Patent Application
20240027380
The present invention relates to methods for non-invasively identifying substandard or counterfeit products, e.g., vaccines, using nuclear magnetic resonance. The methods described herein further provide manufacturers with new approaches to identify parameters to assist users with the identification of substandard or counterfeit products.
Counterfeit and substandard vaccines and biotherapeutics affect mainly regions where medical and distribution infrastructures are inadequate. Domestically, such regions may be remote rural areas and impoverished cities/towns while internationally such regions may be low and middle income countries. In the fight against global pandemics, ensuring equity in medication quality throughout the world is vital.
There is a need for a fast and reliable technique to identify parameters useful to non-invasively identify counterfeit and substandard products by end users with minimal training using instruments that can easily be deployed in the field. Towards that end, the present invention relates to methods of using the transverse relaxation rate of a solvent NMR signal or the longitudinal relaxation rate of a solvent NMR signal to provide the pharmaceutical/biotech industry professionals, law enforcement agencies and other users with more useful parameters to identify counterfeit and substandard products. Advantageously, the methods can be performed noninvasively, without the requirement of opening the vial and/or without destruction of the valuable products contained therein.
In a first aspect, a method of using multi-variate NMR to identify a substandard or counterfeit product is described, said method comprising:
In another aspect, a method of using multi-variate NMR to identify a substandard or counterfeit product in a lot is described, said method comprising:
In still another aspect, a method of using a sedimentation rate to identify a substandard or counterfeit product is described, said method comprising:
In another aspect, a method of obtaining a fingerprint of a product contained in a vial is described, said method comprising:
measuring transverse relaxation rate of solvent R2 over time at two alternating τ-values of a fully dispersed product in said vial, wherein the product is a suspension, and wherein the transverse relaxation rate of solvent R2 at multiple, alternating τ-values is collected throughout a sedimentation process of the product. In some embodiments, the solvent is water and the transverse relaxation rate of water R2(1H2O) is measured. In some embodiments, the two alternating τ-values can be x ms and y ms, wherein the value of x is 1 or less, and the value of y is 10 or more. A non-transitory computer-readable storage medium is also described, storing a computer program thereon which, when run in a computer, causes the computer to carry out the steps of this method.
In yet another aspect, a method of identifying a substandard or counterfeit product is described, said method comprising:
In still another aspect, a method of identifying a substandard or counterfeit product is described, said method comprising:
Other aspects, features and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.
); (
) are the unstressed vials, which served as controls.
); (
) are the unstressed vials, which served as controls.
The present invention generally relates to methods for non-invasively identifying substandard or counterfeit products, e.g., vaccines, using nuclear magnetic resonance. The methods described herein provide manufacturers with new approaches to identify parameters to assist with the identification of substandard or counterfeit products.
As defined herein, a “vial” corresponds to a container, vessel, bottle, syringe, injection pen, or ampoule used to store the product, wherein the vial comprises glass, plastic, ceramic, rubber, elastomeric material, and/or any non-magnetic metal. The vial can have a top including, but not limited to, a screw top, a top that is closed using a cork or plastic stopper, a crimp vial (closed with a rubber stopper and a metal cap), a flip-top or snap cap. The vial can be tubular or have a bottle-like shape with a neck. Other types and shapes of vials used to store products as well as caps are readily understood by the person skilled in the art. The vials can be optically transparent or not optically transparent. There is no need to peel off any label on the vial, whether the label is transparent or not.
As defined herein, a “non-destructive” measurement is defined as a measurement performed without opening the vial or otherwise accessing, harming, or altering the product contained in the vial (for example by withdrawing a portion through a rubber gasket). Alternatively, or in addition to not accessing the contents of a vial, a non-destructive measurement means that no additives or probes or the like (e.g., magnetic particles) are added to the vial prior to the measurement of the longitudinal (T1) or transverse (T2) relaxation time of solvent or the longitudinal (R1) or transverse (R2) relaxation rate of solvent, e.g., water, in the vial. Non-destructive also means that there is no need to make the vials optically transparent and no need to peel off any labels on the vials.
As defined herein, “handling” includes, but is not limited to, manufacturing, fill-finishing, transport, and/or storage of a product.
As defined herein, “substandard” means that the product was not formulated correctly or filled in the vial correctly or has undergone some amount of destruction during handling and should not be used, i.e., administered to a patient. Examples of possible destruction include, but are not limited to: improper formulations, e.g., using the wrong chemical components, incorrect dilutions or contamination; filling errors resulting in a concentration of product that is greater than or less than listed on the vial, e.g., as a result of non-homogeneity or sedimentation; pH changes; unacceptable resuspension; vigorous shaking during transport; accidental heating of the product; accidental freezing of the product; exposure to sunlight; degradation of the product (e.g., aggregation, oxidation or hydrolysis); and repeated freezing and thawing of the product. “Substandard” can also describe a generic of a pioneer or propriety product that is not substantially equivalent to the pioneer or propriety product and should not be used.
As defined herein, a “pharmaceutical product” or “product” can include vaccines and biopharmaceuticals, biologics and small molecules (i.e., non-biologics), in an aqueous medium. The product can further comprise at least one adjuvant, at least one surfactant, at least one water-soluble organic solvent, at least one dispersant, at least one biocide, at least one buffering agent, at least one pH adjusting agent (e.g., acids and/or bases), at least one peptide, at least one polypeptide, at least one protein, at least one antibody or fragment thereof, at least one nucleic acid, at least one oil, or any combination thereof, as readily determined by the person skilled in the art. In some embodiments, the product comprises an emulsion. In some embodiments, the product comprises a vaccine. In some embodiments, the product comprises a solution. In some embodiments, the product comprises a suspension. In some embodiments, the product comprises an emulsion. The product can be transparent or milky or opaque or have color. In some embodiments, the drug product can comprise a protein or a peptide. In some embodiments, the drug product is substantially devoid of any proteins or peptides.
“Substantially devoid” is defined herein to mean that none of the indicated substance is intentionally added to or present in the product. Alternatively, “substantially devoid” can mean that the amount of the indicated substance in the product is less than about 0.1% (w/w), or less than about 0.05% (w/w), or less than about 0.01% (w/w).
It should be appreciated that a “patient” includes any human or other mammal, reptile, bird, fish, or amphibian.
As defined herein, a “biopharmaceutical” includes antibodies, proteins, peptides, nucleic acids, polysaccharides, and combinations thereof. A non-exclusive list of biopharmaceutical products that can be measured for the extent of aggregation includes, but is not limited to, bovine serum albumin; human serum albumin; human γ-globulin; hormones such as insulin, glucagon, gonadotrophins, and growth hormone; haematopoietic growth factors such as erythropoietin; blood factors such as Factor VIII and Factor IX; thrombolytic agents; interferons such as interferon-α, interferon-β, and interferon-γ; interleukin-based products such as interleukin-2; vaccines such as the influenza vaccine; monoclonal antibodies such as adalimumab, rituximab, infliximab, trastuzumab, ustekinumab, denosumab, and golimumab, and including fragments of monoclonal antibodies (e.g., Fc and Fab fragments), variants of monoclonal antibodies, such as single-chain antibodies, bivalent antibodies, and the like, and polyclonal antibody preparations for research or clinical use including therapeutic antibody preparations such as intravenous immunoglobulin (IVIG); tumor necrosis factor; abatacept; alefacept; etanercept; denileukin diftitox; OPTISON; NEUPOGEN; albumin; and ribonuclease A, to name a few.
As defined herein, a “vaccine” can be adjuvanted or not adjuvanted and includes, but is not limited to, an attenuated vaccine comprising an active virus, an inactivated vaccine, toxoid vaccines, a subunit or fragmented vaccine, a conjugate vaccine, an outer membrane vesicle vaccine, a heterologous or heterotypic vaccine, and a genetic vaccine, e.g., mRNA vaccines, viral vector vaccines, DNA vaccines.
As used herein, “counterfeit” products correspond to products sold or provided under a specific brand or generic name that are not manufactured by or on behalf of the owner of said specific brand or generic name. Often the counterfeit compositions are purchased online at a lower cost and are not made according to the exact manufacturing standards of the owner of said specific brand or generic name and/or are less efficacious than the specific brand or generic name product.
As defined herein, a “lot” corresponds to a number of apparently identical vials comprising a product, wherein the vials are provided in the same shipping container or box and/or defined by a manufacturer with a number or code (e.g., a QR code) as being from the same batch of manufactured product. Products in the same lot are assumed to have a uniform character and quality within specified limits. In some embodiments, every vial in a same shipping container or box has the same number or code. In some embodiments, not every vial in a same shipping container or box has the same number or code.
As defined herein, “statistically different” corresponds to a measured value, for example of R2(1H2O) or T2(1H2O), of the alleged counterfeit or substandard product that is (a) greater than 10%, greater than 15%, or greater than 20% of the measured value for the non-substandard product or generic, all other parameters being substantially equal, (b) 6 sigma away from the R2(1H2O) or T2(1H2O) value specified by the manufacturer, wherein 1 sigma is the measurement error, all other parameters being substantially equal, or (c) both (a) and (b).
As used herein, a product “suspected to be substandard or a counterfeit” can correspond to any vial comprising the product in the distribution or supply chain. A product that is “not substandard or a counterfeit” is known and verified by the manufacturer.
It is understood by the person skilled in the art that the “measuring” of the transverse relaxation rate of solvent R2 or longitudinal relaxation rate of solvent R1 may be done by measuring some other parameter and converting to the R2 or R1 value.
As defined herein, “alum” corresponds to aluminum-containing salts comprising one or more of aluminum hydroxide, aluminum phosphate, alum (KAl(SO4)·12H2O), aluminum hydroxyphosphate sulfate, as well as other known or proprietary aluminum salts that can be used as alum adjuvants or in pharmaceutical products comprising aluminum.
As defined herein, the “product comprising aluminum” includes a product with nano- and micron-sized particles comprising aluminum and suspended in a solvent or a mixture of solvents. The alum-containing product can further comprise at least one surfactant, at least one water-soluble organic solvent, at least one dispersant, at least one biocide, at least one buffering agent, at least one pH adjusting agent (e.g., acids and/or bases), with or without antigens, or any combination thereof, as readily determined by the person skilled in the art. Many vaccines are alum-containing products because of the presence of an aluminum adjuvant.
As defined herein, “sophisticated counterfeits” or “sophisticated products” refer to counterfeits that contain one or more components of the real, or non-counterfeit, product. Distinguishing such sophisticated counterfeits from real products is much more challenging than the detection of simple counterfeit products because both comprise similar ingredients.
Recent breakthrough developments in the instrumentation for nuclear magnetic resonance (NMR) spectroscopy and imaging have opened up opportunities to design novel nondestructive analytical techniques for the nanoparticle industry. Of special importance was the introduction of commercially available, relatively inexpensive benchtop and handheld NMR and magnetic resonance imaging (MRI) instruments and relaxometers (Metz, H., et al. 2008. Int. J. Pharm. 364: 170-178). Benchtop NMR instruments enable highly accurate measurements of nuclear spin relaxation times T1 and T2. Moreover, most of these instruments have a permanent or electronically cooled magnet with the variable bore from about 10 mm to about 45 mm and even larger which provides a great flexibility in the nonintrusive measurements of vials of various sizes.
Water proton NMR (wNMR) monitors water, which acts as a reporter for analytes dissolved in it. As a reporter, water has many advantages. First, its concentration far surpasses that of any analyte dissolved in it, by 103-106 fold in most cases. This makes the 1H2O signal easily detectable by NMR instruments. Further, the solute association can be detected through the solvent NMR signal. In addition, water is “endogenous” to all biomanufacturing processes and all pharmaceutical products, including vaccines and biotherapeutics. The high concentration of “endogenous” water makes it possible for wNMR to be performed contact-free in situ. Although only examples of products formulated with water as the solvent are presented in this patent application, the extension of NMR to products formulated with other solvents (e.g., ethanol) is contemplated herein.
wNMR is a useful characterization tool for the pharmaceutical industry, thereby allowing the industry to certify products for release, distribution, and clinical use. For example, wNMR can be used to determine if the product has experienced conditions such as heating or freeze/thaw conditions that make it substandard. Further, wNMR can be used to determine if a product is a counterfeit or a substandard generic of a pioneer or propriety product. Law enforcement agencies can use wNMR to check for counterfeit or substandard products. With this information, the manufacturer of the product can determine if the product can be released, the distributor can determine if the product can be distributed, and/or pharmacy or healthcare providers can determine if the product can be administered to patients. In addition, because the measurement of the wNMR can be determined easily using benchtop and handheld NMR devices, the end user (e.g., healthcare professional) can also measure the wNMR of the product to ensure that the product is not substandard or not a counterfeit. This is particularly advantageous since distribution of products subsequent to manufacturing includes the risk of unacceptable temperature fluctuations (e.g., too hot or too cold), unexpected vibrations, and an unknown length of time between bench and bedside.
The methods described herein are reliable and simple methods to assess whether vaccines and other pharmaceutical products are substandard or counterfeits. The methods enable the assessment of the vaccines and other pharmaceutical products, formulated as aqueous suspensions, without the requirement of opening the vial or product container and without peeling off the label on the vial, all while ensuring that the product contained in the vial can still be administered to a patient if it is determined not to be substandard or a counterfeit. The method is quantitative and comprises measuring the nuclear spin relaxation rate constant, R1 and/or R2, of solvent, e.g., water, as a quality control parameter.
The instant inventors have previously disclosed methods of determining the quality of products using solvent nuclear magnetic resonance (NMR), for example in U.S. Pat. No. 10,267,754 issued on Apr. 23, 2019, U.S. Pat. No. 10,514,347 issued on Dec. 24, 2019, U.S. Pat. No. 10,724,974 issued on Jul. 28, 2020, U.S. Pat. No. 11,119,061 issued on Sep. 14, 2021, U.S. Pat. No. 11,346,908 issued on May 31, 2022, U.S. patent application Ser. No. 16/593,145 filed on Oct. 4, 2019, U.S. patent application Ser. No. 17/030,088 filed on Sep. 23, 2020, U.S. patent application Ser. No. 17/436,169 filed on Sep. 3, 2021, U.S. patent application Ser. No. 17/206,373 filed on Mar. 19, 2021, U.S. patent application Ser. No. 17/401,709 filed on Aug. 13, 2021, and U.S. patent application Ser. No. 17/842,990 filed on Jun. 17, 2022, all of which are incorporated herein by reference in their entireties. The present application is intended to enhance and expand the applicability of the previously filed patents and patent applications, as well as other applications in the related arts.
Broadly, the methods described herein are intended to identify parameters that enhance the ability to detect substandard and counterfeit products. For example, the inventors' own patents and patent applications related to the use of univariate detection, where, for example, the water proton transverse relaxation rate R2(1H2O) of a product in a vial was non-invasively measured at a fixed inter-pulse delay value (τ) (e.g., at 0.5 ms) and compared to a predetermined standard value or range of R2(1H2O) for that product that is not considered substandard or counterfeit. The use of univariate detection is an incredibly powerful technique, however, there are circumstances where the measured R2(1H2O) of a counterfeit or substandard product at a random τ value is not statistically different than that of a non-counterfeit or acceptable (i.e., not substandard) product. The methods described herein aim to overcome this deficiency.
In a first aspect, a method of using multi-variate NMR to identify a substandard or counterfeit product is described, said method comprising identifying a preferred inter-pulse delay (τ) value for a transverse relaxation rate of solvent R2 measurement, comprising:
In some embodiments of the first aspect, a method of using multi-variate NMR to identify a substandard or counterfeit product is described, said method comprising:
In some embodiments, the value of x, y, and z are determined by the user measuring the transverse relaxation rate of solvent R2 of the product in the first vial. In some embodiments, the value of x is 1 or less, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, the value of y is 10 or more, e.g., 10, 15, 20, 25, and the value of z can be any value less than x, for example, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, etc. In some embodiments, y/z is 20 or greater. In some embodiments, y/z is 30 or greater. In some embodiments, y/z is 40 or greater. The greater the value of y/z (which is the number of varied τ values that the transverse relaxation rate of solvent R2 is being measured at), the more complete fingerprint of the product can be obtained. In some embodiments, the value of x is 0.5, the value of y is 10, and the value of z is 0.25, for a total of 40 τ values, or transverse relaxation rate of solvent R2 measurements.
In some embodiments, the product in the first vial is verified by the manufacturer prior to vial filling and prior to obtaining the preferred τ value, using other methods known in the pharmaceutical arts. In some embodiments, the method of the first aspect is carried out non-invasively, wherein the first and second vials are inserted into the core of the NMR without opening the vials or peeling off the label. In some embodiments, instead of the transverse relaxation rate of solvent R2, the longitudinal relaxation rate of solvent R1 is used instead. The method of the first aspect is agnostic to the actual first and second vials. In other words, the vials can have different shapes and sized and/or be made of different materials, as long as they are not paramagnetic or ferromagnetic, and the specific fingerprint will be substantially identical.
Advantageously, the method of the first aspect provides an optimized parameter that permits transporters, law enforcement and medical professionals to more definitively identify counterfeits or substandard products, where previously it was difficult to do so because of the statistically similar transverse relaxation rate of solvent R2 values at a set, and not optimized, τ value. The method of the first aspect is particularly useful for the manufacturer that is familiar with the expected counterfeit or substandard product that the transporters, law enforcement and medical professionals may encounter once the product leaves the manufacturer. For example, a common counterfeit of many products is just water or a saline solution. The manufacturer can identify the preferred τ value, and optionally the fingerprint of the product of the first vial, using the method of the first aspect and provide same in the package insert, on the vial label, on the website, or any of the above. Thereafter, the transporter, law enforcement or medical professional, who may not have a sample of the non-counterfeit or acceptable (i.e., not substandard) product, can measure the transverse relaxation rate of solvent R2 of the vials in a lot at the preferred τ value and more easily identify if they have in their possession a substandard or counterfeit product. A rejected vial should be removed from the distribution chain and not used. Alternatively, the method of the first aspect provides professionals such as manufacturers, transporters, law enforcement and medical professionals to more definitively identify counterfeits or substandard products when they have access to the non-counterfeit or acceptable (i.e., not substandard) product (i.e., the first vial) and they want to determine if a lot of product (i.e., containing a second vial and additional nth vials) is counterfeit or substandard. The first and second fingerprints and the preferred τ value can be obtained, using the method of the first aspect, and thereafter, the transverse relaxation rate of solvent R2 of the vials in the lot at the preferred τ value can be measured to identify if they have in their possession a substandard or counterfeit product. A rejected vial should be removed from the distribution chain and not used.
In a second aspect, a method of using multi-variate NMR to identify a substandard or counterfeit product in a lot is described, said method comprising:
In some embodiments of the second aspect, a method of using multi-variate NMR to identify a substandard or counterfeit product in a lot is described, said method comprising:
In some embodiments, the value of x, y, and z are provided to the user measuring the transverse relaxation rate of solvent R2 of the product in the second vial. In some embodiments, the x, y, and z values are provided on a provided set of instructions or on a website. In some embodiments, the value of x is 1 or less, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, the value of y is 10 or more, e.g., 10, 15, 20, 25, and the value of z can be any value less than x, for example, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, etc. In some embodiments, y/z is 20 or greater. In some embodiments, y/z is 30 or greater. In some embodiments, y/z is 40 or greater. The greater the value of y/z (which is the number of varied τ values that the transverse relaxation rate of solvent R2 is being measured at), the more complete fingerprint of the product can be obtained. In some embodiments, the value of x is 0.5, the value of y is 10, and the value of z is 0.25, for a total of 40 τ values, or transverse relaxation rate of solvent R2 measurements.
In some embodiments, the product in the first vial is verified by the manufacturer prior to vial filling and prior to obtaining the preferred τ value, using other methods known in the pharmaceutical arts. In some embodiments, the method of the second aspect is carried out non-invasively, wherein the second vial is inserted into the core of the NMR without opening the vial or peeling off the label. In some embodiments, instead of the transverse relaxation rate of solvent R2, the longitudinal relaxation rate of solvent R1 is used instead. The method of the second aspect is agnostic to the actual first and second vials. In other words, the vials can have different shapes and sized and/or be made of different materials, as long as they are not paramagnetic or ferromagnetic, and the fingerprint will be substantially identical.
In the second aspect, the first product is not substandard or a counterfeit. In some embodiments, the user does not have a vial comprising the first product available, just the relevant parameters, data and figures provided by the manufacturer. Advantageously, the method of the second aspect permits transporters, law enforcement and medical professionals to identify substandard or counterfeit products when details about the product in a lot are previously unknown. For example, a lot or container of vials comprising an alleged product (i.e., the second product) can be obtained by law enforcement. They can determine if some, or all, of the vials in the lot are substandard or counterfeit by obtaining a second fingerprint of the second product and comparing it to the provided fingerprint of the first product (e.g., as provided by the manufacturer). A preferred τ value can be obtained and thereafter the user can check all of the other vials of the second product in the lot. A substandard or counterfeit product in the lot is identified when the transverse relaxation rate of solvent R2 is statistically different than the transverse relaxation rate of solvent R2 of the first product at the preferred τ value, which can be obtained from the fingerprint of the first product. A rejected vial should be removed from the distribution chain and not used.
In some embodiments, the method of the first and second aspect are applicable to products comprising solutions, emulsions, and settled suspensions (i.e., dosage forms that do not settle).
In a third aspect, a method of using a sedimentation rate to identify a substandard or counterfeit product is described, said method comprising:
In some embodiments, the product of the first vial and the second vial is a dispersed suspension. In some embodiments, the product of the first vial and the second vial comprises an aluminum adjuvant. In some embodiments, the aluminum adjuvant comprises alum particles. In some embodiments, the method of the third aspect further comprises: fully dispersing the product contained in the first vial, wherein the product is a suspension; immediately inserting the first vial in a nuclear magnetic resonance (NMR) instrument; measuring a transverse relaxation rate of solvent R2 at a single τ value over time t; and determining the sedimentation rate of the product in the first vial using a sedimentation kinetics profile (R2(solvent, t)) of the product. In some embodiments, the method of the third aspect further comprises: fully dispersing the product contained in the first vial, wherein the product is a suspension; immediately inserting the first vial in a nuclear magnetic resonance (NMR) instrument; measuring a transverse relaxation rate of water R2(1H2O) at a single τ value over time t; and determining the sedimentation rate of the product in the first vial using a sedimentation kinetics profile (R2(1H2O, t)) of the product. In some embodiments, the method of the third aspect further comprises: fully dispersing the product contained in the second vial, wherein the product is a suspension; immediately inserting the second vial in a nuclear magnetic resonance (NMR) instrument; measuring a transverse relaxation rate of solvent R2 at a single τ value over time t; and determining the sedimentation rate of the product in the second vial using a sedimentation kinetics profile (R2(solvent, t)) of the product. In some embodiments, the method of the third aspect further comprises: fully dispersing the product contained in the second vial, wherein the product is a suspension; immediately inserting the second vial in a nuclear magnetic resonance (NMR) instrument; measuring a transverse relaxation rate of water R2(1H2O) at a single τ value over time t; and determining the sedimentation rate of the product in the second vial using a sedimentation kinetics profile (R2(1H2O, t)) of the product.
In some embodiments, the method of the third aspect is carried out non-invasively, wherein the first and second vials are inserted into the core of the NMR without opening the vials or peeling off the label. In some embodiments, instead of the transverse relaxation rate of solvent R2, the longitudinal relaxation rate of solvent R1 is used instead. The method of the third aspect is agnostic to the actual first and second vials. In other words, the vials can have different shapes and sizes and/or be made of different materials, as long as they are not paramagnetic or ferromagnetic, and the sedimentation rates can still be compared.
In a fourth aspect, a fingerprint of a product contained in a vial is obtained, said method comprising measuring transverse relaxation rate of solvent R2 over time at two alternating τ-values of a fully dispersed product in said vial, wherein the product is a suspension, and wherein the transverse relaxation rate of solvent R2 at multiple, alternating τ-values is collected throughout a sedimentation process of the product. In some embodiments, the two alternating τ-values can be x ms and y ms, wherein the value of x is 1 or less, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and the value of y is 10 or more, e.g., 10, 15, 20, 25. The data R2(1H2O, t, τ), forms a fingerprint for a sophisticated product. In some embodiments, the transverse relaxation rate of water Rs(1H2O) is measured instead.
In some embodiments, the method of the fourth aspect is used to identify a substandard or counterfeit product, said method comprising:
In some embodiments, the method of the fourth aspect permits the selection of a preferred τ-value for subsequent sedimentation methods, e.g., the method of the third aspect. An example of this is shown in
In some embodiments, the method of the fourth aspect is carried out non-invasively, wherein the first and second vials are inserted into the core of the NMR without opening the vials or peeling off the label. In some embodiments, instead of the transverse relaxation rate of solvent R2, the longitudinal relaxation rate of solvent R1 is used instead. The method of the fourth aspect is agnostic to the actual first and second vials. In other words, the vials can have different shapes and sizes and/or be made of different materials, as long as they are not paramagnetic or ferromagnetic, and the R2(1H2O, t, τ) can still be obtained and compared. In some embodiments, the two alternating τ-values can be x ms and y ms, wherein the value of x is 1 or less, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and the value of y is 10 or more, e.g., 10, 15, 20, 25. The data R2(1H2O, t, τ), forms a fingerprint for sophisticated a product. In some embodiments, the transverse relaxation rate of water R2(1H2O) is measured. Advantageously, the method of the fourth aspect involving multi-variate NMR can produce fingerprints of a product for product authentication and counterfeit detection.
In some embodiments, the method of the fourth aspect further comprises: fully dispersing the product contained in a vial, wherein the product is a suspension; immediately inserting a vial in a nuclear magnetic resonance (NMR) instrument; and measuring a transverse relaxation rate of solvent R2 at the two alternating τ values. In some embodiments, the method of the fourth aspect further comprises: fully dispersing the product contained in a vial, wherein the product is a suspension; immediately inserting the vial in a nuclear magnetic resonance (NMR) instrument; and measuring a transverse relaxation rate of water R2(1H2O) at the two alternating τ values.
In some embodiments, the method of the fourth aspect further comprises determining the ratio of R2(y ms)/R2(x ms), as a function of time, for a first vial and comparing it to the same ratio, as a function of time, for a second vial. The results provide another way to distinguish sophisticated products from one another, e.g., as shown in
In a fifth aspect, multi-variate NMR is used to capture the dependence of the transverse relaxation rate of solvent R2(solvent, t), but also the dependence of the transverse relaxation rate of solvent R2(solvent, τ), synergistically providing a complex fingerprint of an individual product, wherein the product comprises a dispersed suspension, e.g., a vaccine formulation.
In some embodiments, the method of the fifth aspect is used to identify a substandard or counterfeit product, said method comprising:
In some embodiments, the multiple τ-values are obtained by varying pulse sequence inter-pulse delay (τ) values from x ms to y ms in increments of z ms, wherein z could be any value, but preferably <x and cannot be >y, at a set temperature. In some embodiments, the value of x, y, and z are provided to the user measuring the transverse relaxation rate of solvent R2 of the product in the second vial. In some embodiments, the x, y, and z values are provided on a provided set of instructions or on a website. In some embodiments, the value of x is 1 or less, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, the value of y is 10 or more, e.g., 10, 15, 20, 25, and the value of z can be any value less than x, for example, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, etc. In some embodiments, y/z is 20 or greater. In some embodiments, y/z is 30 or greater. In some embodiments, y/z is 40 or greater. The greater the value of y/z (which is the number of varied τ values that the transverse relaxation rate of solvent R2 is being measured at), the more complete fingerprint of the product can be obtained. In some embodiments, the value of x is 0.5, the value of y is 10, and the value of z is 0.25, for a total of 40 τ values, or transverse relaxation rate of solvent R2 measurements.
In some embodiments, the method of the fifth aspect is carried out non-invasively, wherein the first and second vials are inserted into the core of the NMR without opening the vials or peeling off the label. In some embodiments, instead of the transverse relaxation rate of solvent R2, the longitudinal relaxation rate of solvent R1 is used instead. The method of the fifth aspect is agnostic to the actual first and second vials. In other words, the vials can have different shapes and sizes and/or be made of different materials, as long as they are not paramagnetic or ferromagnetic, and the R2 versus τ can still be obtained and compared. In some embodiments, the transverse relaxation rate of water R2(1H2O) is measured.
In some embodiments, the method of the fifth aspect further comprises: fully dispersing the product contained in a vial, wherein the product is a suspension; immediately inserting a vial in a nuclear magnetic resonance (NMR) instrument; and measuring a transverse relaxation rate of solvent R2 at multiple τ values. In some embodiments, the method of the fifth aspect further comprises: fully dispersing the product contained in a vial, wherein the product is a suspension; immediately inserting the vial in a nuclear magnetic resonance (NMR) instrument; and measuring a transverse relaxation rate of water R2(1H2O) at multiple τ values.
The methods described herein are a reliable and simple method to assess whether a product contained in a vial is substandard or is a counterfeit or substandard generic of a pioneer or propriety product. The methods described herein are qualitative and/or quantitative and comprise determining the nuclear spin relaxation rate constant of solvent, e.g., R1(1H2O) and/or R2(1H2O), as a quality control parameter.
In addition to being noninvasive, additional advantages of low field solvent NMR includes, but is not limited to, low cost instrumentation (e.g., a desktop or handheld NMR), simple and rapid data acquisition and analysis, and minimal technical expertise requirement. It should be appreciated that the measurements can occur destructively as well, whereby the vial is opened, if needed. Further, the method described herein can utilize high field NMR, if needed.
The present subject matter may be a system, a method, and/or a computer program product. In some embodiments, the computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present subject matter.
In some embodiments, the computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a RAM, a ROM, an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.
In some embodiments, computer readable program instructions described herein can be downloaded to respective computing/processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and/or a wireless network, or Near Field Communication. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and/or edge servers. A network adapter card or network interface in each computing/processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing/processing device.
In some embodiments, computer readable program instructions for carrying out operations of the present subject matter may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C++, Javascript or the like, and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The computer readable program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present subject matter.
In some embodiments, the computer readable program instructions may be provided to a processor of a computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the flowchart and/or block diagram block or blocks. In some embodiments, the computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and/or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function/act specified in the flowchart and/or block diagram block or blocks.
In some embodiments, the computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other programmable apparatus, or other device implement the functions/acts specified in the flowchart and/or block diagram block or blocks.
The features and advantages of the invention are more fully shown by the illustrative examples discussed below.
Two small molecule drugs formulations as oil-in-water emulsions were used to illustrate the detection of counterfeit pharmaceutical products formulated as aqueous solutions using wNMR. The two emulsions drugs were DIPRIVAN and PROPOFOL; DIPRIVAN is the innovator drug and PROPOFOL is its FDA-approved generic. The active pharmaceutical ingredient (API) in both drugs is 2,6-diisopropylphenol.
For the sake of this experiment, DIPRIVAN and PROPOFOL will serve as each other's complex counterfeit. For emulsions, simple counterfeits like water or physiological saline, can be visually distinguished from an actual emulsion drug product. Accordingly, in the present experiment, a lipid emulsion product, INTRALIPID, a nutritional supplement for intravenous delivery, was used to serve as a simple counterfeit of DIPRIVAN, since they are visually similar in their white opacity.
a. Using Univariate wNMR to Detect Simple Counterfeit Emulsion Products
Water proton transverse relaxation time T2(1H2O) measured using CPMG pulse sequence was extracted by fitting experimental echo decay data to Formula (1):
I(t)=I0×exp(−t/T2(1H2O)) (1)
where I(t) is the water proton echo signal intensity at time t, I0 is the initial water proton echo signal at t=0, and t is the echo delay time. Extracted water proton relaxation time T2(1H2O) values were converted to water proton relaxation rate R2(1H2O) using Formula (2):
R2(1H2O)=1/T2(1H2O) (2)
The person skilled in the art would understand that other methods and NMR instruments can be used to determine T2(1H2O), and the CPMG pulse sequence used herein is not intended to limit the determination of T2(1H2O), and ultimately R2(1H2O).
b. Using Type I Multi-Variate wNMR to Detect Simple Counterfeit Emulsion Products
In Type I multi-variate wNMR, R2(1H2O) is measured at multiple τ-values, where the inter-pulse delay, τ, is an adjustable parameter in the CPMG pulse sequence used to measure R2(1H2O). In Type I measurements, R2(1H2O, τ) is performed on emulsions noninvasively, in the original vial. In this multi-variate wNMR experiment, the CPMG pulse sequence inter-pulse delay, τ, was varied from 0.5 ms to 10 ms by increments of 0.25 ms (˜40 different τ-values per emulsion) at a set sample temperature of 25° C. in the probe cavity of a VT-NMR.
c. Using Univariate wNMR to Detect Sophisticated Counterfeit Vaccines
Table 1 lists a single R2(1H2O) for two 2,6-diisopropylphenol emulsion products, brand name, DIPRIVAN, and generic, PROPOFOL, which for the purpose of this study, we considered the sophisticated complex counterfeit. All measurements were done noninvasively using the CPMG pulse sequence, at the same inter-pulse delay time τ (0.5 ms), at 25° C., in the probe cavity of VT-NMR. It can be seen that for these two emulsions, the R2(1H2O) values differ by more than the measurement error, which means a single R2(1H2O) may be used to distinguish between them.
d. Using Type I Multi-Variate wNMR to Detect Sophisticated Counterfeit Vaccines
In Type I multi-variate wNMR, R2(1H2O) is measured at multiple τ-values, where the inter-pulse delay, τ, is an adjustable parameter in the CPMG pulse sequence used to measure R2(1H2O). In Type I measurements, R2(1H2O, τ) is performed on emulsions noninvasively, in the original vial. Two marketed emulsion products with close values of water proton transverse relaxation rates, R2(1H2O), at a single τ-value (0.5 ms) were selected for the experiment. In this multi-variate wNMR experiment, the CPMG pulse sequence inter-pulse delay, τ, was varied from 0.5 ms to 10 ms by increments of 0.25 ms (˜40 different τ-values per emulsion) at a set sample temperature of 25° C. in the probe cavity of a VT-NMR.
These results clearly demonstrate the fingerprinting potential of τ-dispersion profiles and confirm the capability of wNMR technology to differentiate between real and counterfeit emulsion products, including sophisticated counterfeits. Further, in actual practice, multi-variate wNMR may be used to optimize univariate wNMR for counterfeit detection (e.g., through optimizing the inter-pulse separation τ in the CPMG pulse sequence).
e. Other Types of Multi-Variate wNMR
With emulsion products, in addition to single exponential fitting, double exponential fitting of the data reveals more information about the oil and water components of the emulsion. In certain cases of counterfeits, double exponential fitting can provide another layer of information about a product. In addition, R1(1H2O) analysis may also be used instead of R2(1H2O) analysis.
In this example, non-adjuvanted vaccines and biotherapeutics are used to illustrate wNMR capability to detect counterfeit pharmaceutical products formulated as aqueous solutions.
a. Detecting Simple Counterfeit Vaccine Products Using Univariate wNMR
Physiological saline serves as a simple counterfeit to vaccines formulated as solutions.
In summary, a single R2(1H2O) (univariate wNMR) can readily distinguish vaccine products formulated as aqueous solutions from a simple counterfeit such as physiological saline.
b. Detecting Simple Counterfeit Biotherapeutic Products Using Univariate wNMR
Physiological saline serves as a simple counterfeit to biotherapeutics formulated as solutions. For comparison of real biotherapeutics with the simple counterfeits, the water proton relaxation rate R2(1H2O) of two insulin glargine drug products in pre-filled pens, LANTUS and BASAGLAR, and three filgrastim drug products in sealed vials, NEUPOGEN, GRANIX, and NIVESTYM, were measured. For each of the above products, simple counterfeits were created by placing the corresponding amount of the sterile saline (0.9% NaCl) solution in the respective drug container. Specifically, one LANTUS pen and one BASAGLAR pen were emptied and refilled with 3 mL of saline. A sealed glass vial filled with 1 mL of saline, which was similar to the ones used for NEUPOGEN, GRANIX, and NIVESTYM, served as a simple counterfeit for these drug products. All measurements were done noninvasively using the CPMG pulse sequence, at the same interpulse delay time τ (0.5 ms), at 5° C., in the probe cavity of VT-NMR. All measurements were done in triplicate, and the error bars show the standard deviations of three consecutive measurements.
Similar to the results with insulin products, a comparison of R2(1H2O) for filgrastim drug products shows detectable differences between the real biotherapeutics and a simple counterfeit in a similar vial, which means a single R2(1H2O) may be used to distinguish between them.
In summary, a single R2(1H2O) (univariate wNMR) can readily distinguish biotherapeutic products formulated as aqueous solutions from a simple counterfeit such as physiological saline.
c. Using Univariate wNMR to Detect Sophisticated Counterfeit Products Formulated as Solutions
Two sets of biotherapeutics were used to demonstrate that a single R2(1H2O) (univariate wNMR) may detect sophisticated counterfeit biotherapeutics that are formulated as aqueous solutions. Each set of products contains one innovator biotherapeutic and its two biosimilars. In each set, the three products serve as sophisticated counterfeits for each other. Note that this is a very stringent test for counterfeit detection because biosimilars are recognized by the FDA to be highly similar to their innovator products.
All measurements were done noninvasively using the CPMG pulse sequence, at the same inter-pulse delay time τ (0.5 ms), at 5° C., in the probe cavity of our VT-NMR. All measurements were done in triplicate, and the error bars show the standard deviations of three consecutive measurements. For each of these products, 10 units per product from the same batch were analyzed, in prefilled pens for insulin and glass vials for filgrastim.
The first set is the innovator insulin product LANTUS and its two biosimilar products BASAGLAR, and insulin glargine-yfgn. In
The second set is the innovator filgrastim product NEUPOGEN and its two biosimilar products, GRANIX and NIVESTYM.
d. Using Multi-Variate wNMR to Detect Sophisticated Counterfeit Products Formulated as Solutions
Multi-variate wNMR may be used for detecting counterfeit pharmaceutical products formulated as solutions. For solution products, the most straightforward form of multi-variate wNMR is the τ-dispersion profile of R2(1H2O), i.e., R2(1H2O) measured at different τ values, with τ being an adjustable parameter in the CPMG pulse sequence used to measure R2(1H2O). For solution products, their R2(1H2O) values may be very close to each other in certain τ region but much more different from each other in other τ regions. This is the rationale for using multi-variate R2(1H2O, τ) to differentiate solution counterfeits from the real solution products.
In
The results in
Substandard products refer to authentic pharmaceutical products that do not meet release specifications. There are various types of substandard products as a result of manufacturing and handling errors. In this example, mock substandard products are generated by heating or freeze/thaw (F/T) stress, two of the most common types of handling errors that may result in substandard pharmaceutical products.
a. Detection of Substandard Pharmaceutical Products Damaged by Heat Stress Using Univariate wNMR
Most biologics are sensitive to heat and therefore require a cold chain for their distribution. In this example, wNMR is used to detect heat-damaged pharmaceutical products.
Two vaccines were analyzed, (a) ENGERIX-B, presented in a glass vial containing 1.0 mL of the vaccine, and formulated using the aluminum hydroxide adjuvant ALHYDROGEL (0.5 mg/mL of Al(III)), and (b) TDVAX, presented in a glass vial containing 0.5 mL of the vaccine, and formulated using the aluminum hydroxyphosphate adjuvant ADJU-PHOS (1.06 mg/mL of Al(III)). Unstressed products serve as the reference for comparison for the two vaccines. Six vials of each vaccine were analyzed: before stress (vials 1, 2, 3, 7, 8, 9), and after heat stress 55° C. for 24 hours (vials 1, 2, 3). The remaining 3 vials from each set (vials 7, 8, 9) served as unstressed controls and were kept at 4-5° C.
All measurements were done noninvasively using the CPMG pulse sequence, at the same interpulse delay time τ (0.5 ms), at 5° C., in the probe cavity of VT-NMR. All measurements were done in triplicate, and the error bars show the standard deviations of three consecutive measurements.
An even larger difference between unstressed and heat-stressed samples was observed for TDVAX.
These results demonstrate the capability of univariate wNMR to detect pharmaceutical products that have been damaged by heat.
b. Detection of Substandard Vaccine Products Damaged by F/T-Induced Damage Using Univariate wNMR
Three aluminum-adjuvanted vaccines, DAPTACEL, ENGERIX-B and VAQTA, were subjected to F/T stress. Each vaccine contained Al3+ in quantities of 0.33 mg per dose/vial (0.66 mg/mL) for DAPTACEL as aluminum phosphate, 0.5 mg per dose/vial (0.5 mg/mL) for ENGERIX-B as aluminum hydroxide, and 0.45 mg per dose/vial (0.45 mg/mL) for VAQTA as amorphous aluminum hydroxyphosphate sulfate. Of the ten vials, vials #1-5 were kept at 5° C. to serve as controls while vials #6-10 were subjected to three cycles of the following sequence: −18° C. for 18 h followed by 5° C. for 2 h.
After each cycle, the R2(1H2O) of all ten vials of each product in their fully suspended state was measured. The results are shown in
The results show that univariate R2(1H2O) can reliably detect vials of aluminum-adjuvanted vaccines that have experienced actual freeze/thaw instead of mere exposure to subzero temperatures.
c. Detection of Substandard Drug Products Damaged by F/T-Induced Damage Using Univariate wNMR
Two emulsion products, DIPRIVAN and PROPOFOL, were subjected to one F/T cycle of −30° C. for 16 h followed by room temperature overnight. The product vials were measured before and after the F/T cycle.
d. Detecting F/T-Induced Damage Using Multi-Variate wNMR
Substandard pharmaceutical products may also be detected using multi-variate wNMR. This capability was demonstrated using F/T-damaged aluminum-adjuvanted vaccines. Note that F/T-damaged aluminum adjuvanted vaccines can be detected using univariate wNMR, as shown in the above sections, however the usefulness of multi-variate wNMR in detecting F/T-damaged products is significant. Sedimentation kinetics of aluminum-adjuvanted vaccines, i.e., R2(1H2O, t) or Type II, served as the multi-variate wNMR for detecting F/T-induced damage.
Three aluminum-adjuvanted vaccines, DAPTACEL, ENGERIX-B and VAQTA, were subjected to three cycles of −18° C. for 18 h and 5° C. for 2 h. The sedimentation kinetics of one non-stressed vial (vial #1) and one stressed vial (vial #8) was monitored via R2(1H2O). The stressed vial (vial #8) of DAPTACEL and ENGERIX-B froze after each F/T cycle while the stressed vial (vial #8) of VAQTA did not freeze after any of the three F/T cycles.
R2(1H2O, t) is clearly different for vials that experienced F/T versus vials that did not experience F/T. It is worth noting that the stressed vial (vial #8) of VAQTA did not experience any actual freeze/thaw in spite of being exposed to −18° C. The two vials of VAQTA, stressed and unstressed, show almost identical R2(1H2O, t) profiles. The results show that R2(1H2O, t) can reliably detect vaccine vials that have experienced actual freeze/thaw damage rather than mere exposure to subzero temperatures.
Here, aluminum-adjuvanted vaccines illustrate that wNMR is useful to detect counterfeit pharmaceutical products formulated as suspensions.
Suspensions and emulsions have complex hydrodynamic behavior, which might complicate the very last step of product manufacturing, the fill-finish step. For example, alum particles tend to sediment in water, which may lead to uneven filling of vials from the batch.
a. Detecting Simple Counterfeit Suspensions Using Univariate wNMR
In summary, univariate wNMR, in the form of a single R2(1H2O) value measured per pharmaceutical product, can readily distinguish aluminum-adjuvanted vaccines from simple counterfeits such as physiological saline.
b. Detecting Sophisticated Counterfeit Vaccines Using Univariate wNMR
Table 3 lists a single R2(1H2O) for eight aluminum adjuvanted vaccines. It can be seen that for any given pair of these eight vaccines, their R2(1H2O) values differ by more than the measurement error, which means a single R2(1H2O) may be used to distinguish between a pair of aluminum-adjuvanted vaccines.
However, for some pairs, their R2(1H2O), although statistically different, are close in values. For example, R2(1H2O) values for DAPTACEL and TENIVAC are 2.239±0.012 s−1 and 2.056±0.005 s−1, respectively. Multi-variate wNMR was performed for selected pairs to determine if its possible to more readily distinguish sophisticated vaccines (and counterfeits) from one another.
b. Detecting Sophisticated Counterfeit Vaccines Using Type I Multi-Variate wNMR
In Type I multi-variate wNMR, R2(1H2O) is measured at multiple τ-values, where τ is an adjustable parameter in the CPMG pulse sequence used to measure R2(1H2O). In Type I measurements, R2(1H2O, τ) is performed on fully settled suspension vaccines, noninvasively, in the original vial/syringe. Pairs of the vaccines with close values of R2(1H2O at a single τ-value (0.5 ms) in the settled state were selected. In these multi-variate wNMR experiments, the CPMG pulse sequence inter-pulse delay, τ, was varied from 0.5 ms to 10 ms with a 0.25 ms increment (˜40 different τ-values per vaccine) at a set sample temperature of 5° C. in the probe cavity of a VT-NMR.
These results clearly demonstrate the fingerprinting potential of τ-dispersion profiles and confirm the capability of wNMR technology to differentiate between real and complex counterfeit vaccines.
c. Type II Multi-Variate wNMR: Sedimentation Kinetics
In Type II multi-variate wNMR, R2(1H2O) is measured over time on (initially) a fully dispersed suspension at a single τ value (full dispersion is achieved by vigorous shaking of the vial or pre-filled syringe, as instructed by the package inserts for aluminum-adjuvanted vaccines). As the particles in the suspension settle, the R2(1H2O) changes with time. R2(1H2O, t) is the sedimentation kinetics profile of a suspension.
The sedimentation rate, ksed, of DAPTACEL was 22.683±1.056 min−1 compared to ksed of TENIVAC of 9.819±0.416 min−1, which are significantly different from each other, despite their identical adjuvant concentration. This is an illustration of how counterfeit suspension products could be distinguished from one another using Type II multi-variate wNMR.
d. Type III Multi-Variate wNMR: τ-Dispersion+Sedimentation Kinetics
In Type III multi-variate wNMR, the R2(1H2O) is measured over time on fully dispersed suspensions. However, in contrast to Type II, where R2(1H2O) is measured over time monitoring sedimentation kinetics at a single τ-value, in Type III, the R2(1H2O) is measured over time at multiple τ-values. The data, R2(1H2O, t, τ), forms a sophisticated fingerprint for a product.
The simplest Type III profile is R2(1H2O) of a fully dispersed suspension measured over time at two alternating τ-values, where the R2(1H2O) at multiple τ-values are collected during and throughout a single sedimentation process of a suspension product in an alternating pattern.
PREVNAR 13 and PREVNAR 20 are pneumococcal 13- and 20-valent conjugate vaccines, respectively, both containing 0.25 mg/mL of Al(III) as aluminum phosphate (ADJU-PHOS) adjuvant. Both vaccines are presented in pre-filled needless syringes containing 0.5 mL of a vaccine. Since these vaccines differ only by a number of antigens of S. pneumoniae serotypes—13 in PREVNAR 13 and 20 in PREVNAR 20—these two vaccines can serve as close counterfeits of each other.
The sedimentation kinetics of PREVNAR 13 and PREVNAR 20 was compared using the CPMG pulse sequence with monitoring at alternating τ-values −0.5 ms and 10 ms. Importantly, the sedimentation process was monitored with the syringes positioned tip-up in the NMR tube inside the probe of the VT-NMR. The total time of sedimentation kinetics monitoring was ˜24 hours. All measurements were performed at 4° C.
As seen from
In more sophisticated Type III multi-variate wNMR, R2(1H2O) of a fully dispersed suspension is measured over time at multiple τ values. In the examples below, τ was varied from 0.5 ms to 10 ms at 0.25 ms steps. For aluminum-adjuvanted vaccines, all τ-dispersion measurements started with suspended drug products, noninvasively, in the original vial/syringe. Again, in these experiments, pairs of the vaccines with close values of R2(1H2O) at single τ-value (0.5 ms) in settled state were selected. Measurement at a single τ-value takes ˜1 min, and total experiment duration is ˜55 min. In Type III experiments, multi-variate wNMR captures not only the dependence of R2(1H2O, t), but also the dependence of R2(1H2O, τ) synergistically providing a complex fingerprint of an individual vaccine formulation.
Remarkable differences between vaccine pairs could be seen when measured in the suspended states (
These results clearly demonstrate the fingerprinting potential of Type III multi-variate wNMR and confirm the capability of wNMR technology to differentiate between real and complex counterfeit vaccines.
e. Other Types of Multi-Variate wNMR
If needed, other types of multi-variate wNMR profile may also be constructed, such as τ values may be scanned from 0.5 ms to 10 ms at 0.25 ms steps and then decrease from 1.5 ms to 0.5 ms at 0.25 ms steps. Such cycles could be repeated multiple times to obtain a more complex picture of the sedimentation vs. τ profiles which might be unique for different drug products.
Additional types of wNMR, R1-R2 (also called T1-T2) correlation profiles for all types of products (solutions, emulsions and suspensions), R2/R1 ratio over time for suspensions at a single τ or variable τ, may be used for counterfeit detection.
Although the invention has been variously disclosed herein with reference to illustrative embodiments and features, it will be appreciated that the embodiments and features described hereinabove are not intended to limit the invention, and that other variations, modifications and other embodiments will suggest themselves to those of ordinary skill in the art, based on the disclosure herein. The invention therefore is to be broadly construed, as encompassing all such variations, modifications and alternative embodiments within the spirit and scope of the claims hereafter set forth.
This application is filed under the provisions of 35 U.S.C. § 111(a) and claims priority to U.S. Provisional Patent Application No. 63/369,333 filed on Jul. 25, 2022 in the name of Yihua (Bruce) Y U, et al., and entitled “Noninvasive Detection of Counterfeit and Substandard Vaccines and Biotherapeutics,” which is hereby incorporated by reference herein in its entirety.
This invention was made with government support under Grant Number 70NANB21H085 awarded by the National Institute for Innovation in Manufacturing Biopharmaceuticals. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63369333 | Jul 2022 | US |
