoparticle vehicle measurement / characterization device.

[0239] The nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device is, for example, a lipid-based drug delivery vehicle analyzer or a lipid-based drug delivery vehicle measurement / characterization device. The lipid-based drug delivery vehicle analyzer or a lipid-based drug delivery measurement / characterization device is for (or configured for) determining, for instance, a drug load quantity of the lipid-based drug delivery vehicle, or determining a drug load-to- lipidic content / concentration ratio of the lipid-based drug delivery vehicle, or determining a lipidic component ratios of the lipid-based drug delivery vehicle.

[0240] The nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device is, for example, a lipid-based nanoparticle analyzer or a lipid-based nanoparticle measurement / characterization device for determining a lipid- based nanoparticle concentration in a solution or in a formulation.

[0241] The lipid-based nanoparticle vehicle may comprise or consist of, for example, a (solid) lipid nanoparticle and / or a liposome, or a lipid emulsion.

[0242] The nanoparticle vehicle may, for example, comprise or consist of a lipid-based nanoparticle as mentioned above. The nanoparticle vehicle may alternatively comprise or consist of a polymeric nanoparticle such as a polymeric nanocapsule or nanosphere, or a polymersome, a dendrimer, or a polymer micelle.

[0243] The nanoparticle vehicle may alternatively comprise or consist of an inorganic nanoparticle such as a silica nanoparticle, a quantum dot, an iron-based or iron oxide nanoparticle or gold nanoparticle.

[0244] The nanoparticle vehicle may alternatively comprise or consist of an exosome, a microvesicle, a lentivirus, an adeno-associated virus (AAV) or an adenovirus.

[0245] The particle or carrier component of the nanoparticle vehicle may, for example, be made of or comprise various materials, including but not limited to:

[0246] • Lipidic formulations or a Lipidic composition

[0247] • Polymer formulations or a Polymer composition

[0248] • Liposome formulations or a Liposome composition • Other nanoparticle formulations or nanoparticle composition

[0249] The payload fraction or component of the nanoparticle vehicle may, for example, be made of or comprise:

[0250] • mRNA

[0251] • siRNA

[0252] • DNA

[0253] • Other therapeutic payloads or cargo

[0254] • Active agricultural ingredients

[0255] • Active pharmaceutical ingredients

[0256] The present disclosure also relates, for example, to a nanoparticle vehicle analysis method or measurement / characterization method.

[0257] The present disclosure also relates to a nanoparticle vehicle analysis method or measurement / characterization method, for example, for: determining a payload quantity or payload concentration of the nanoparticle vehicles, and / or determining a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or determining a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or determining a nanoparticle vehicle concentration in a solution or formulation.

[0258] The analysis method or measurement / characterization method can be carried out using the above mentioned nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device.

[0259] The apparatus may produce two spectra of a liquid sample that may, for example, be measured simultaneously. The first is the “extinction spectrum”, where the sample is measured in a transmission geometry and the reduction in light intensity is a result of sample absorbance and scattering. This is the equivalent spectrum produced by a traditional UV-Vis spectrophotometer. The second is the “absorption spectrum”, where the sample is measured in an integrating-cavity geometry and the reduction in light intensity is a result of sample absorbance only.

[0260] The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device may include, for example, at least one processor or calculation means; and at least one memory or storage means including a computer program comprising instructions which, when the computer program is executed by the processor or calculation means, cause the analyzer or measurement / characterization device to carry out all or a portion of the steps of the analysis method or measurement / characterization method, or the method according of any one of below claims.

[0261] The present disclosure also concerns a computer program comprising instructions which, when the program is executed by a computer, processor or calculation means cause the computer, processor or calculation means to carry out the analysis method or measurement / characterization method. The present disclosure also concerns a computer- readable data carrier having stored thereon said computer program.

[0262] Further details of non-limiting and exemplary embodiments of the analysis methods or measurement / characterization methods of the present disclosure are now provided, which include payload loading NPVs, and fast particle concentration methods and measurements.

[0263] A.L Quantifying the loading of payload in an NPV formulation using the extinction coefficient of the payload

[0264] To obtain the payload-loading of an NPV sample using the extinction coefficient of the payload:

[0265] 1 . Dilute the sample, as necessary, in an appropriate buffer, to bring the absorbance within the working range of the integrating-sphere spectrophotometer.

[0266] 2. Measure the buffer solution as a “blank”.

[0267] 3. Measure the scattered-corrected absorbance of the NPV sample, using the buffer as the blank.

[0268] 4. Record the scatter-corrected absorbance value of the payload at the wavelength of interest (e.g. 260 nanometers for mpayload).

[0269] 5. To obtain the extinction coefficient of the payload, determine it directly by measuring a known amount of payload, or a concentration-series of neat payload solutions, in a UV-Vis instrument, or look up its value in the literature.

[0270] 6. Depending on the nature of the NPV and payload, there are two methods to determine payload loading: a. Method A (Direct Beer-Lambert law): If payload and the NPV do not absorb in the same wavelength regions: i. Use the Beer-Lambert law to compute the concentration of the payload in the NPV sample by where cpayioad is the payload concentration, cis the measured scatter- corrected absorbance of the NPV sample at the wavelength of interest (corrected for any dilution factor applied in Step 1 ), A, and 6D(A) is the molar extinction coefficient of the payload at the wavelength of interest. b. Method B: If the payload and the NPV do absorb in the same wavelength regions, use a spectral deconvolution method, such as multivariate curve resolution, to determine the isolated scatter-corrected absorbance spectra of the payload and NPV respectively and repeat steps 6(a)(i) and using the absorbance values from the isolated payload spectrum.

[0271] 7. If 100% of the payload is encapsulated in the NPV, the determined payload concentration equals the payload loading. If the encapsulation of the payload is less than 100%, the loading value should be scaled accordingly.

[0272] A more detailed example is given below:

[0273] The commonly accepted extinction coefficient of RNA at 260 nm is In other words, an absorption of 1 (optical density, OD, of 1 , in a 1cm cuvette) at 260 nm would correspond to a concentration of Assuming there are no other constituents with significant absorption at 260nm, the mRNA concentration can therefore be extracted from the absorption at 260 nm from

[0274] If a dilution was carried out, the above loading corresponds to the diluted sample and the original loading can be obtained by multiplying by the dilution factor.

[0275] As an example, Figures 12(a) and 12(b) are the absorption spectra (integrating sphere mode, i.e. scatter-corrected-absorbance) for payload-loaded and unloaded LNPs measured in the Cloudspec. For the mRNA NPV, we see that X Taking into account the 20x dilution, this corresponds to a payload loading:

[0276] It is also clear from the spectrum of the empty NPVs that they do not contribute to This means the payload concentration can be directly quantified from the of the CS scatter-corrected absorbance spectra.

[0277] A.IL Quantifying the loading of payload and / or lipidic fraction in NPV formulations using the extinction coefficients of the payload and the lipidic fractions

[0278] To obtain the payload-loading and / or lipidic fraction of an NPV sample using the extinction coefficients of the payload and the lipidic fractions: 1 . Dilute the sample, as necessary, in an appropriate buffer, to bring the absorbance within the working range of the integrating-sphere spectrophotometer.

[0279] 2. Measure the buffer solution as a “blank”.

[0280] 3. Measure the scattered-corrected absorbance of the NPV sample, using the buffer as the blank.

[0281] 4. Record the scatter-corrected absorbance value(s) of the payload at the wavelength of interest (e.g. 260 nanometers for mRNA) and the NPV, multiplying the measured absorbance values by the dilution factor used in Step 1 .

[0282] 5. To obtain the extinction coefficient of the payload, determine it directly by measuring a known amount of payload, or a concentration-series of neat payload solutions, in a UV-Vis instrument, or look up its value in the literature

[0283] 6. To obtain the extinction coefficient of the NPV fraction, determine it directly by measuring a known amount of NPV fraction, or a concentration-series of neat NPV fraction solutions, in a UV-Vis instrument, or look up its value in the literature

[0284] 7. Depending on the nature of the NPV and payloads, there are two methods to determine payload and NPV concentrations: a. Method A (Direct Beer-Lambert law): If payload and the NPV do not absorb in the same wavelength regions: i. Use the Beer-Lambert law to compute the concentration of the payload in the NPV sample by where isthe payload concentration is the measured scatter- corrected absorbance of the NPV sample at the wavelength of interest (corrected for any dilution factor applied in Step 1) and is the molar extinction coefficient of the payload at the wavelength of interest. ii. Use the Beer-Lambert law to compute the concentration the NPV fraction by c where is the NPV fraction concentration is the measured scatter-corrected absorbance of the NPV sample at the wavelength of interest (corrected for any dilution factor applied in Step 1), , and is the molar extinction coefficient of the NPV fraction at the wavelength of interest. b. Method B: If the payload and the NPV do absorb in the same wavelength regions, use a spectral deconvolution method, such as multivariate curve resolution, to determine the isolated scatter-corrected absorbance spectra of the payload and NPV respectively and repeat steps 7(a)(1) and 7(a)(ii) using the absorbance values from the isolated spectra.

[0285] 8. If 100% of the payload is encapsulated in the NPV, the determined payload concentration equals the payload loading. If the encapsulation of the payload is less than 100%, the loading value should be scaled accordingly.

[0286] 9. The payload to NPV fraction ratio can be calculated by dividing by c

[0287] A.111. Quantifvino the relative lipidic component fractions in NPV formulations usino the extinction coefficients of the individual lipidic fractions

[0288] To obtain the relative lipidic component fractions of an NPV sample using the extinction coefficients the individual lipidic fractions:

[0289] 1 . Dilute the sample, as necessary, in an appropriate buffer, to bring the absorbance within the working range of the integrating-sphere spectrophotometer.

[0290] 2. Measure the buffer solution as a “blank”.

[0291] 3. Measure the scattered-corrected absorbance of the NPV sample, using the buffer as the blank.

[0292] 4. Record the scatter-corrected absorbance value(s) of each lipidic component fraction at their respective wavelengths of interest.

[0293] 5. To obtain the extinction coefficient of the lipidic component fractions, determine them directly by measuring a known amount of lipidic component fractions or a concentration-series of neat lipidic component fraction solutions, in a UV-Vis instrument, or look up their values in the literature.

[0294] 6. Use the Beer-Lambert law to compute the concentration of each lipidic component fraction in the sample by where is the component concentration, A is the measured scatter-corrected absorbance of the NPV sample at the wavelength of interest (corrected for any dilution factor applied in Step 1 ), and is the molar extinction coefficient of the component fraction at the wavelength of interest.

[0295] B.1 Quantifying the particle concentration of NPVs in a formulation using the extinction coefficient of the payload and the particle size

[0296] To obtain the concentration of particles of an NPV sample using the extinction coefficient of the payload, the extinction coefficient of the lipid fraction and particle size information: Dilute the sample, as necessary, in an appropriate buffer, to bring the absorbance within the working range of the integrating-sphere spectrophotometer. Measure the buffer solution as a “blank”. Measure the scattered-corrected absorbance of the NPV sample, using the buffer as the blank. Record the scatter-corrected absorbance value(s) of the lipid fraction and the payload at their respective wavelengths of interest. To obtain the extinction coefficient of the lipid fraction, determine it directly by measuring a known amount of lipid fraction or concentration-series of neat solutions of the lipid fraction in a UV-Vis instrument, or look up its values in the literature. To obtain the extinction coefficient of the payload, determine it directly by measuring a known amount of payload or concentration-series of neat payload solutions in a UV-Vis instrument, or look up its value in the literature. Depending on the nature of the NPV and payloads, there are two methods to determine payload and NPV concentrations: a. Method A (Direct Beer-Lambert law): If payload and the NPV do not absorb in the same wavelength regions: i. Use the Beer-Lambert law to compute the concentration of the payload in the NPV sample by where isthe payload concentration, is the measured scatter- corrected absorbance of the NPV sample at the wavelength of interest (corrected for any dilution factor applied in Step 1), and is the molar extinction coefficient of the payload at the wavelength of interest. ii. Use the Beer-Lambert law to compute the concentration of the lipid fraction by where is the lipid fraction concentration, is the measured scatter-corrected absorbance of the NPV sample at the wavelength of interest (corrected for any dilution factor applied in Step 1), and s the molar extinction coefficient of the lipid fraction at the wavelength of interest. b. Method B: If the payload and the lipid fraction do absorb in the same wavelength regions, use a spectral deconvolution method, such as multivariate curve resolution, to determine the isolated scatter-corrected absorbance spectra of the payload and lipid respectively and repeat steps 7(a)(i) and 7(a)(ii) using the absorbance values from the isolated spectra. 8. Determine the size of the particles by an external method, such as Dynamic Light Scattering, Particle Tracking Analysis or other appropriate techniques.

[0297] 9. For example, assuming the lipid fraction per particle is known, compute the number of lipid molecules in each particle, based on the size determined in step 8.

[0298] 10. Compute, for example, the particle concentration cP[particles per mL] by cP= cNPV / Np, where NPis the lipid fraction per particle, in moles per particle.

[0299] B.11 To obtain the concentration of particles (bv particle size) of an NPV sample usino the particle scatterino and particle size information:

[0300] 1 . Dilute the sample, as necessary, in an appropriate buffer, to bring the absorbance within the working range of the integrating-sphere spectrophotometer.

[0301] 2. Measure the buffer solution as a “blank”.

[0302] 3. Measure the scattered-corrected absorbance spectrum of the NPV sample, using the buffer as the blank.

[0303] 4. Measure the extinction spectrum of the NPV sample, using the buffer as the blank.

[0304] 5. Calculate the scattering spectrum of the particle by subtracting the scatter-corrected absorbance from the extinction spectrum.

[0305] 6. Determine the size of the particles by an external method, such as Dynamic Light Scattering, Particle Tracking Analysis or other appropriate techniques.

[0306] 7. Compute the theoretical wavelength-dependent scattering cross section, oscat, of the NPVs with Mie Theory, using the particle size from step 6 and the wavelengthdependent dielectric function of the particle components as inputs to the Mie Theory calculation.

[0307] 8. Compute the theoretical scattering spectrum (Optical Density O.D) of the particles by O. Dtheo= oscat* cP, where cPis the particle concentration [moles per liter],

[0308] 9. Determine the NPV particle concentration by adjusting cPto obtain the best fit between O. Dttieoand the measured scattering spectrum.

[0309] 10. Convert cP[moles per liter] to cP[particles per mL] by cP[particles per mL] = cP[moles per liter]* N * 0.001, where N = Avogadro’s number.

[0310] Implementations described herein are not intended to limit the scope of the present disclosure but are just provided to illustrate possible realizations.

[0311] While the invention has been disclosed with reference to certain preferred embodiments, numerous modifications, alterations, and changes to the described embodiments, and equivalents thereof, are possible without departing from the scope of the invention as set out in the appended claims. Accordingly, it is intended that the invention not be limited to the described embodiments and be given the broadest reasonable interpretation in accordance with the language of the appended claims. The features of any one of the above described embodiments may be included in any other embodiment described herein.

[0312] The person skilled in the art will readily appreciate that various modifications and alterations may be made to the above described methods and apparatus. The modifications may be made without departing from the scope of the appended claims. For example, the present invention is not just directed to measure lipid nanoparticles carrying RNA but other delivery vehicles carrying other payloads. Different wavelengths and shapes of integrating cavity may alternatively be used.

[0313] Embodiments of the present invention are set out in the following numbered clauses.

[0314] A1 . Nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device including: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette within the integrating cavity, the cuvette being configured to contain a liquid sample or solution including at least one or a plurality of nanoparticle vehicles; wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the or each light inlet port being configured to receive light from at least one light source and the or each light outlet port being configured to deliver light to a spectrometer; wherein the lipid-based drug delivery vehicle analyzer or the lipid-based drug delivery vehicle measurement / characterization device is configured to operate in a diffusely reflecting mode in which light from the light source follows a light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the at least one or the plurality of nanoparticle vehicles contained in the liquid sample or solution.

[0315] A2. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to clause A1 , wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is for or is configured for:

[0316] - determining a payload quantity or payload concentration of the nanoparticle vehicles, and / or

[0317] - determining a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or

[0318] - determining a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or determining a nanoparticle vehicle concentration in a solution or formulation.

[0319] A3. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is:

[0320] - a nanoparticle vehicle payload quantity or payload concentration analyzer or measurement / characterization device, and / or

[0321] - a nanoparticle vehicle payload-to-carrier content / concentration ratio analyzer or measurement / characterization device, and / or

[0322] - a nanoparticle vehicle ratio or fractional carrier component content analyzer or measurement / characterization device, and / or

[0323] - a nanoparticle vehicle solution concentration analyzer or measurement / characterization device.

[0324] A4. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, wherein the nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device is, for example, a lipid-based nanoparticle vehicle analyzer or a lipid-based nanoparticle vehicle measurement / characterization device.

[0325] A5. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, wherein the nanoparticle vehicle analyzer or the nanoparticle vehicle measurement / characterization device is a lipid-based drug delivery vehicle analyzer or a lipid-based drug delivery vehicle measurement / characterization device.

[0326] A6. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to the previous clause, wherein the lipid- based drug delivery vehicle analyzer or a lipid-based drug delivery measurement / characterization device is for or is configured for determining a drug load quantity of the lipid-based drug delivery vehicle, or determining a drug load-to-lipidic content / concentration ratio of the lipid-based drug delivery vehicle, or determining a lipidic component ratios of the lipid-based drug delivery vehicle.

[0327] A7. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, wherein the nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device is a lipid-based nanoparticle analyzer or a lipid- based nanoparticle measurement / characterization device for or configured for determining a lipid-based nanoparticle concentration in a solution or in a formulation, or for or configured for determining a lipid-based drug delivery vehicle concentration in a solution or in a formulation.

[0328] A8. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, wherein the lipid-based nanoparticle vehicle comprises or consists of a (solid) lipid nanoparticle and / or a liposome.

[0329] A9. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, further including a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided; wherein when the light path adjuster is in a first configuration, the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is in a transmission mode in which light from the light source follows a first light path from the or one of the light inlet port(s) to the liquid sample such that the light from the light source irradiates the liquid sample directly before the light transmitted by the sample is transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample; and when the light path adjuster is in a second configuration, the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is in the diffusely reflecting mode in which light from the light source follows a second light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid or liquid matrix contained in the liquid sample. A10. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the previous clauses arranged such that, light is transmitted: a. directly from the inlet port onto the wall or walls of the integrating cavity; and / or b. directly from the inlet port, onto and through the sample and subsequently onto the wall or walls of the integrating cavity.

[0330] A11 . The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the previous clauses wherein the inlet port used in the first configuration is directly opposed from the outlet port used in the first configuration such that, when in the first configuration, the first light path extends directly across the integrating cavity.

[0331] A12. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the previous clauses, further comprising the light source.

[0332] A13. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses, further comprising a controller, or a controller configured to control the light path adjuster to selectively adjust the path of light through the apparatus.

[0333] A14. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A13 wherein the controller is an integral part of the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device and is in direct communication with the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device.

[0334] A15. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A13 wherein the controller is remote from the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device and is configured to be in wireless communication with a transceiver of nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device.

[0335] A16. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A13 to A15 wherein the controller is configured to control the spectrometer, and in particular is configured to process the light received by the spectrometer for wavelength analysis of the light to provide extinction and / or absorbance spectrum of the liquid sample contained in the cuvette. A17. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the previous clauses further comprising the spectrometer.

[0336] A18. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A13 to A17 wherein the controller is configured to control one or more of: a. switching between the first and second configurations; b. acquiring spectra from the integrating cavity; c. choosing operating conditions ; d. displaying spectra on a display of the apparatus, or of the controller, or in communication with the apparatus or controller; e. saving data on a memory of the apparatus, or of the controller, or in communication with the apparatus or controller; f. a user-interface of the apparatus, or of the controller, or in communication with the apparatus or controller, that interacts with the apparatus and allows a user to control the position of the light path adjustment mechanism.

[0337] A19. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the light path adjuster comprises at least one movable optical element configured to manipulate light incident on the optical element from the light source, the light path adjuster being configured to adjust the movable optical element to selectively provide the first and second light paths.

[0338] A20. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A19 wherein the optical element is adjustable by moving the optical element with respect to the integrating cavity from a first position in which the light travels along the first light path, and a second position in which the light travels along the second light path.

[0339] A21 . The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A20 wherein the integrating cavity comprises orthogonal longitudinal, vertical, transverse axes, and any one or more of the following positional characteristics of the optical element may be adjusted with respect to any one or more of the axes: a. longitudinal position; b. vertical position; c. transverse position d. orientation; e. inclination.

[0340] A22. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A19 to A21 wherein a plurality of movable optical elements are provided.

[0341] A23. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A19 wherein the movable optical element is selected from: a. a prism; b. a lens; c. a mirror; d. a diffraction gratin e. a fibre optic cable; f. the light source.

[0342] A24. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A19 to A23 wherein the light path adjuster comprises at least one fixed optical element which is not adjustable with respect to the integrating cavity.

[0343] A25. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A24 wherein the fixed optical element is configured to manipulate the light from the light source prior to the light inlet port.

[0344] A26. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A24 or clause A25 wherein the fixed optical element is configured to manipulate the light from the light outlet port.

[0345] A27. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A24 to A26 wherein the fixed optical element is selected from: a. a prism; b. a lens; c. a mirror; d. a diffraction grating; e. a fibre optic cable; f. the light source.

[0346] A28. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the light path adjuster comprises at least one electronic controller operative to effect selective operation of one or more light sources, to selectively provide the first and second light path. A29. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A28 comprising at least first and second light sources, the controller being configured to control each light source independently. A30. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the light path adjuster is positioned between the light source and the light inlet port.

[0347] A31 . The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the light path adjuster is positioned between the spectrometer and the light outlet port.

[0348] A32. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein a plurality of light path adjustment mechanisms are provided.

[0349] A33. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein a plurality of light inlet ports are provided, the light path adjuster being configured to provide the first light path by directing light from the light source through a first light inlet port, and to provide the second light path by directing light from the light source through a second light inlet port.

[0350] A34. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein a plurality of light outlet ports are provided, the first light path directing light from the integrating cavity through a first light outlet port, and the second light path directing light from the integrating cavity through a second light outlet port.

[0351] A35. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the integrating cavity comprises any one of: a. a diffusely reflecting spherical integrating cavity; b. a cylindrical cavity; c. a cuboidal or square cavity.

[0352] A36. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the integrating cavity comprises an internal coating configured to provide any one or more of: a. specular reflectance; b. diffuse reflectance; c. reflectance in the UV light spectrum; d. reflectance in the visible light spectrum; e. reflectance in the infra-red spectrum.

[0353] A37. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the light source comprises any one or more of: a. a quartz-halogen source; b. an LED ; c. a laser; d. any polychromatic source.

[0354] A38. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the shape of the cuvette is one or more of: a. square; b. plate-like; c. cylindrical; d. spherical.

[0355] A39. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses being configured to carry out a UV-VIS spectrometry.

[0356] A40. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses further comprising a sample holder configured to retain a cuvette containing liquid sample within the integrating cavity.

[0357] A41 . The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the preceding clauses wherein the light source comprises first and second LED light sources, and the light path adjuster comprises a controller configured to control the first and second LED light sources such that when in the first configuration, the first LED light source is controlled to provide light on the first light path, and when in the second configuration the second LED light source is controlled to provide light on the second light path.

[0358] A42. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A40 wherein light from each LED light source is delivered to the integrating cavity via a respective fiber optic cable. A43. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of clause A41 or A42 wherein each LED light source delivers light to a respective light inlet port.

[0359] A44. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A41 to A43 wherein each light path delivers light through a respective light outlet port.

[0360] A45. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A41 to A44 wherein the first LED light source is associated with a collimation lens positioned between the first LED light source and the light inlet port associated with that LED light source

[0361] A46. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of clauses A41 to A45 further comprising first and second outlet ports, and a beam splitter configured to selectively allow light from the first and second outlet ports to be transmitted to the spectrometer.

[0362] A47. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the previous clauses wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is: a (solid) lipid nanoparticle drug delivery vehicle drug load quantity analyzer or (solid) lipid nanoparticle drug delivery vehicle drug load quantity measurement / characterization device; or a (solid) lipid nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio analyzer or (solid) lipid nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio measurement / characterization device; or a (solid) lipid nanoparticle drug delivery vehicle lipidic component ratio analyzer or (solid) lipid nanoparticle drug delivery vehicle lipidic component ratio measurement / characterization device.

[0363] A48. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of any one of the previous clauses wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is a liposome drug delivery vehicle analyzer or measurement / characterization device.

[0364] A49. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device of the previous clause wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is: a liposome nanoparticle drug delivery vehicle drug load quantity analyzer or liposome nanoparticle drug delivery vehicle drug load quantity measurement / characterization device; or a liposome nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio analyzer or a liposome nanoparticle drug delivery vehicle drug load-to-lipidic content / concentration ratio measurement / characterization device; or a liposome nanoparticle drug delivery vehicle lipidic component ratio analyzer or liposome nanoparticle drug delivery vehicle lipidic component ratio measurement / characterization device.

[0365] B50. Nanoparticle vehicle analysis or measurement / characterization method comprising:

[0366] - providing the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to anyone of the previous clauses, or providing an apparatus configured to measure or determine at least a scatter-corrected absorbance measurement or a scatter-corrected absorbance spectrum; and / or

[0367] - obtaining / receiving at least one or a plurality of scatter-corrected absorbance measurements / data or scatter-corrected absorbance spectrum measurements / data for a liquid sample or solution containing a plurality of nanoparticle vehicles.

[0368] B51 . Method according to the previous clause, further comprising using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using an extinction coefficient of the payload of the nanoparticle vehicles at an absorption wavelength of interest, a quantity of a loading of the payload of the nanoparticle vehicles of the liquid sample or solution.

[0369] B52. Method according to the previous clause, wherein the Beer-Lambert law is used to determine a payload concentration of the nanoparticle vehicles of the liquid sample or solution as follows: where is the payload concentration, is the obtained or measured scatter- corrected absorbance of the nanoparticle vehicle liquid sample at the absorption wavelength of interest (eventually corrected for any dilution factor applied) is wavelength and is the molar extinction coefficient of the payload at the absorption wavelength of interest.

[0370] B53. Method according to the previous clause B51 or B52, further including applying a spectral deconvolution method to determine an isolated scatter-corrected absorbance or absorbance spectra of the payload and carrier component / fraction respectively if the payload and the carrier component / fraction absorb in the same wavelength regions.

[0371] B54. Method according to clause B51 or B52, further comprising applying a scaling value to the determined payload concentration based on a level or amount of payload capsulation inside the nanoparticle vehicle.

[0372] B55. Method according to clause B50, further comprising using the at least one scatter- corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using an extinction coefficient of the payload and an extinction coefficient of the carrier component of the nanoparticle vehicles at an absorption wavelength of interest, a quantity ratio of the payload to carrier component / fraction of the nanoparticle vehicles of the liquid sample or solution.

[0373] B56. Method according to the previous clause, wherein the Beer-Lambert law is used to determine a payload concentration of the nanoparticle vehicles of the liquid sample or solution as follows: where is the payload concentration, is the obtained or measured scatter- corrected absorbance of the nanoparticle vehicle liquid sample at the absorption wavelength of interest (eventually corrected for any dilution factor applied) is wavelength and is the molar extinction coefficient of the payload at the absorption wavelength of interest; and wherein the Beer-Lambert law is used to determine the carrier component / fraction concentration of the nanoparticle vehicles of the liquid sample or solution as follows: where cNPVis the carrier component / fraction concentration of the nanoparticle vehicle, 4(A)SCis the obtained or measured scatter-corrected absorbance of the nanoparticle vehicle liquid sample at the absorption wavelength of interest (eventually corrected for any dilution factor applied), A is wavelength and 6NPK(A) is the molar extinction coefficient of the carrier component / fraction of the nanoparticle vehicle at the absorption wavelength of interest.

[0374] B57. Method according to the previous clause B55 or B56, further including applying a spectral deconvolution method to determine an isolated scatter-corrected absorbance or absorbance spectra of the payload and carrier component / fraction respectively if the payload and the carrier component / fraction absorb in the same wavelength regions. B58. Method according to clause B55 or B56, further comprising applying a scaling value to the determined payload concentration based on a level or amount of payload capsulation inside the nanoparticle vehicle.

[0375] B59. Method according to clause B55 or B56, further comprising determining a quantity ratio of the payload to carrier component / fraction of the nanoparticle vehicles of the liquid sample or solution by dividing by

[0376] B60. Method according to clause B50, further comprising using the at least one scatter- corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using extinction coefficients of constituent carrier elements of the carrier component / fraction of the nanoparticle vehicle, a quantity ratio of the constituent carrier elements of the carrier component / fraction of the nanoparticle vehicles.

[0377] B61 . Method according to the previous clause, wherein the Beer-Lambert law is used to determine, for each constituent carrier element of the carrier component / fraction of the nanoparticle vehicle, a constituent carrier element concentration of the carrier component / fraction of the nanoparticle vehicle of the liquid sample or solution as follows: where cfis a constituent carrier element concentration, A(A)SCis the obtained or measured scatter-corrected absorbance of the nanoparticle vehicle liquid sample at the absorption wavelength of interest (eventually corrected for any dilution factor applied), A is wavelength and 6^(2) is the molar extinction coefficient of the constituent carrier element of the carrier component / fraction of the nanoparticle vehicle at the absorption wavelength of interest. B62. Method according to clause B50, further comprising using the at least one scatter- corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data and a measured or determined nanoparticle vehicle size to determine a nanoparticle vehicle concentration of the nanoparticle vehicles in the liquid sample or solution.

[0378] B63. Method according to the previous clause, wherein the Beer-Lambert law is used to determine a payload concentration of the nanoparticle vehicles of the liquid sample or solution as follows: where cpayioadis the payload concentration, is the obtained or measured scatter- corrected absorbance of the nanoparticle vehicle liquid sample at the absorption wavelength of interest (eventually corrected for any dilution factor applied), A is wavelength and is the molar extinction coefficient of the payload at the absorption wavelength of interest; and wherein the Beer-Lambert law is used to determine the carrier component / fraction concentration of the nanoparticle vehicle of the liquid sample or solution as follows: where cNPVis the carrier component / fraction concentration, is the obtained or measured scatter-corrected absorbance of the nanoparticle vehicle liquid sample at the absorption wavelength of interest (eventually corrected for any dilution factor applied), A is wavelength and is the molar extinction coefficient of the carrier component / fraction of the nanoparticle vehicle at the absorption wavelength of interest.

[0379] B64. Method according to the previous clause B62 or B63, further including applying a spectral deconvolution method to determine an isolated scatter-corrected absorbance or absorbance spectra of the payload and carrier component / fraction respectively if the payload and the carrier component / fraction absorb in the same wavelength regions. B65. Method according to clause B62 or B63, further comprising providing constituent carrier element fraction values of the carrier component / fraction of the nanoparticle vehicle, and determining a number of molecules in the carrier component / fraction of the nanoparticle vehicle using the a measured or determined nanoparticle vehicle size.

[0380] B66. Method according to any one of the previous clauses B62 to B65, further determining the particle concentration cP[particles per mL] according to where N is the lipid fraction per particle, in moles per particle.

[0381] B67. Method according to clause B50, further comprising using at least one scattering spectrum or scattering spectrum data and measured or determined nanoparticle vehicle size to determine a nanoparticle vehicle concentration of the nanoparticle vehicles in the liquid sample or solution.

[0382] B68. Method according to clause B57, further comprising

[0383] - obtaining at least one or a plurality of extinction measurements / data or extinction spectrum measurements / data for the liquid sample or solution containing the plurality of nanoparticle vehicles; - determining at least one scattering spectrum or scattering spectrum data using (i) the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data and (ii) the determined at least one extinction measurement / data or extinction spectrum measurement / data;

[0384] - determining a wavelength-dependent scattering cross section, oscat, of the nanoparticle vehicles using a measured or determined nanoparticle vehicle size and wavelength-dependent dielectric function of the particle components;

[0385] - determining a scattering spectrum (Optical Density O.D) of the particles according to O. Dtheo= oscat* cP, where cPis the particle concentration [moles per liter],

[0386] B69. Method according to the previous clause, further including

[0387] - converting cP[moles per liter] to cP[particles per mL] by cP[particles per mL] = cP[moles per liter]* N * 0.001, where N = Avogadro’s number.

[0388] B70. Method according to any one of the previous clauses, wherein the nanoparticle vehicles are lipid-based nanoparticle vehicles, and the carrier component / fraction of the nanoparticle vehicle comprises at least one lipid, and the constituent carrier element comprises at least one lipid.

[0389] B71 . Method according to the previous clauses, wherein the payload of the lipid-based nanoparticle vehicles comprises an oligonucleotide such as mRNA, siRNA, DNA.

[0390] B72. Method according to any one of the previous clauses, wherein the nanoparticle vehicles are lipid-based drug delivery vehicles.

[0391] B73. Method according to any one of the previous clauses, wherein the nanoparticle vehicles comprises or consist of a (solid) lipid nanoparticles and / or liposomes.

[0392] C74. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of clauses A1 to A49, further including at least one processor or calculation means; and at least one memory or storage means including a computer program comprising instructions which, when the computer program is executed by the processor or calculation means, cause the analyzer or measurement / characterization device to carry out the steps of the method according of any one of clauses B50 to B73.

[0393] C75. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to the previous clause, wherein the controller includes the at least one processor or calculation means; and the at least one memory or storage means including the computer program. D76. A computer program comprising instructions which, when the program is executed by a computer, processor or calculation means cause the computer, processor or calculation means to carry out the method according of any one of clauses B50 to B73.

[0394] E77. A computer-readable data carrier having stored thereon said computer program according to the previous clause.

[0395] F78. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, wherein the nanoparticle vehicle comprises a polymeric nanoparticle or an inorganic nanoparticle.

[0396] F79. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to the previous clause, wherein the polymeric nanoparticle comprises a polymeric nanocapsule or nanosphere, a polymersome, a dendrimer, or a polymer micelle.

[0397] F80. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to the previous clause B78, wherein the inorganic nanoparticle comprises a silica nanoparticle, a quantum dot, an iron-based or iron oxide nanoparticle, or a gold nanoparticle.

[0398] F81 . The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any one of the previous clauses, wherein the nanoparticle vehicle comprises an exosome, a microvesicle, a lentivirus, an adeno-associated virus (AAV) or an adenovirus.

[0399] G82. The nanoparticle vehicle analysis or measurement / characterization method according to any one of the previous clauses, wherein the nanoparticle vehicle comprises a polymeric nanoparticle or an inorganic nanoparticle.

[0400] G83. The nanoparticle vehicle analysis or measurement / characterization method according to the previous clause, wherein the polymeric nanoparticle comprises a polymeric nanocapsule or a polymeric nanosphere, or a polymersome, or a dendrimer, or a polymer micelle.

[0401] G84. The nanoparticle vehicle analysis or measurement / characterization method according to the previous clause G82, wherein the inorganic nanoparticle comprises a silica nanoparticle, a quantum dot, an iron-based or iron oxide nanoparticle, or a gold nanoparticle.

[0402] G85. The nanoparticle vehicle analysis or measurement / characterization method according to any one of the previous clauses, wherein the nanoparticle vehicle comprises an exosome, a microvesicle, a lentivirus, an adeno-associated virus (AAV) or an adenovirus. ANNEX

[0403] Content of International Patent Application No. PCT / NZ2017 / 050131 published as WO2018 / 070882 A1.

[0404] A SPECTROMETER APPARATUS FOR MEASURING SPECTRA OF A EIQUID SAMPEE USING AN INTEGRATING CAVITY

[0405] Field of the Invention

[0406] This invention relates to a spectrometer apparatus for measuring spectra of a liquid sample using an integrating cavity and in some embodiments, the invention relates to a UV-vis spectrometer apparatus for measuring turbid liquids.

[0407] Background

[0408] Standard UV-VIS spectroscopy is performed by shining a light source through a sample and measuring the transmitted light as a function of wavelength. The sample is generally a liquid that is contained within a square cuvette placed with the cuvette faces being perpendicular to the light beam. The transmitted light is then converted into an absorption spectrum which gives a measure of the absorbing power of the sample at every wavelength used. Absorbance can be used as a measure of the concentration of dissolved species (absorbance is proportional to concentration, known as the Beer-Lumbert Lnw) or to identify the chemical content of a solution based on absorbance peaks of species at known wavelengths.

[0409] UV-VIS spectrometers are a standard instrument in analytical chemistry and can be used for both quantitative and qualitative analysis of liquids. UV-VIS spectrometers measure the spectrum of light directly transmitted by the sample, and determine the absorption spectrum based on the assumption that the only loss of light occurs due to absorption in the sample. This leads to the general requirement of brilliantly clear sample liquids in UV-VIS spectrometers.

[0410] In the more general case including turbid liquids, light is lost due to scattering by the sample, and UV-VIS spectrometers will measure the extinction spectrum instead of the absorption spectrum. In short: extinction=scattering+ absorption.

[0411] The intensity of light scattered generally is wavelength dependent, leading to a scattering spectrum. In UV-VIS spectrometers absorption and scattering spectra are superimposed and cannot be disentangled without separate knowledge of one of the two constituent spectra. In strongly scattering liquids (e.g. milk, paint, blood, wine) the light reaching the detector is diminished to a degree which renders the absorption spectrum component virtually indiscernible from the measured extinction spectrum, even if the scattering spectrum was known. For scattering / turbid samples standard UV-VIS is therefore of very limited general applicability, and if used, nonetheless requires sample pre-processing (e.g. filtration, centrifugation or other methods to remove the scattering species). Dilution of the sample is generally not helpful because it reduces both scattering and absorbance of the sample in the same proportion. In summary, there is a significant range of samples where UV-VIS either does not work or timeconsuming processing is required in order to allow analysis of cloudy solutions. Moreover, it can be impossible to separate out the relative contribution of scattering and absorption using standard UV-VIS spectroscopy.

[0412] Object of the Invention

[0413] It is therefore an object of the invention to provide a spectrometer apparatus which overcomes or at least ameliorates one or more disadvantages of the prior art, or alternatively to at least provide the public with a useful choice.

[0414] Further objects of the invention will become apparent from the following description.

[0415] Summary of Invention

[0416] Accordingly in one aspect the invention may broadly be said to consist in a spectrometer apparatus for measuring spectra of a liquid sample, the apparatus comprising: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette containing liquid sample within the integrating cavity, wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the or each light inlet port being configured to receive light from a light source and the or each light outlet port being configured to deliver light to a spectrometer; the apparatus further comprising a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided; wherein when the light path adjuster is in a first configuration, the apparatus is in a transmission mode in which light from the light source follows a first light path from the or one of the light inlet port(s) to the liquid sample such that the light from the light source irradiates the liquid sample directly before the light transmitted by the sample is transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample; and when the light path adjuster is in a second configuration, the apparatus is in a diffusely reflecting mode in which light from the light source follows a second light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the liquid sample.

[0417] Such a spectrometer apparatus may in particular be used to obtain spectra being the absorption and extinction spectra of the sample, whereby using a suitable calibration procedure implemented by one or more electronic data processors yields absorbance and extinction spectra that are defined for a given path length through the sample,

[0418] By providing an apparatus which can be used in each of the above configurations it is possible to obtain quantitative spectra where the path length of light through the sample in each configuration is well defined so that the data obtained in each configuration are relatable.

[0419] The apparatus may be configured such that, when in the second configuration, light from the second light path is transmitted: a) directly from an inlet port onto the wall or walls of the integrating cavity; and / or b) directly from an inlet port, onto and through the sample and subsequently onto the wall or walls of the integrating cavity.

[0420] Thus, when in the second configuration, the second light path may be transmitted from the inlet port either first through the sample or directly onto the cavity wall or walls. With either variant, the apparatus is configured such that the outlet port that is used in the second configuration does not look at the inlet port. In other words, the outlet port used in the second configuration “faces” the walls of the integrating cavity. An outlet port for example can be at 90° to an inlet port, or any other position on the integrating cavity. The relative position of the inlet port and outlet port used in the second configuration is such that the spectrometer does not collect the incident light or the light directly transmitted from the sample.

[0421] Preferably, when in the first configuration the inlet port is directly opposed from the outlet port such that , the first light path extends directly across the integrating cavity.

[0422] In another aspect of the invention there is provided a spectrometer apparatus for measuring spectra of a liquid sample, in particular where the spectra obtained are the absorption and extinction spectra of the sample, the apparatus comprising: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette containing liquid sample within the integrating cavity, wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the light inlet port being configured to receive light from a light source and the light outlet port being configured to deliver light to a spectrometer; the apparatus further comprising a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided; wherein when the light path adjuster is in a first configuration, the apparatus is in a transmission mode in which light from the light source follows a first light path from the light inlet port to the liquid sample such that the light from the light source irradiates the liquid sample directly before the light transmitted by the sample is collected via the light outlet port positioned directly opposite the inlet port and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample; and when the light path adjuster is in a second configuration, the apparatus is in a diffusely reflecting mode in which light from the light source follows a second light path from the inlet port into the integrating cavity, and is incident onto either the reflective inner wall or walls of the integrating cavity or directly onto the liquid sample; wherein the light transmitted and / or scattered by the sample is transmitted through the outlet port, the apparatus being configured such that light directly transmitted and / or reflected by the sample is reflected by the inner wall or walls of the cavity before being transmitted through the outlet port, and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the liquid sample.

[0423] Preferably, using a suitable calibration procedure yields absorbance and extinction spectra that are defined for a given path length through the sample,

[0424] A preferred implementation of the second configuration is to position the outlet port such that it directly faces an area of the cavity wall that the light from the inlet port does not directly illuminate.

[0425] The apparatus, used in both configurations and with a suitable calibration procedure, yields both the extinction and absorption spectrum of the liquid sample, where the path length through the sample in both said configurations is well defined, such that the spectra obtained give wavelength-dependent extinction and absorption coefficients of the sample respectively across the wavelength range of the light illuminating the sample.

[0426] The apparatus may comprise one or more integral light source(s), or the light source may be configured to be connected to one or more separate light source(s).

[0427] The apparatus may further comprise an integral or remote controller configured to control the light path adjuster to selectively adjust the path of light through the apparatus.

[0428] The controller is preferably configured to control the spectrometer, and in particular is configured to process the light received by the spectrometer for wavelength analysis of the light to provide the extinction and / or absorbance spectrum of the liquid sample contained in the cuvette. The spectrometer may be integral with the apparatus.

[0429] The controller or controllers may be configured to control one or more of: a) switching between the first and second configurations; b) acquiring spectra from the integrating cavity; c) choosing operating conditions; d) displaying spectra on a display of the apparatus, or of the controller, or in communication with the apparatus or controller; e) saving data on a memory of the apparatus, or of the controller, or in communication with the apparatus or controller; f) a user-interface of the apparatus, or of the controller, or in communication with the apparatus or controller, that interacts with the apparatus and allows a user to control the position of the light path adjustment mechanism.

[0430] The light path adjuster may comprise at least one movable optical element configured to manipulate light incident on the optical element from the light source, the light path adjuster being configured to adjust the movable optical element to selectively provide the first and second light paths.

[0431] The optical element may be adjustable by moving the optical element with respect to the integrating cavity from a first position in which the light travels along the first light path, and a second position in which the light travels along the second light path.

[0432] The integrating cavity comprises orthogonal longitudinal, vertical, transverse axes, and any one or more of the following positional characteristics of the optical element may be adjusted with respect to any one or more of the axes: a) longitudinal position; b) vertical position; c) transverse position d) orientation; e) inclination.

[0433] A plurality of movable optical elements may be provided.

[0434] The movable optical element is preferably selected from any one or combination of: a prism; a lens; a mirror; a diffraction grating; a fibre optic cable; the light source; a shutter.

[0435] The light path adjuster may additionally or alternatively comprise at least one fixed optical element which is not adjustable with respect to the integrating cavity. The fixed optical element may be configured to manipulate the light from the light source prior to the light inlet port. The fixed optical element may be configured to manipulate the light from the light outlet port.

[0436] The fixed optical element may be selected from any one or combination of: a) a prism; b) a lens; c) a mirror; •0 a diffraction grating; e) a fibre optic cable; f) the light source.

[0437] The light path adjuster may comprise at least one electronic controller operative to effect selective operation of one or more light sources, to selectively provide the first and second light path.

[0438] The apparatus may comprise at least first and second light sources, the controller being configured to control each light source independently. The light sources could be switched on and off in a blinking or sequential fashion wherein in configuration one the first light source is switched on and in configuration two the second light source is on with the first off. The light sources may be controlled such that both or all light sources can be switched off, to acquire a dark spectrum.

[0439] The light path adjuster may be positioned: a) between the light source and the light inlet port and / or b) between the spectrometer and the light outlet port.

[0440] A plurality of light path adjustment mechanisms may be provided.

[0441] A plurality of light inlet ports may be provided, the light path adjuster being configured to provide the first light path by directing light from the light source through a first light inlet port, and to provide the second light path by directing light from the light source through a second light inlet port.

[0442] A plurality of light outlet ports may be provided, the first light path directing light from the integrating cavity through a first light outlet port, and the second light path directing light from the integrating cavity through a second light outlet port.

[0443] The integrating cavity may comprise any one of: a) a diffusely reflecting spherical integrating cavity; b) a cylindrical cavity; c) a cuboidal or square cavity.

[0444] It will be appreciated that the integrating cavity may be any other shape or combination of shapes.

[0445] The integrating cavity may comprise an internal coating configured to provide any one or more of: a) specular reflectance; b) diffuse reflectance; c) reflectance in the UV light spectrum; d) reflectance in the visible light spectrum; e) reflectance in the infra-red spectrum.

[0446] The light source may comprise any one or more of: a) a quartz-halogen source; b) an LED; c) a laser; d) any polychromatic source.

[0447] The shape of the cuvette may be: a) square; b) plate-like; c) cylindrical; d) spherical;

[0448] The apparatus may be a UV-VIS spectrometer apparatus.

[0449] The apparatus may further comprise a sample holder configured to retain a cuvette containing liquid sample within the integrating cavity.

[0450] The light source may comprise first and second LED light sources, and the light path adjuster comprises a controller configured to control the first and second LED light sources such that when in the first configuration, the first LED light source is controlled to provide light on the first light path, and when in the second configuration the second LED light source is controlled to provide light on the second light path.

[0451] Light from each LED light source may be delivered to the integrating cavity via a respective fibre optic cable. Each LED light source may deliver light to a respective light inlet port. Each light path delivers light through a respective light outlet port.

[0452] The first LED light source may be associated with a collimation lens positioned between the first LED light source and the light inlet port associated with that LED light source

[0453] The apparatus may further comprise first and second outlet ports, and a beam splitter configured to selectively allow light from the first and second outlet ports to be transmitted to the spectrometer.

[0454] According to another aspect of the invention there is provided a spectrometer apparatus for measuring spectra of a liquid sample, the apparatus comprising: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette containing liquid sample within the integrating cavity, wherein the integrating cavity comprises a first light inlet port and a second light inlet path at least one light outlet port, the first light inlet port being configured to receive light from a first LED light source and the second light inlet port being configured to receive light from a second LED light source, at least one light outlet port being provided and configured to deliver light to a spectrometer; the apparatus further comprising a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided; wherein when the light path adjuster is in a first configuration, the apparatus is in a transmission mode in which light from the first LED light source follows a first light path from the first light inlet port to the liquid sample such that the light from the first LED light source irradiates the liquid sample directly before the light transmitted by the sample is transmitted through the light outlet port and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample; and when the light path adjuster is in a second configuration, the apparatus is in a diffusely reflecting mode in which light from the second LED light source follows a second light path from the second inlet port into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the, or another, light outlet port and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the liquid sample.

[0455] The spectrometer apparatus may be configured to measure spectra of a liquid sample selected from any one or more of the following: a. Water; b. Wine; c. A beverage; d. An edible liquid or partially liquid product.

[0456] According to a further aspect of the invention there is provided a method of measuring spectra of a liquid sample using the apparatus of any of the other aspects of the invention, comprising steps of: a. activating the light source; b. controlling the light path adjuster to be in the transmission mode or the diffusely reflecting mode; and c. conducting wavelength analysis of the light transmitted through the light outlet port via the spectrometer for wavelength analysis of the light to provide an absorbance and / or extinction spectrum of the liquid sample contained in the cuvette.

[0457] Detailed Description of the Drawings

[0458] A number of embodiments of the invention will now be described by way of example with reference to the drawings in which:

[0459] Figure 1 is a schematic view of example components of a spectrometer apparatus in accordance with the invention;

[0460] Figures 2a and 2b are schematic views of a first embodiment of a spectrometer apparatus in accordance with the invention, in first and second configurations;

[0461] Figures 3a and 3b are schematic views of a second embodiment of a spectrometer apparatus in accordance with the invention, in first and second configurations;

[0462] Figure 4 is a schematic view of a third embodiment of a spectrometer apparatus in accordance with the invention, simultaneously illustrating first and second configurations of the apparatus;

[0463] Figure 5 is a schematic view of a fourth embodiment of a spectrometer apparatus in accordance with the invention, simultaneously illustrating first and second configurations of the apparatus;

[0464] Figure 6 is a schematic view of a fifth embodiment of a spectrometer apparatus in accordance with the invention, simultaneously illustrating first and second configurations of the apparatus; and

[0465] Figure 7 is a schematic view of a sixth embodiment of a spectrometer apparatus in accordance with the invention, simultaneously illustrating first and second configurations of the apparatus.

[0466] Detailed Description

[0467] Throughout the description like reference numerals will be used to refer to like features in different embodiments.

[0468] With reference to Figure 1 , a spectrometer apparatus 1 for measuring spectra of a liquid sample is provided which is configured to be able to measure multiple optical properties of a liquid sample, of which the properties are the wavelength dependent extinction and absorption coefficients of the liquid.

[0469] The apparatus 1 comprises an integrating cavity 3 comprising reflective inner walls 5, and configured to retain a cuvette 7 containing liquid within the integrating cavity 3, with light from a light source 9 being delivered into the cavity 3 via different light paths 15, 17 entering the cavity 3, the different light paths 15 , 17 being selectively adjustable via a light path adjuster 13. The light path adjuster 13 is used to deliver the light into the cavity 3 through at least one inlet port Pl , P2 along different paths depending on the configuration of the light path adjuster 13.

[0470] The apparatus 1 further comprises at least one light outlet port P3, P4 configured to deliver light to a spectrometer 1 1. In some examples, an output light path adjuster 13B is provided that controls the path of light from the integrating cavity 3 to the spectrometer 1 1.

[0471] In the first configuration, the apparatus 1 is in a transmission mode, where the input path adjuster 13 is positioned such that the light from the light source 9 entering the cavity 3 through an inlet port Pl so as to directly illuminate the liquid contained in the cuvette 7 and the outlet light path adjuster 13B is configured such that the light collected through an outlet port P3, and sent to the spectrometer 1 1 so that a proportion of light from the light source 9 is directly transmitted by the sample after illuminating the sample. In this configuration, the extinction spectrum of the sample is obtained.

[0472] In the second configuration, the apparatus 1 is in a diffusely reflecting mode, where the inlet light path adjuster 13 is positioned such that the light from the light source 9 entering the cavity 3 through an inlet port P2 can either directly illuminate the liquid contained in the cuvette 7 or can be incident on the cavity wall 5 and be diffusely reflected within the cavity 3 before interacting with the liquid sample. Furthermore in this second configuration, the outlet light path adjuster 13B is configured such that the light transmitted and / or reflected by the sample and collected through outlet port P4 and sent to the spectrometer 1 1 has undergone at least one reflection from the cavity walls 5 before entering the outlet port P4. In this configuration, the absorption spectrum of the sample is obtained, free from the effects of scattering by the liquid sample.

[0473] The means of switching between configuration modes is provided by one or more electronic controllers that select the configuration of both the inlet light path adjuster 13 and the outlet light path adjuster 13B (if provided), to obtain either the extinction or absorption spectrum of the liquid sample depending on the configuration mode that is selected.

[0474] The apparatus 1 , and method of use of the apparatus 1 , allows the measurement of the extinction and absorption spectrum of a liquid sample using a single apparatus and without movement of the liquid sample.

[0475] Referring now to Figures 2a, 2b, a first embodiment of a spectrometer apparatus 1 for measuring spectra of a liquid sample comprises an integrating cavity 3 comprising a reflective inner wall or walls 5, and configured to retain a cuvette 7 containing liquid sample within the integrating cavity 3. The integrating cavity 3 comprises at least one light inlet port Pl , P2 and at least one light outlet port P3, P4, the light inlet port(s) Pl , P2 being configured to receive light from a light source 9 and the light outlet port(s) P3, P4 being configured to deliver light to a spectrometer 1 1.

[0476] The apparatus 1 further comprises a light path adjuster 13 configured to selectively adjust a path of light through the integrating cavity 3 such that at least two distinct light paths 15, 17 are provided.

[0477] When the light path adjuster 13 is in a first configuration, the apparatus 1 is in a transmission mode in which light from the light source 9 follows a direct light path 15 from the, or one of the, light inlet ports Pl , to the liquid sample such that the light from the light source 9 irradiates the liquid sample directly before being transmitted through the, or one of the, light outlet ports P3, P4 and received by the spectrometer 11 for wavelength analysis of the light to provide an extinction spectrum of the liquid sample in the cuvette 7.

[0478] When the light path adjuster 13 is in a second configuration, the apparatus 1 is in a diffusely reflecting mode in which light from the light source 9 follows a light path 17 from the, or one of the, inlet ports Pl , P2 into the integrating cavity 3, and is either: a) incident directly onto the reflective inner wall or walls 5 of the integrating cavity 3 and is diffusely reflected within the integrating cavity 3, such that the light from the light source 9 irradiates the liquid sample indirectly; or b) incident directly (not shown) onto the liquid sample 7 such that the light from the light source 9 irradiates the liquid sample directly and the light transmitted and / or reflected by the sample is diffusely reflected within the integrating cavity

[0479] The light is subsequently transmitted through the, or one of the, light outlet ports P3, P4 and received by the spectrometer 1 1 for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the cuvette 7.

[0480] The apparatus 1 , and method of use of the apparatus, allows the measurement of the extinction and absorption spectrum of a liquid sample using a single apparatus and without movement of the liquid sample. The method consists of placing a liquid sample, which may be contained in a standard 1 cm square cuvette 7, in an integrating cavity 3 and delivering light to the sample either in a transmission or diffusely reflecting configuration. In the first configuration, the light transmitted by the sample is sent to a spectrometer 1 1 and an extinction spectrum is obtained, while in the second configuration light is diffusely reflected within the cavity 3 and interacts with the sample, so that the light scattered by the sample is not lost. In the second configuration the light may initially interact with the sample, or be incident directly on the walls of the cavity. The spectrum collected by the spectrometer 1 1 in the second configuration can then be related to the absolute absorption spectrum with suitable calibration and modelling. Switching between measurement configurations is provided via one or more adjustable optical elements L1 -L5, Ml - M4, configured to manipulate the light from the light source 9 prior to the light entering the integrating cavity 3. Such optical elements can comprise one or more shutters and / or moveable mirrors that control the light path through the integrating cavity 3, and as such allow both the extinction and absorption spectrum of the liquid to be obtained using a single apparatus 1.

[0481] The apparatus 1 suspends or supports a sample cuvette 7 within an integrating cavity 3, whereby the latter has a specific light inlet / outlet port configuration which, in combination with one or more optical elements, allows two distinct light-paths to be provided through the integrating cavity 3 between the light source 9 and spectrometer 1 1 , and in particular the light detector of or connected to such a spectrometer.

[0482] The skilled person will appreciate that the first and second light paths through the integrating cavity 3 may be provided in a number of different ways, and by varying one or more of at least the following: a. The number of, and / or position of inlet ports; b. The number of, and / or position of outlet ports; c. The number of, and / or position of, and / or type of, movable optical elements; d. The number of, and / or position of, and / or type of any auxiliary fixed optical elements that may be used; e. The relative position of the integrating cavity with respect to the light source and / or the spectrometer. In practice the use of the apparatus 1 provides one or more of the following advantages:

[0483] • A method for performing standard UV-VIS measurements as in any other device available on the market with standard cuvettes.

[0484] • The ability to switch to an absorbance mode to remove any effects of scattering.

[0485] • Retrieval of both the extinction and absorbance spectra immediately, from the perspective of the user.

[0486] • Measurement of absorption and extinction spectra in a single instrument and without user intervention.

[0487] • Convenient sample replacement through a cavity port, akin to replacement in a standard UV-VIS instrument

[0488] • Provides a means to determine the absolute absorbance of turbid / scattering media

[0489] Different Inlet Ports

[0490] With reference to the first example of Figures 2a and 2b, light is transmitted from the light source 9 into the integrating cavity 3 along first light path 15 through one of two light inlet ports P, P2. When the apparatus 1 is in the first configuration, light enters through first light inlet port Pl , and is directly incident on the liquid sample in the cuvette 7. The light transmitted by the liquid sample is collected via first light outlet port P3 and is processed in the same way a standard UV- VIS measurement would be done, by measuring the wavelength dependent extinction spectrum of the sample which determines the wavelength dependent extinction coefficient of the sample.

[0491] In the second configuration, the light from the light source is sent through P2 along second light path 17 and is directly incident on the reflective walls 5 of the cavity 3 first. The surface of the walls 5 of the cavity 3 is , to a good approximation, a perfect diffuse reflector (lambertian surface). The incident light thus spreads diffusely in the cavity 3 and illuminates and interacts with the sample. Light may be absorbed by the sample, but light scattered by the sample remains part of the diffuse illumination present in the cavity 3.

[0492] In the second configuration, the light is then collected via second light outlet port P4 that is specifically positioned such that as much as possible of the light directly transmitted or reflected by the sample does not enter outlet port P4 before it is reflected from the cavity walls 5, and is processed by the spectrometer 1 1 , allowing the true absorbance spectrum of the sample to be determined, without spectral light loss due to scattering. Switching between extinction and absorbance modes is done via the light path adjuster without needing to change the sample position or any other optics of the apparatus.

[0493] The light path adjuster 13 thus adjusts the light received by the integrating cavity 3 from the light source 9 to provide a first light path 15 in which light is directly incident in the liquid sample and not on the walls 5 of the cavity 3, and a second light path 17 in which light is directly incident on the walls 5 of the cavity 3 but not on the liquid sample. In the example of Figures l a, lb, the light path adjuster 13 comprises optical elements in the form of two transversely spaced part, angled set of inlet mirrors Ml , M2 between the light source 9 and cavity 3, and a corresponding pair of transversely spaced part, angled set of outlet mirrors M3, M4 between the cavity 3 and the spectrometer 1 1. In this example, the cavity 3 comprises two transversely space apart light inlet ports Pl, P2, and comprises two transversely space apart light outlet ports P3, P4. In this example, a plurality of lens L1 -L5 are provided in different positions along the first and second light paths 15, 17. The light path adjuster also comprises a movable shutter S I configured to open and close first outlet port P3.

[0494] One inlet mirror Ml and one outlet mirror M4 are both movable along the transverse axis of the cavity 3, whilst second inlet mirror M2 and second outlet mirror M3 are fixed and not movable. In the first configuration, both sets of mirrors are in a position in which they do not impede a notional path from the light source 9, first inlet port Pl , the liquid sample, and the first outlet port P3. In this position light from the light source 9 is transmitted along a direct light path 15 and is directly incident on the liquid sample.

[0495] In parallel, when mirror Ml is out of the first light path 15, shutter S I is simultaneously open, allowing light transmitted by the sample to exit the cavity 3 from the extinction light outlet port P3. Moveable outlet mirror M3 is also simultaneously positioned out of the first light path 15 such that the light exiting P3 can be focused directly onto the spectrometer 1 1 via lens L5.

[0496] In configuration 2, the moveable inlet mirror Ml is placed in the light path between the light source 9 and the first inlet port Pl, with mirror Ml being positioned at 45° to the light path such that the light is directed to the fixed mirror M2 which consequently allows the light to be focused into the absorption light inlet port P2 via the focusing lens L2. In this configuration, the light is incident directly onto the interior wall 5 of the cavity 3 and is diffusely reflected within the cavity 3. The light within the cavity 3 is then collected via the outlet port P4 using lens L4 and sent to the spectrometer. Light is prevented from exiting the cavity 3 via the first outlet port P3 because this has been closed by movable shutter S I .

[0497] In practice the use of the apparatus 1 provides one or more of the advantages stated above.

[0498] The apparatus 1 may comprise, or be in communication with, an electronic controller / software configured to perform the measurement i.e. reference and sample measurement, acquisition time, integration time and display of obtained extinction, absorbance and scattering spectra.

[0499] Same Inlet Port

[0500] Referring now to Figure 3a and 3b, a second embodiment of apparatus 1 is provided with like features being given like references. In this example, the apparatus 1 is similar to that of Figure 2, but a single light inlet port Pl is provided. The light path adjuster 13 comprises a combined pinhole-lens system comprising pinhole PN1 and focusing lens L2 placed between the light source 9 and the inlet port Pl and the moveable shutter S I placed after the outlet ports P3 and P4. In the first configuration, shown in Figure 3b the light path adjuster 13 is configured such that such that pinhole PN1 is aligned with the incoming light path and the light entering the inlet port Pl is essentially collimated and in this position light from the light source 9 is transmitted along a direct light path 15 and is directly incident on the liquid sample. In parallel, when pinhole PN1 is in the light path, shutter S2 is simultaneously closed allowing light transmitted by the sample to exit the cavity 3 from the extinction light outlet port P3. Moveable outlet mirror M3 is also simultaneously positioned out of the first light path 15 such that the light exiting P3 can be focused directly onto the spectrometer 1 1 via lens L5.

[0501] In configuration 2, the light path adjuster is 13 is positioned such that the input pinhole PN1 is out of the light path and the focusing lens L2 is in the light path and the incident light from the light source 9 is focused onto the inlet port P l such that the light is transmitted along a direct light path to the sample but because it has been focused to a point at the inlet port position, the light is divergent such that the light illuminates the entire transverse width of the sample cuvette. In parallel, when focusing lens L2 is in the light path, the shutter S2 is simultaneously open, covering the outlet port P3 with moveable mirror M4 positioned at 45° to the light path, In this configuration, light scattered, transmitted and reflected by the sample is diffusely reflected within the cavity 3 which then allows light that has been diffusely reflected within the cavity 3 to exit the cavity 3 from the absorption outlet port P4. This light is then collected via the outlet port P4 using lens L4 and sent to the spectrometer via mirror M3 and moveable mirror M4.

[0502] Shutter Selection Avoiding Sample

[0503] Referring now to Figure 4, a third embodiment of apparatus 1 is provided with like features being given like references. In this example, the movable inlet mirror Ml has been replaced by an inlet shutter S2, and a second fixed inlet mirror Ml . The outlet mirrors M3, M4 are together transversely movable from a position as shown in Figure 2 in which angled outlet mirror M3 is in the light path of outlet port P3 so as to direct light from first light path 15 onto second outlet mirror M4 and onto spectrometer 1 1. Outlet shutter S I comprises a shutter aperture which is aligned with outlet port P3 in this first configuration. Inlet shutter S2 comprises a pair of transversely spaced apart shutter apertures. In the first configuration the shutter S2 is positioned such that one of the shutter apertures is aligned with inlet port P l, but with inlet port P2 closed. Angled, fixed inlet mirrors Ml , M2 direct light to inlet port P l .

[0504] In the second configuration inlet shutter S2 is moved transversely such that inlet port Pl is closed and inlet port P2 aligned with one of the inlet shutter S2 apertures such that light from light source 9 is transmitted directly into inlet port P2. Outlet mirrors M3, M4 are moved transversely so that mirror M3 is not in the light path between outlet port P4 and spectrometer 1 1.

[0505] Shutter Selection Straight Through Sample

[0506] With reference to Figure 5, fourth embodiment of apparatus 1 is provided with like features being given like references. In this example, the apparatus 1 is similar to that of Figure 4, but no inlet mirrors are provided. The inlet shutter S2 is provided adjacent an inlet lens L6. Transverse adjustment of the position of the inlet shutter S2 aligns one or other shutter aperture with the inlet lens L6 and the light source. One inlet shutter aperture is relatively small, and the other is relatively large. By adjusting which aperture is aligned with the light source, in combination with lens L6, it is possible for both light paths 15, 17 to be directly incident on the liquid sample, with the first light path passing through the sample and exiting the cavity via outlet port P3, and the second light path also passing through the liquid sample but diffusing into contact with the walls 5 of the cavity 3 before exiting cavity 3 via second outlet port P4, when outlet shutter S I closes first outlet port P3.

[0507] Referring now to Figure 6, a fifth embodiment of apparatus 1 is provided with like features being given like references. In this example, the apparatus 1 is similar to that of Figure 3a and 3b but the manipulation of the optical path for two different configurations is provided via off-axis parabolic (OAP) mirrors instead of lenses and flat mirrors. There are furthermore two inlet ports P l , P2 provided in this embodiment. In this example, the OAP Ml comprises a mirror, placed between the light source 9 and the first inlet port P l , with a hole drilled through the center, parallel to the incident light path, while OAP M2 has no hole drilled and redirects light with an angle, in this example, of 60°, between the light source 9 and the second inlet port P2. The light path adjuster comprises two moveable shutters S I , S2 on the inlet and outlet side of the integrating cavity 3, that move in parallel and depending on their position, block light incoming and outgoing from either ports Pl and P3 simultaneously, or P2 and P4 simultaneously.

[0508] In the first configuration, the light path adjuster is positioned such that the light reflected and focused from OAP M2 is blocked from entering the cavity 3 via second inlet port P2, such that only the light passing through the hole in OAP Ml enters the cavity 3 via first inlet port P l , and is transmitted along a direct light path 15. This light is directly incident on the liquid sample 7. In parallel, on the outlet side of the cavity 3, the shutter S2 of the light path adjuster is positioned such that second outlet port P4 is closed and light diffusely reflected within the cavity 3 does not reach the spectrometer 1 1. In parallel, first outlet port P3 is open, such that the light transmitted by the sample 7 can exit the first light outlet port P3, transmitted through the hole drilled in OAP M3 parallel to the light path, and can be focused directly onto the spectrometer 1 1 via lens L5.

[0509] In the second configuration, the shutter S I of the light path adjuster is positioned such that the light passing through the hole in OAP Ml is blocked from entering the cavity 3 via inlet port P l . As such, the divergent light reaching OAP Ml is collimated and redirected 90° by OAP Ml onto OAP M2 from which it is then focused and redirected at 60° to the to a point at second inlet port P2. The light entering the cavity 3 is then divergent such that the light illuminates the entire transverse width of the sample cuvette, while not allowing any light to be directly transmitted onto the first light inlet port P l . In parallel, on the outlet side of the cavity 3, the shutter S2 of the light path adjuster is positioned such that outlet port P3 is closed and light directly transmitted by the sample 7 does not reach the spectrometer 1 1. In parallel, outlet port P4 is open, such that the light scattered, transmitted and reflected by the sample 7 is diffusely reflected within the cavity 3 after which it leaves the cavity 3 via second outlet port P4. This divergent light is then collected via OAP M4, collimated and redirected at 90° by OAP M4, onto OAP M3 from which it is redirected at 90° and focused directly onto the spectrometer 1 1 by OAP M3.

[0510] Referring now to Figure 7, a sixth embodiment of apparatus 1 is provided with like features being given like references. In this example, the manipulation of the optical path for two different configurations is provided via a pair of fibre optic cables 21 , 23, each of which is associated with a respective light source 25, 27, and with a respective inlet port P l , P2. Each light source 25, 27 may comprise a respective LED source 25, 27 which with an associated LED electronic controller 29 comprise the light adjuster in this example, whereby the provision of light to inlet port P l or P2 is controlled by suitable activation and deactivation of the LED sources 25, 27 by the controller 29.

[0511] In this example, the fibre optic cable 21 supplies light directly to first inlet port Pl . Fibre optic cable 23 supplies light to second inlet port P2 via a collimation lens 30.

[0512] An outlet mirror 32 and beam splitter 33 are provided between outlet ports P3, P4 and the spectrometer 1 1 and are configured to allow selectively allow light from first and second outlet ports P3, P4 to reach spectrometer 1 1 in dependence upon in which configuration the apparatus is operating.

[0513] In the first configuration, the apparatus 1 is in a transmission mode in which the light path adjuster, namely the controller 29 is controlled such that light is provided from LED source 25, via first fibre optic cable 21 to inlet port P l . Light entering the cavity 3 through inlet port P l directly illuminates the liquid contained in the cuvette 7 and the outlet light path adjuster, namely outlet mirror 31 and splitter 33, are configured such that the light collected through outlet port P3, and sent to the spectrometer 1 1 , includes a proportion of light from the first LED source 25 is directly transmitted by the sample after illuminating the sample. In this configuration, the extinction spectrum of the sample is obtained.

[0514] In the second configuration, the apparatus 1 is in a diffusely reflecting mode, where the controller 29 controls second LED source 27 to provide light via second fibre optic cable 23 to the second inlet port P2. Light from the LED source 25 entering the cavity 3 through inlet port P2 can either directly illuminate the liquid contained in the cuvette 7 or can be incident on the cavity wall 5 and be diffusely reflected within the cavity 3 before interacting with the liquid sample. In this second configuration, the outlet mirror 31 and / or splitter 33 are configured such that the light transmitted and / or reflected by the sample and collected through second outlet port P4 and sent to the spectrometer 1 1 has undergone at least one reflection from the cavity walls 5 before entering the outlet port P4. In this configuration, the absorption spectrum of the sample is obtained, free from the effects of scattering by the liquid sample. The use of independently controllable LED light sources each of which feed a particular inlet port P l , P2 may result in a somewhat simpler apparatus which requires less separate movable and / or fixed optical elements to control the light entering sphere 3 , and to allow the apparatus to operate in the first and second configurations.

[0515] In this embodiment, inlet port P2 is non-parallel with inlet port P l , such that light enters the cavity via inlet port P2 at an angle inclined to the major axes of the cavity. The position / angle of the port P2 should be chosen so as to minimise the chance for any fresnel reflections from the cuvette 7 exiting through the transmission port P3, P4 upon first reflection when the light hits the cuvette 7. The angle of the light path through port P2 can be selected accordingly.

[0516] The movable and / or fixed optical elements may, in an apparatus 1 , be selected from: a. a prism; b. a lens; c. a mirror; d. a diffraction grating; e. a fibre optic cable; f. the light source.

[0517] Example Components

[0518] Provided below is, a non-limiting outline of example components that can be used with some examples of apparatus 1 :

[0519] • Light source 9: A tungsten halogen lamp providing light for excitation from 350-900 nm, purchased from ThorLabs.

[0520] • Moveable Mirrors (Ml , M4, in the example of Figures 1 and 2): Standard optical mirrors mounted 45° to the light path, that can be translated into and out of the beam path for choosing either the first or second configurations. Purchased from ThorLabs.

[0521] • Fixed Mirrors (M2, M4, in the example of Figures 1 and 2): Standard optical mirrors mounted 45° to the light path that can be translated into and out of the beam path for choosing either the first or second configurations. Purchased from ThorLabs.

[0522] • Delivery Lens (L2, in the example of Figures 1 and 2): Standard convex lens of defined focal length used for the second configuration to focus the incoming light through inlet port P2 onto the cavity walls 5 for absorption measurements. Purchased from ThorLabs.

[0523] • Integrating Cavity 3: 50 mm internal diameter spherical integrating cavity with diffusely reflecting inner walls. The sphere has four ports (P1 -P4) drilled in the walls for light delivery and collection and a custom drilled sample port on the north pole for suspending the cuvette 7 in the centre of the cavity 3. The integrating cavity 3 is purchased from Avian Technologies. The sphere geometry may be bespoke, to suit the application with which apparatus 1 is used. The cavity 3 may be non-spherical, and could be cylindrical or cuboidal. The coating of walls 5 may have different types of surface reflectivity, including specular and diffuse reflectance or combinations thereof in the UV, visible, or infrared region or combinations thereof.

[0524] • Sample Holder / Cuvette 7: The cuvette 7 is held in the apparatus 1 via a holder that clamps around the cuvette 7 and also allows the cuvette 7 to be suspended within the cavity 3 at a fixed position. The following cuvette geometries may be provided: standard (1 cm square), thin or plate-like(10 x 1 mm), cylindrical, spherical (combinations are possible too, e.g. cylindrical with a flat region)

[0525] • USB Spectrometer 11: Analyzes the intensity of the light leaving the cavity 3 as a function of wavelength, allowing a spectrum to be obtained and displayed on, for example, a computer screen. This may be a standalone device powered and interfaced via USB connection to a controller in the form of a laptop / computer. Light detection may be as per a standard spectrometer with dispersive optics and detection via CMOS, CCD, diode-array, or scanning-monochromator.

[0526] • Electronics: The movable mirrors are driven by stepper motors, and controlled by programmable micro-controller with stepper motor driver board. Both micro-controller and the USB spectrometer are attached to a controller such as a mini-computer internal to the apparatus 1. The purpose of the mini-computer is two-fold, i) it facilitates communication with the spectrometer 1 1 and with the motor driver, and ii) it provides a web-based graphical user interface. This facilitates interaction with the apparatus 1 in that there is no need for the user to install special software, and no need for the developer to maintain operating-system dependent custom software.

[0527] • Light sources: standard UV-VIS (i.e. Halogen, Xenon, Deuterium lamps), LEDs of any sort, lasers, combinations of all these; and any polychromatic source with attached monochromator for wavelength selection.

[0528] • Delivery optics: assemblies of standard optical components such as lenses, mirrors, shutters, diffraction gratings, optical fibers, or any combinations thereof.

[0529] • Light-path switching: Motorised linear stage(s) and / or shutter(s).

[0530] Parameters / Variables

[0531] There are a number of physical and geometrical parameters / variables which are factors in the design and operation of an apparatus 1 as described above, which include any one or more of the following:

[0532] • Cavity Surface reflectivity p is the ratio of reflected to incident light rays. For the operation of the cavity in line with apparatus 1 , is the reflectivity must be close to unity, i.e. the walls 5 comprise highly reflective material. The apparatus 1 further requires the reflectivity to be strongly diffuse (Lambertian).

[0533] • Port fraction / is the ratio of the surface area of all cavity ports P1 -P4 to the total surface area of the walls 5 of the cavity 3. A ray of light randomly traversing the cavity 3 thus has a chance / to escape.

[0534] • Enhancement factor M: approximately encodes the number of diffuse cavity surface reflections a ray will undergo before either absorbed by the walls of the cavity or leaving via a port. In the ideal case of an empty spherical cavity we have M =1■

[0535] • Chance to hit the sample p: a purely geometric factor, states the probability for a ray which diffusely reflected off the cavity surface to interact with the sample cuvette.

[0536] • Path-length L is the average length of the path a ray of light takes within the sample volume. L is large if M and i are large.

[0537] Apparatus Calibration / Measurements / Control Overview

[0538] The following factors form the basis for the apparatus 1 in order to obtain error free spectra: Relating to absorbance measurements:

[0539] • The controller determines the absolute absorption cross-section of samples inserted into an integrating cavity; this requires accurate calibration of measureable intensities against known standards.

[0540] • Input port positions for absorbance: There are two options for the placement of this port: o i) Avoiding direct illumination of the sample improves reproducibility of measurements as it is less sensitive on the exact geometric replacement of the sample cuvette. The disadvantage of this approach is that some light reaches the detector (determined by p) without interacting with the sample, even for a fully absorbing sample, which limits the range of measurable optical density. o ii) Alternatively all incident rays can be made to pass through the sample. This solves the problem of saturating absorbance and allows the measurement of strongly absorbing samples. In this case the detection port needs to collect from a section of the cavity wall which does not receive light from direct or reflected illumination.

[0541] • Detection port positions for absorbance: The field of view of the detection port must not intersect the sample, instead it should gather light only from the cavity surface. This minimizes the dependence of the measurement on the scattering properties of the sample.

[0542] • Geometric optimization of the setup: the average pathlength in the sample, L, can be approximated by the ratio of the sample volume and the cavity volume, rV = Vsampie / Vcavity: multiplied by the average chord length in the cavity, c =4VCavity / AcaVity (where AcaVityis the surface area of the cavity), and by the enhancement factor M. The approximate pathlength L = rV cM governs the lower limits of the detectable optical density; for example, for low-absorbance samples it is desirable to maximise L: i) M becomes maximal for a cavity surface reflectivity p —> 1 and cavity port fraction / -^-O, ii) rV increases with the relative sample volume and approaches one as the sample fills the sphere entirely, Hi) c is maximal for a spherical cavity. A spherical cavity filled entirely by the sample, with maximal surface reflectivity and minimal port openings may be an optimal setup for detection of ultra-low concentrations.

[0543] • It is not straight-forward to choose a combination of parameters (cavity and sample geometries, port locations, numerical apertures, etc.) which fit the requirements of validity, reproducibility, and user-convenience. The design choices may be a non-trivial compromise. For example, the apparatus 1 described above is suited for standard cuvettes, including cuvettes with short optical pathlength for strongly absorbing liquids.

[0544] Relating to combined extinction-absorbance measurements:

[0545] • extinction measurements are performed inside an integrating cavity; this comes with geometric constraints in that the sample walls must be perpendicular to the incident beam, which requires a square or flat-walled cuvette. Cuvettes with curved surfaces (e.g. cylindrical) are also possible, but would require specialised optics to counter the refractive effects.

[0546] • The numerical aperture available in both delivery and detection needs to be constrained in order to avoid diffuse illumination of the sample and to minimize detection of multiple-scattering light.

[0547] • Combined delivery and detection optics capable of switching between the absorption and extinction pathways are required. The arrangement of these pathways must ensure that they do not affect each other.

[0548] Apparatus Calibration / Measurements / Control Example Detail

[0549] Detail of an example calibration method that could be used to calibrate a spectrometer apparatus as described above, is set out in the attached Appendix.

[0550] The spectrometer apparatus may be configured to measure spectra of a liquid sample selected from any one or more of the following: a. Water; b. Wine; c. A beverage; d. An edible liquid or partially liquid product; e. Paint; f. Water, such as seawater; g. Nanoparticles; h. Emulsions; i. Blood

[0551] In one example the spectrometer apparatus may therefore be a wine testing apparatus. The above list is non-limiting.

[0552] Unless the context clearly requires otherwise, throughout the description, the words “comprise”, “comprising”, and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of “including, but not limited to”.

[0553] Although this invention has been described by way of example and with reference to possible embodiments thereof, it is to be understood that modifications or improvements may be made thereto without departing from the scope of the invention. The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features. Furthermore, where reference has been made to specific components or integers of the invention having known equivalents, then such equivalents are herein incorporated as if individually set forth. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0554] Appendix - Example Calibration Method

[0555] Calibration Procedure for Combined Extinction and Absorbance Spectrometer

[0556] Background interest has an C4(A) but is embedded within a liquid

[0557] In a standard UV-VIS transmission measurement, the that has a non zero es(A) / 0, then the concentration extinction, E’(A), of a sample being measured is related of the analyte cannot be determined via an extinction to the extinction coefficient, ru of the sample and the measurement due to the contribution of scattering. The path length through the sample, K, by the Beer-Lambert solution to this problem is to embed the sample within an Law, given as integrating cavity as described in the original invention, to remove the effects of scattering on the measured signal.

[0558] E(A) = — log10T(A) = K«E , (1) Integrating Cavity Path Length

[0559] In a non-standard transmission configuration, such as where T(A) is the rati* of transmitted to incident light at that shown below, where the sample is embedded within a given wavelength. If the extinction is due to a partican integrating cavity such that the light interacting with ular analyte (such as a dye molecule) in the sample, the the sample is diffusely reflected within the cavity by the extinction coefficient is then proportional t* the concencavity walls, the path length in the sample is no longer tration of analytes, c and the molar extinction coefficient simply defined as the thickness of the cuvette because of the analyte, effixy Thus when the path length is well the light may pass many times through sample and at known then the measured extinction, E(X) is directly different angles before leaving the cavity and entering proportional to the sample extinction coefficient, which the spectrometer. As such, it is no longer valid to use can be used t* compute the concentration of the analyte Equation 2 to determine the molar absorption coefficient through of the sample (limiting ourselves here to case 1 for the sake of argument) . With C,S(A) = ® and writing E’(A) as AR(A), the measured absorbance in the cavity is instead

[0560] E(A) = KceE(A), (2) given by

[0561] This illustrates the quantitative power of UV-VIS measurements, because for a standard UV-VIS setup, the A(A)MEAS = Keff(-4R.(A))^4R.(A), (4) path length is well defined by the length of the cuvette used, which is in general 1 cm. In reality extinction is where Keff(A(A)) is the effective cavity path length and the sum of scattering and absorption of the sample and AR (A) is the “real absorbance” signal that would be meathus Q?(A)canbe separated into two contributions: one sured in a standard transmission measurement for a path from absorption and one from scattering. These are then length of 1 cm. Keff (^4R(A)) is explicitly a function of called the molar absorption coefficient, ?A(A)and the wavelength (due to the non-flat reflectivity of the cavity scattering coefficient es(X) respectively. This leads to the material) and AR (A). Rearranging Equation 4 gives Beer-Lambert expression of

[0562] (5)

[0563] B(A) = Kc(en(A) + es(A) ) - (3)

[0564] This allows three general cases where the sample can be: showing that it is straightforward t* determine the cavity path Keff (AR(A)) as a function of wavelength and sam¬

[0565] 1. absorbing only, i.e. L4(A) 0 and e,s(A) = 0 ple (real) absorbance by measuring a range of absorbing samples in both transmission and absorption mode (as is described in the original invention) and taking the rati* of A(A)MEAS t* AR(A). 0 method for determining the cavity path length is required s* that the measured absorbance in this configu¬

[0566] For the majority of samples, case 1 applies, where ration, UMEAS , can be converted into the equivalent abt£(X) = £A(A) >and Equation 2 can be used t* determine sorbance that would be measured in a 1 cm path length the concentration of the analyte or the molar absorption transmission setup. Consequently, once the path length coefficient. However when case 3 is applicable, standard is known, A MEAS can be corrected for the increase (or UV-VIS configuration cannot be used as it is very decrease) in path length compared to the transmission difficult to separate the contributions from scattering setup and Equation 2 can be applied to determine the and absorbance independently. Thus if the analyte of absorption coefficient (or concentration) of the analyte. Integrating Cavity Calibration dye in each measurement is indicated. While the spectra

[0567] The approach out lined for determining the effective cavlook similar in both configurations, there is a clear ity path length is implemented below using the setup difference in the magnitudes of the measured spectra at described in the orginal invention and using the dye each concentration. As expected from the Beer Lambert Eosin B as the analyte. A concentration series of the Law, in the extinction configration (not shown), with dye is measured to determine the path length across a no scattering present, the (J4R(A)) scales linearly with range of sample absorbances. In all cases, (AR(A)) is the absorption coefficient. In the cavity configration how“real” absorbance measured in the transmission mode ever, it is clear that magnitude quickly deviates from and -A(A)MEAS *Sthe measured absorbance in the abbeing linearly proportional to concentration, owing to sorbance mode and Keff (-^(A)) is the absorption and the non linear response of the cavity path length to wavelength dependent path length in the cavity. The deincreasing absorbance. tails of the measurements are described in the Methods section. series in transmission mode.

[0568] Using the data in Figures 1 and 2, Keff(AR(A)) can

[0569] The A(A)MEAS 'U I^ (AR(A)) spectra obtained using the be calculated at any wavelength by taking the ratio of invention described for an Eosin B concentration series -A(A)MEAS and J4R(A) as given by Equation 4. The path are shown in Figures 1 and 2, where the concentration of length factor at 517 nm obtained from the Eosin data is plotted in Figure 3 against measured absorbance

[0570] Wavelength [nm]

[0571] Cavity Path Length in the Presence of Scattering

[0572] FIG. 8. A (A)MEAS spectra of 7.8 / iM Eosin in solutions of increasing scattering coefficients, where the dilution of the

[0573] The effects of scattering on A(A)M (and thus M(AR(A))) silica particles from stock is indicated in the legend. when the sample has a non-zero scattering coefficient are shown for two cases in Figures 5 8. It is clear that in Red 3 respectively, with different absorption spectra were

[0574] FIG 5. A (A)MEAS spectra of 488 nM Eosin in solutions of measured in the cavity spectromter. The dyes absorb increasing scattering coefficients, where the dilution of the at significantly different wavelengths and thus are ideal silica particles from stock is indicated in the legend. to test the validity of the cah bration method. Using a suitable mathematical model that computes Keff(A(A)) based on the Eosin calibration data, the measured ab- serbance, J4(A)M, can be recalibrated to yield the caliAPPENDIX A - MEASUREMENT PROCEDURE brated absorbance, which, as shown in the figures is almost identical t» the real absorbance, A(A)R (obtained The following steps are used to perform a full measurevia the transmission mode measurment). These two cases ment of a sample using the invention: illustrate the validity of the proposed approach for returning the equivalent absorption spectrum in transmis1. The user sets the spectrum integration time and sion mode from the measured absorbance in absorbance the total number, N , of spectral acquisitions remode. Thus for a sample with arbitrary scattering coefquired for the measurement. For each step (referficient, the approach is still valid and will correctly yield ence, sample and dark) , the average spectrum is the sample’s real absorbance spectrum, J4(A)R, along computed by taking the average of the N spectra with its extinction spectrum, E'(A)R simultaneously. acquired.

[0575] 2. The software is used to set the instrument to ab- removed and replaced with the 2 mL of the sample solution, i.e. the at the lowest concentration

[0576] FIG. 8. A(A)R spectrum of 1.25p M Bluel in H2t along with used. The cuvette is replaced into the cavity in the the raw absorbance spectrum, A(A)M, scaled by a factor of 4 same position as the reference solution. for comparison, and the calibrated version of A(A)M-

[0577] 7. In extinction mode, the intensity of the light transmitted by the sample is measured, registered by the software as Zg\A)

[0578] FIG. 9. A(A)R spectrum of 1.25p M Red 3 in H2t along with the raw absorbance spectrum, A(A)M, scaled by a factor of 4 En(X) = -log for comparison, and the calibrated version of A(A)M- and Scattering solutions of 300 nm silica particles were prepared by dilution from a stock solution of 50 mgmL-1m (7) aqueous solutions as received from the supplier. Sact- tering solutions were then mixed with equal volumes of Eosin B to achieve the desired final dye+scattering conwhere n is the sample number in the dilution series, centration. Implicit in this approach is the assumption meaning n = 1 is the lowest concentration and n = n is that the dye molecule and the scattering particles do the highest concentration. not interact, either through electrostatic adsorption of the molecules to the particle surface or through chemical

[0579] For the extinction and absorption spectra shown (zero interaction. For this reason, it is desirable to use a scattering case), each spectrum is then post processed •lye that has the same charge as the particle surface; by first subtracting a constant background. In the case in the case here, Eosin B is negatively charged and the of Eosin B , this is done by taking the average value of of silica particles have a COOH surface group that will each spectrum in the 700 to 750 nm, where the dye does be negatively charged in solution, so there should be not absorb. Each spectrum is then smoothed to remove no interaction between both species, as evidenced by noise by using a Savitzky-Golay filter with a box width the similarity between the dye absorbance spectrum in of 61 and a polynomial of order 2. The measured absorpdissolved in H20 and in the silica solutions. All samples tion spectrum is then converted into the real absorption were prepared immediately prior to measurement in spectrum as outlined above using Equation 5, yeilding 2 the instrument. After each sample was measured in a spectra, namely the extinction E(X) and the real absorpdilution series, cuvettes were washed thoroughly with tion, Tlfl(A) spectra. water, then ethanol, then water as required and a new water reference was taken if needed.

[0580] APPENDIX B - EXPERIMENTAL DETAILS

[0581] Eosin B, sourced from SigmaAldrich, samples were prepared by diluting a stock of 500 / / M in water. The REFERENCES stock solution was prepared from the powder as received.

[0582] CLAIMS

[0583] 1. A spectrometer apparatus for measuring spectra of a liquid sample, the apparatus comprising: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette containing liquid sample within the integrating cavity, wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the or each light inlet port being configured to receive light from at least one light source and the or each light outlet port being configured to deliver light to a spectrometer; the apparatus further comprising a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided; wherein when the light path adjuster is in a first configuration, the apparatus is in a transmission mode in which light from the light source follows a first light path from the or one of the light inlet port(s) to the liquid sample such that the light from the light source irradiates the liquid sample directly before the light transmitted by the sample is transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample; and when the light path adjuster is in a second configuration, the apparatus is in a diffusely reflecting mode in which light from the light source follows a second light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the liquid sample.

[0584] 2. The apparatus of claim 1 arranged such that, when in the second configuration, light from the second light path is transmitted: a. directly from the inlet port onto the wall or walls of the integrating cavity; and / or b. directly from the inlet port, onto and through the sample and subsequently onto the wall ficor walls of the integrating cavity.

[0585] 3. The apparatus of claim 1 or claim 2 wherein the inlet port used in the first configuration is directly opposed from the outlet port used in the first configuration such that, when in the first configuration, the first light path extends directly across the integrating cavity.

[0586] 4. The apparatus of any one of claims I to 3 further comprising the light source. 5. The apparatus of any one of the preceding claims further comprising a controller configured to control the light path adjuster to selectively adjust the path of light through the apparatus.

[0587] 6. The apparatus of claim 5 wherein the controller is an integral part of the apparatus and is in direct communication with the apparatus.

[0588] 7. The apparatus of claim 5 wherein the controller is remote from the apparatus and is configured to be in wireless communication with a transceiver of the apparatus.

[0589] 8. The apparatus of any one of claims 5 to 7 wherein the controller is configured to control the spectrometer, and in particular is configured to process the light received by the spectrometer for wavelength analysis of the light to provide the extinction and / or absorbance spectrum of the liquid sample contained in the cuvette.

[0590] 9. The apparatus of claim 5 further comprising the spectrometer.

[0591] 10. The apparatus of any one of claims 5 to 9 wherein the controller is configured to control one or more of: a. switching between the first and second configurations; b. acquiring spectra from the integrating cavity; c. choosing operating conditions; d. displaying spectra on a display of the apparatus, or of the controller, or in communication with the apparatus or controller; e. saving data on a memory of the apparatus, or of the controller, or in communication with the apparatus or controller; f. a user-interface of the apparatus, or of the controller, or in communication with the apparatus or controller, that interacts with the apparatus and allows a user to control the position of the light path adjustment mechanism.

[0592] 1 1. The apparatus of any one of the preceding claims wherein the light path adjuster comprises at least one movable optical element configured to manipulate light incident on the optical element from the light source, the light path adjuster being configured to adjust the movable optical element to selectively provide the first and second light paths.

[0593] 12. The apparatus of claim 1 1 wherein the optical element is adjustable by moving the optical element with respect to the integrating cavity from a first position in which the light travels along the first light path, and a second position in which the light travels along the second light path.

[0594] 13. The apparatus of claim 12 wherein the integrating cavity comprises orthogonal longitudinal, vertical, transverse axes, and any one or more of the following positional characteristics of the optical element may be adjusted with respect to any one or more of the axes: a. longitudinal position; b. vertical position; c. transverse position d. orientation; e. inclination.

[0595] 14. The apparatus of any one of claims 1 1 to 13 wherein a plurality of movable optical elements are provided.

[0596] 15. The apparatus of claim 1 1 wherein the movable optical element is selected from: a. a prism; b. a lens; c. a mirror; d. a diffraction grating; e. a fibre optic cable; f. the light source.

[0597] 16. The apparatus of any one of claims 1 1 to 15 wherein the light path adjuster comprises at least one fixed optical element which is not adjustable with respect to the integrating cavity.

[0598] 17. The apparatus of claim 16 wherein the fixed optical element is configured to manipulate the light from the light source prior to the light inlet port.

[0599] 18. The apparatus of claim 16 or claim 17 wherein the fixed optical element is configured to manipulate the light from the light outlet port.

[0600] 19. The apparatus of any one of claims 16 to 18 wherein the fixed optical element is selected from: a. a prism; b. a lens; c. a mirror; d. a diffraction grating; e. a fibre optic cable; f. the light source.

[0601] 20. The apparatus of any one of the preceding claims wherein the light path adjuster comprises at least one electronic controller operative to effect selective operation of one or more light sources, to selectively provide the first and second light path.

[0602] 21. The apparatus of claim 20 comprising at least first and second light sources, the controller being configured to control each light source independently.

[0603] 22. The apparatus of any one of the preceding claims wherein the light path adjuster is positioned between the light source and the light inlet port.

[0604] 23. The apparatus of any one of the preceding claims wherein the light path adjuster is positioned between the spectrometer and the light outlet port.

[0605] 24. The apparatus of any one of the preceding claims wherein a plurality of light path adjustment mechanisms are provided.

[0606] 25. The apparatus of any one of the preceding claims wherein a plurality of light inlet ports are provided, the light path adjuster being configured to provide the first light path by directing light from the light source through a first light inlet port, and to provide the second light path by directing light from the light source through a second light inlet port.

[0607] 26. The apparatus of any one of the preceding claims wherein a plurality of light outlet ports are provided, the first light path directing light from the integrating cavity through a first light outlet port, and the second light path directing light from the integrating cavity through a second light outlet port.

[0608] 27. The apparatus of any one of the preceding claims wherein the integrating cavity comprises any one of: a. a diffusely reflecting spherical integrating cavity; b. a cylindrical cavity; c. a cuboidal or square cavity.

[0609] 28. The apparatus of any one of the preceding claims wherein the integrating cavity comprises an internal coating configured to provide any one or more of: a. specular reflectance; b. diffuse reflectance; c. reflectance in the UV light spectrum; d. reflectance in the visible light spectrum; e. reflectance in the infra-red spectrum.

[0610] 29. The apparatus of any one of the preceding claims wherein the light source comprises any one or more of: a. a quartz-halogen source; b. an LED ; c. a laser; d. any polychromatic source.

[0611] 30. The apparatus of any one of the preceding claims wherein the shape of the cuvette is : a. square; b. plate-like; c. cylindrical; d. spherical;

[0612] 31. The apparatus of any one of the preceding claims being a UV-VIS spectrometer apparatus.

[0613] 32. The apparatus of any one of the preceding claims further comprising a sample holder configured to retain a cuvette containing liquid sample within the integrating cavity.

[0614] 33. The apparatus of any one of the preceding claims wherein the light source comprises first and second LED light sources, and the light path adjuster comprises a controller configured to control the first and second LED light sources such that when in the first configuration, the first LED light source is controlled to provide light on the first light path, and when in the second configuration the second LED light source is controlled to provide light on the second light path.

[0615] 34. The apparatus of claim 33 wherein light from each LED light source is delivered to the integrating cavity via a respective fibre optic cable.

[0616] 35. The apparatus of claim 33 or 34 wherein each LED light source delivers light to a respective light inlet port.

[0617] 36. The apparatus of any one of claims 33 to 35 wherein each light path delivers light through a respective light outlet port.

[0618] 37. The apparatus of any one of claims 33 to 36 wherein the first LED light source is associated with a collimation lens positioned between the first LED light source and the light inlet port associated with that LED light source

[0619] 38. The apparatus of any one of claims 33 to 37 further comprising first and second outlet ports, and a beam splitter configured to selectively allow light from the first and second outlet ports to be transmitted to the spectrometer.

[0620] 39. A spectrometer apparatus for measuring spectra of a liquid sample, in particular where the spectra obtained are the absorption and extinction spectra of the sample, the apparatus comprising: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette containing liquid sample within the integrating cavity, wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the light inlet port being configured to receive light from a light source and the light outlet port being configured to deliver light to a spectrometer; the apparatus further comprising a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided; wherein: when the light path adjuster is in a first configuration, the apparatus is in a transmission mode in which light from the light source follows a first light path from the light inlet port to the liquid sample such that the light from the light source irradiates the liquid sample directly before the light transmitted by the sample is collected via the light outlet port and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample; and when the light path adjuster is in a second configuration, the apparatus is in a diffusely reflecting mode in which light from the light source follows a second light path from the inlet port into the integrating cavity, and is incident onto either the reflective inner wall or walls of the integrating cavity or directly onto the liquid sample; wherein light transmitted and / or scattered by the sample is transmitted through the outlet port, the apparatus being configured such that light directly transmitted by the sample is reflected by the inner wall or walls of the cavity before being transmitted through the outlet port, and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the liquid sample.

[0621] 40. A spectrometer apparatus for measuring spectra of a liquid sample, the apparatus comprising: an integrating cavity comprising a reflective inner wall or walls, and configured to receive a cuvette containing liquid sample within the integrating cavity, wherein the integrating cavity comprises a first light inlet port and a second light inlet path at least one light outlet port, the first light inlet port being configured to receive light from a first LED light source and the second light inlet port being configured to receive light from a second LED light source, at least one light outlet port being provided and configured to deliver light to a spectrometer; the apparatus further comprising a light path adjuster configured to selectively adjust a light path through the integrating cavity such that at least two distinct light paths are provided; wherein when the light path adjuster is in a first configuration, the apparatus is in a transmission mode in which light from the first LED light source follows a first light path from the first light inlet port to the liquid sample such that the light from the first LED light source irradiates the liquid sample directly before the light transmitted by the sample is transmitted through the light outlet port and received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum of the liquid sample; and when the light path adjuster is in a second configuration, the apparatus is in a diffusely reflecting mode in which light from the second LED light source follows a second light path from the second inlet port into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the, or another, light outlet port and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the liquid sample.

[0622] 41. The spectrometer apparatus of any one of the preceding claims configured to measure spectra of a liquid sample selected from any one or more of the following: a. Water; b. Wine; c. A beverage; d. An edible liquid or partially liquid product.

[0623] 42. A spectrometer apparatus substantially as described herein and as shown in the accompanying drawings. 43. A method of measuring spectra of a liquid sample using the apparatus of any one of claims 1 to 41 , comprising steps of: a. activating the light source; b. controlling the light path adjuster to be in the transmission mode or the diffusely reflecting mode; and c. conducting wavelength analysis of the light transmitted through the light outlet port via the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the liquid sample contained in the cuvette.

[0624]

[0625] Figure 1

[0626]

[0627] Figure 2 b

[0628]

[0629] Figure 4

[0630] Figure 5

Claims

CLAIMS:1 . A method of determining at least one property of a therapeutic payload and / or nanoparticle delivery vehicles, the therapeutic payload and nanoparticle delivery vehicles forming a diffusely scattering liquid suspension sample, the method comprising: locating the sample within an integrating cavity; directing probe light to a first port of the integrating cavity; receiving a portion of the probe light from a second port of the integrating cavity following scattering from the sample; measuring one or more spectral features of the probe light received at the second port; and determining the at least one property of the therapeutic payload and / or nanoparticle delivery vehicles in the liquid suspension from the measured one or more spectral features of the probe light received at the second port.

2. The method of claim 1 , wherein determining the at least one property of the therapeutic payload and / or nanoparticle delivery vehicles in the liquid suspension comprises determining one or more absorbance spectral features of the respective therapeutic payload and / or nanoparticle delivery vehicles from the measured one or more spectral features of the probe light.

3. The method of claim 2, wherein determining one or more absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles comprises applying a calibration transform to the one or more measured spectral features of the probe light to determine the absorbance spectral features of the respective therapeutic payload and / or nanoparticle delivery vehicles.

4. The method of claim 3, wherein the calibration transform is based on a calibration of the integrating sphere and / or an absorption coefficient of the respective therapeutic payload and / or nanoparticle delivery vehicles at the wavelength of the probe light.

5. The method of any preceding claim, wherein the at least one property of the therapeutic payload and / or nanoparticle delivery vehicles comprises a measure related to the quantity or concentration of the payload and / or nanoparticle delivery vehicles in the sample.

6. The method of any preceding claim, wherein the at least one property of the therapeutic payload comprises the absorbance spectrum of the payload and / or nanoparticle delivery vehicles.

7. The method of claim 6, further comprising determining from the absorbance spectrum of the payload and / or nanoparticle delivery vehicles one or more characteristics relating to the respective payload and / or nanoparticle delivery vehicles and identifying the payload and / or nanoparticle delivery vehicles from the characteristics.

8. The method of any preceding claim, wherein probe light enters the integrating cavity through the first port in a first axial direction and is received at the second port in a second axial direction, wherein the second axial direction is offset from the first axial direction such that probe light received at the second port is scattered from the integrating cavity and / or sample.

9. The method of any preceding claim, further comprising directing probe light between third and fourth ports of the integrating cavity, the fourth port arranged opposing a third port such that the fourth port receives probe light transmitted directly from the third port, measuring the transmitted light and determining an extinction value for probe light received at the fourth port.

10. The method of claim 9, further comprising quantifying an amount of scattering of the sample of suspension based on the extinction value and determined absorbance spectral features.11 . The method of claim 8 or claim 9, wherein the third port is the first port such that the fourth port receives probe light transmitted directly from the first port.

12. The method of claim 8 or claim 9, wherein the fourth port is the second port such that the second port receives probe light transmitted directly from the third port.

13. The method of any preceding claim, wherein the probe light includes one or more wavelengths in the UV-visible-NIR spectrum, such as including light covering a range between 240nm to 750nm, 240nm to 650nm, at around 260 nm, at a discrete wavelength between 240nm and 320nm, or to scan a range of wavelengths between 240nm to 750nm, 240nm to 650nm, or between 240nm and 320nm.

14. The method of any preceding claim, wherein the nanoparticle delivery vehicle carryies the therapeutic payload and comprises one or more of: a lipid-based nanoparticle carrying RNA, antibody-drug conjugates, viral vectors such as lentiviruses and AAVs, liposomes, exosomes, polymeric nanoparticles, liposome-based systems such as lipoplexes, metal nanoparticles, SPIONs, polymeric micelles, dendrimers, nanoemulsions, silica particles and nanogels.

15. The method of any preceding claim, wherein the nanoparticle delivery vehicles is a lipid-based nanoparticle and the payload comprises RNA such as mRNA, SiRNA, ssRNA.

16. The method of any preceding claim, wherein the payload comprises one or more of: oligonucleotides, DNA, pharmaceuticals, vaccines, oncological treatments and gene editing treatments, and / or small molecule drugs.

17. Nanoparticle vehicle analysis or measurement / characterization method comprising: obtaining / receiving at least one or a plurality of scatter-corrected absorbance measurements / data or scatter-corrected absorbance spectrum measurements / data for a liquid sample or solution containing a plurality of nanoparticle vehicles, the method comprising the method of any preceding claim, and wherein the liquid sample or solution comprises the diffusely scattering liquid suspension sample, and wherein the step of obtaining / receiving at least one or a plurality of scatter-corrected absorbance measurements / data or scatter-corrected absorbance spectrum measurements / data comprises measuring one or more spectral features of the probe light.

18. The method of claim 17, wherein determining at least one property of the therapeutic payload in the liquid suspension comprises using the at least one scatter- corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using an extinction coefficient of the payload of the nanoparticle vehicles at an absorption wavelength of interest, a quantity of a loading of the payload of the nanoparticle vehicles of the liquid sample or solution.

19. The method of claim 17, wherein determining at least one property of the therapeutic payload in the liquid suspension comprises using the at least one scatter- corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using an extinction coefficient of the payload and an extinction coefficient of the carrier component of the nanoparticle vehicles at an absorptionwavelength of interest, a quantity ratio of the payload to carrier component / fraction of the nanoparticle vehicles of the liquid sample or solution.

20. The method of claim 17, wherein determining at least one property of the therapeutic payload in the liquid suspension comprises using the at least one scatter- corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data to determine, using extinction coefficients of constituent carrier elements of the carrier component / fraction of the nanoparticle vehicle, a quantity ratio of the constituent carrier elements of the carrier component / fraction of the nanoparticle vehicles.21 . The method of claim 17, wherein determining at least one property of the therapeutic payload in the liquid suspension comprises using the at least one scatter- corrected absorption measurement / data or scatter-corrected absorption spectrum measurement / data and a measured or determined nanoparticle vehicle size to determine a nanoparticle vehicle concentration of the nanoparticle vehicles in the liquid sample or solution.

22. The method of claim 17, wherein determining at least one property of the therapeutic payload in the liquid suspension comprises using at least one scattering spectrum or scattering spectrum data and measured or determined nanoparticle vehicle size to determine a nanoparticle vehicle concentration of the nanoparticle vehicles in the liquid sample or solution.

23. Apparatus for determining at least one property of a therapeutic payload and / or nanoparticle delivery vehicles, the therapeutic payload and nanoparticle delivery vehicles forming a diffusely scattering liquid suspension, the apparatus comprising: an integrating cavity configured to receive a sample of the liquid suspension into a sample space; a light source arranged to direct probe light through a first port into the integrating cavity; a detector for measuring probe light received at a second port of the integrating cavity, the second port arranged such that the light received is following scattering from the sample in the sample space; and an analyser configured to determine the at least one property of the therapeutic payload and / or nanoparticle delivery vehicles in the liquid suspension from the one or more spectral features of the probe light received at the second port.

24. The apparatus of claim 23, wherein the analyser is further configured to determine one or more absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles from the measured one or more spectral features of the probe light, and the at least one property of the respective therapeutic payload and / or nanoparticle delivery vehicles is determined from the one or more absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles.

25. The apparatus of claim 23, wherein the analyser is configured to determine the absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles by applying a calibration transform to the one or more measured spectral features of the probe light to determine the absorbance spectral features of the therapeutic payload and / or nanoparticle delivery vehicles.

26. The apparatus of claim 25, wherein the calibration transform is based on a calibration of the integrating sphere and / or an absorption coefficient of the respective therapeutic payload and / or nanoparticle delivery vehicles at the wavelength of the probe light.

27. The apparatus of any of claims 23-26, wherein the light source is configured to direct probe light to enter the integrating cavity through the first port in a first axial direction and the second port is arranged to receive probe light scattered from the integrating sphere and / or sample at the second port in a second axial direction, wherein the second axial direction is offset from the first axial direction.

28. The apparatus of claim 27, wherein the integrating sphere further comprises third and fourth ports, the fourth port arranged opposing a third port to receive probe light transmitted from the third port, the analyser further configured to determine an extinction value for probe light received at the fourth port.

29. The apparatus of claim 28, wherein the analyser is further configured to quantify an amount of scattering of the sample of suspension based on the extinction value and the determined absorbance spectral features.

30. The apparatus of claim 28 or claim 29, wherein the third port is the first port such that the fourth port is configured to receive probe light transmitted from the first port.31 . The apparatus of claim 28 or claim 29, wherein the fourth port is the second port such that the second port is configured to receive probe light transmitted from the third port.

32. The apparatus of any of claims 23 to 31 , wherein the probe light includes one or more wavelengths in the UV-visible-NIR spectrum, such as including light covering a range between 240nm to 750nm, 240nm to 650nm, at around 260 nm, at a discrete wavelength between 240nm and 320nm, or to scan a range of wavelengths between 240nm to 750nm, 240nm to 650nm, or between 240nm and 320nm.

33. The apparatus of any of claims 23 to 32, wherein the at least one property of the therapeutic payload is a measure of the quantity or concentration of the payload present in the sample.

34. The apparatus of any of claims 23 to 33, wherein the analyser is configured to determine at least one quantitative property of the nanoparticle delivery vehicle carrying a therapeutic payload, wherein the nanoparticle delivery vehicle carrying the therapeutic payload comprises one or more of: a lipid-based nanoparticle carrying RNA, antibody-drug conjugates, viral vectors such as lentiviruses and AAVs, liposomes, exosomes, polymeric nanoparticles, liposome-based systems such as lipoplexes, metal nanoparticles, SPIONs, polymeric micelles, dendrimers, nanoemulsions, silica particles and nanogels.

35. The apparatus of any of claims 23 to 34, wherein the analyser is configured to determine at least one quantitative property of the nanoparticle delivery vehicle carrying a therapeutic payload, wherein the nanoparticle delivery vehicles is a lipid-based nanoparticle and the payload comprises RNA such as mRNA, SiRNA, ssRNA.

36. The apparatus of any of claims 23 to 35, wherein the analyser is configured to determine at least one quantitative property of the nanoparticle delivery vehicle carrying a therapeutic payload, wherein the payload comprises one or more of: oligonucleotides, DNA, pharmaceuticals, vaccines, oncological treatments and gene editing treatments, and / or small molecule drugs.

37. Nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device comprising the apparatus of any of claims 23 to 36, wherein the integrating cavity comprises a reflective inner wall or walls, the integrating cavity configured to receive a cuvette, the cuvette being configured to contain a liquid sample orsolution including at least one or a plurality of nanoparticle vehicles, the liquid sample or solution comprising the diffusely scattering liquid sample; wherein the integrating cavity comprises at least one light inlet port and at least one light outlet port, the or each light inlet port being configured to receive light from the light source and the or each light outlet port being configured to deliver light to a spectrometer, wherein the spectrometer comprises the detector; wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is optionally a lipid-based drug delivery vehicle analyzer or a lipid-based drug delivery vehicle measurement / characterization device, and is configured to operate in a diffusely reflecting mode in which light from the light source follows a light path from the or one of the inlet port(s) into the integrating cavity, is incident onto the reflective inner wall or walls of the integrating cavity and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through the or one of the light outlet port(s) and received by the spectrometer for wavelength analysis of the light to provide an absorbance spectrum of the at least one or the plurality of nanoparticle vehicles contained in the liquid sample or solution.

38. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to claim 36, wherein the nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device is for or is configured for:- determining a payload quantity or payload concentration of the nanoparticle vehicles, and / or- determining a payload-to-carrier content / concentration ratio of the nanoparticle vehicle, and / or- determining a ratio or fractional content of constituent carrier components of the nanoparticle vehicles, and / or determining a nanoparticle vehicle concentration in a solution or formulation.

39. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to claim 37 or claim 38, wherein the nanoparticle vehicle analyzer or a nanoparticle vehicle measurement / characterization device is a lipid-based nanoparticle analyzer or a lipid-based nanoparticle measurement / characterization device for or configured for determining a lipid-based nanoparticle concentration in a solution or in a formulation, or for or configured fordetermining a lipid-based drug delivery vehicle concentration in a solution or in a formulation.

40. The nanoparticle vehicle analyzer or nanoparticle vehicle measurement / characterization device according to any of claims 37 to 39, wherein the lipid-based nanoparticle vehicle comprises or consists of a (solid) lipid nanoparticle and / or a liposome.

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