Patent Description:
The systems and methods disclosed herein are also performed in conjunction with a processor, controller, microcontroller, or computer system, such as the computer system of <FIG>.

mAbs are proteins made by immune cells. These antibodies are monoclonal in that a certain antibody will only bind to a single binding site or paratope. Antibodies, in general, are used by an organism's immune system to mark foreign or diseased tissue for removal. Conversely, in autoimmune diseases, antibodies mistakenly attach to normal tissue causing that tissue also to be removed or damaged.

mAbs can be produced in large numbers in the laboratory for many different species. These laboratory produced mAbs have turned out to be highly effective and well-tolerated biologic drugs for humans. They have found use in fighting many different types of cancers and autoimmune diseases and are currently being tested in other diseases including Alzheimer's. According to <NPL> (hereinafter the "Cotham Paper"), upward of <NUM> therapeutic mAb candidates and their derivatives were in clinical drug development as late as <NUM>.

<FIG> is an exemplary diagram <NUM> of an IgG mAb. The mAb of <FIG> includes two identical long heavy chains <NUM> and two identical shorter light chains <NUM>. Each of heavy chain <NUM> and light chain <NUM> includes constant and variable portions. The amino acid sequence of constant or fixed portions of mAbs does not have many variations. For example, the IgG class of mAb has four different subclasses. For each of these four different subclasses, the constant portions of the IgG mAb may vary. So there are just four different variations of the constant portions for the entire IgG class.

In contrast, the variable portions of mAbs can vary significantly and give mAbs their unique ability to find and attach to billions of different types of foreign or diseased tissue. These variable portions include the binding sites or paratopes of mAbs. Although the human genome includes less than <NUM>,<NUM> genes, the body is able to produce antibodies with billions of different variable portions, or, more specifically, billions of different paratopes. Susumu Tonegawa received the Nobel Prize for Physiology or Medicine in <NUM> for discovering that different portions of genes can be selected and combined to produce these billions of different variable portions.

Each heavy chain <NUM> of the IgG mAb in <FIG> includes constant portions <NUM>, <NUM>, and <NUM> and one variable portion <NUM>. Each light chain <NUM> includes one constant portion <NUM> and one variable portion <NUM>. The variable portion <NUM> of each heavy chain <NUM> includes binding site <NUM>, and the variable portion <NUM> of each light chain <NUM> includes binding site <NUM>.

The increased use of therapeutic mAbs has increased the need to experimentally identify their amino acid sequence. Mass spectrometry/mass spectrometry (MS/MS) is often used to determine the amino acid sequence of peptides and proteins. Recently, top-down and middle-down MS/MS methods have been used to identify the sequence of known therapeutic mAbs.

For example, <NPL> (hereinafter the "First Fornelli Paper") proposed a top-down MS/MS approach to sequence intact mAbs. This approach was chosen in order to characterize mAbs and their post-translational modifications (PTMs).

<FIG> is a schematic diagram <NUM> showing the top-down MS/MS approach used in the First Fornelli Paper. In step <NUM> of <FIG>, a sample is prepared of many copies of a known intact mAb in solution. In step <NUM>, liquid chromatography (LC) is performed on the solution to separate and desalt the mAb.

In step <NUM>, MS/MS is performed on the separated mAb. For example, the separated mAb is ionized using ion source device <NUM>, and the mAb precursor ions are fragmented using electron transfer dissociation (ETD) in linear ion trap (LTQ) <NUM>. The First Fornelli Paper describes that electron capture dissociation (ECD) and ETD are ion activation techniques that allow polypeptide fragmentation with reduced PTM loses. Finally, the precursor and product ions are detected using orbitrap Fourier Transform mass spectrometer (FT-MS) <NUM>, producing a plurality of product ion spectra <NUM>.

In step <NUM>, analysis of fragmentation patterns in plurality of product ion spectra <NUM> is performed using top-down MS/MS analysis software. Searches are performed against a custom sequence database <NUM> incorporating the known sequences of both the light and heavy chains of the known mAb. From these searches, the First Fornelli Paper reported a matching sequence <NUM> with about <NUM>% coverage of the intact known mAb.

<NPL> (hereinafter the "Second Fornelli Paper") proposed a middle-down MS/MS approach to sequence mAbs. The Second Fornelli Paper describes that the success of mAbs as therapeutics has required that these mAbs be well characterized structurally to ensure their safety, efficiency, batch-to-batch consistency, and stability. As a result, the <NUM>% sequence coverage of the method of the First Fornelli Paper had to be improved. To do this, the Second Fornelli Paper proposed chemically reducing a mAb into large fragments or subunits before MS/MS analysis. This use of reduced mAb subunits is referred to as middle-down MS/MS analysis.

<FIG> is a schematic diagram <NUM> showing the middle-down MS/MS approach used in the Second Fornelli Paper. In step <NUM> of <FIG>, a sample is prepared of many copies of a known intact mAb in solution. In step <NUM>, however, immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digestion of the mAb and reduction of the digested mAb using a disulfide bond reducing agent <NUM> are performed before LC. In step <NUM>, LC is performed on mAb subunits to separate and desalt the mAb subunits.

In step <NUM>, MS/MS is performed on the separated mAb subunits. For example, the separated mAb subunits are ionized using ion source device <NUM>, and the mAb subunit precursor ions are fragmented using electron transfer dissociation (ETD) in linear ion trap (LTQ) <NUM>. Finally, the precursor and product ions are detected using orbitrap FT-MS <NUM>, producing a plurality of product ion spectra <NUM>.

In step <NUM>, analysis of fragmentation patterns in plurality of product ion spectra <NUM> is performed using MS analysis software. Searches are performed against a database <NUM> incorporating the known sequences of both the light and heavy chains of the known mAb. From these searches, the Second Fornelli Paper reported a matching sequence <NUM> with about <NUM>% coverage of the intact known mAb.

Like the Second Fornelli Paper, the Cotham Paper describes that the therapeutic efficacy of mAbs is regulated by structural integrity with regard to the primary sequence and the presence and abundance of PTMs. As a result, the Cotham Paper finds that characterization of the antibody primary sequence in addition to PTM identification and site localization is critical to ensure mAb safety and efficacy. The Cotham Paper reviews the methods of the First Fornelli Paper and the Second Fornelli Paper and proposes a middle-down approach, like the Second Fornelli Paper, but with the ETD fragmentation replaced by <NUM> ultraviolet photodissociation (UVPD).

<FIG> is a schematic diagram <NUM> showing the middle-down MS/MS approach used in the Cotham Paper. In step <NUM> of <FIG>, a sample is prepared of many copies of a known intact mAb in solution. In step <NUM>, IdeS digestion of the mAb and reduction and denaturation of the digested mAb are performed before LC. In step <NUM>, LC is performed on the mAb subunits.

In step <NUM>, MS/MS is performed on the separated mAb subunits. For example, the separated mAb subunits are ionized using ion source device <NUM>, and the mAb subunit precursor ions are fragmented using UVPD in higher-energy collisional dissociation (HCD) cell <NUM>. Finally, the precursor and product ions are detected using orbitrap FT-MS <NUM>, producing a plurality of product ion spectra <NUM>.

In step <NUM>, plurality of product ion spectra <NUM> is analyzed using MS analysis software. Using the method of <FIG>, the Cotham Paper reported approximately <NUM>% overall sequence coverage of the known mAb.

In step <NUM>, analysis of fragmentation patterns of a single spectrum <NUM> averaged from plurality of product ion spectra <NUM> is performed using MS analysis software. Searches are performed against a database <NUM> incorporating the known sequences of both the light and heavy chains of the known mAb. From these searches, the Cotham Paper reported a matching sequence <NUM> with about <NUM>% coverage of the intact known mAb.

The First Fornelli Paper, the Second Fornelli Paper, and the Cotham Paper are all directed to identifying a known mAb in a sample solution. As described by the Second Fornelli Paper, these methods can be used in mAb drug development to show that the manufactured mAbs have similar physicochemical characteristics, functional properties, and clinical efficiency to those of the innovator product.

The methods of the First Fornelli Paper, the Second Fornelli Paper, and the Cotham Paper are not directed to sequencing unknown mAbs. In other words, these methods are not directed to mAb drug discovery. Determining the sequence of an unknown mAb is a much more difficult problem than identifying or confirming the sequence of a known mAb in a solution. It is a much more difficult problem, because, as described above, the variable portion of a mAb can have billions of different possible sequences.

As a result, there is a need for systems and methods to determine the sequence of an unknown mAb. More specifically, there is a need for systems and methods to determine the sequence of the variable portion of an unknown mAb.

Mass spectrometry (MS) is an analytical technique for detection and quantitation of chemical compounds based on the analysis of m/z values of ions formed from those compounds. MS involves ionization of one or more compounds of interest from a sample, producing precursor ions, and mass analysis of the precursor ions.

Tandem mass spectrometry or mass spectrometry/mass spectrometry (MS/MS) involves ionization of one or more compounds of interest from a sample, selection of one or more precursor ions of the one or more compounds, fragmentation of the one or more precursor ions into product ions, and mass analysis of the product ions.

Both MS and MS/MS can provide qualitative and quantitative information. The measured precursor or product ion spectrum can be used to identify a molecule of interest. The intensities of precursor ions and product ions can also be used to quantitate the amount of the compound present in a sample.

Electron-based dissociation (ExD), ultraviolet photodissociation (UVPD), infrared photodissociation (IRMPD) and collision-induced dissociation (CID) are often used as fragmentation techniques for tandem mass spectrometry (MS/MS). ExD can include, but is not limited to, electron capture dissociation (ECD) or electron transfer dissociation (ETD). CID is the most conventional technique for dissociation in tandem mass spectrometers.

As described above, in top-down and middle-down proteomics, an intact or digested protein is ionized and subjected to tandem mass spectrometry. ECD, for example, is a dissociation technique that dissociates peptide and protein backbones preferentially. As a result, this technique is an ideal tool to analyze peptide or protein sequences using a top-down and middle-down proteomics approach. "<NPL>, suggests a technique for rapid characterization of variable regions of monoclonal antibodies (mAb). Several intact mAbs were analyzed on a Thermo-Fisher LTQ-Orbitrap high-resolution mass spectrometer (MS) by in-source fragmentation. A series of b ions corresponding to N-terminal residues of both heavy chain and light chain were observed. To further characterize the variable regions, these b ions were isolated and fragmented by collision-induced dissociation in the linear trap, followed by mass analysis in the orbitrap.

A system, method, and computer program product are disclosed for sequencing one or more variable portions of an unknown mAb. The system includes a genome database, a separation device, an ion source device, a mass spectrometer with one or more dissociation devices, and a processor.

The processor instructs the separation device to separate an unknown intact mAb or reduced mAb subunits of a known mAb class from a sample. The processor instructs the ion source device to ionize the unknown intact mAb or reduced mAb subunits. The mass spectrometer includes a dissociation device and a mass analyzer. The dissociation device is preferably an ECD device. The processor instructs the mass spectrometer to fragment the ionized unknown intact mAb or reduced mAb subunits using the dissociation device and to mass analyze resulting product ions using the mass analyzer, producing one or more product ion spectra.

The processor calculates theoretical product ion peaks for one or more constant portions of the known mAb class. The processor removes the calculated theoretical product ion peaks from the one or more product ion spectra, producing one or more difference product ion spectra. The processor applies de novo sequencing to the one or more difference product ion spectra, producing one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunits. The processor searches (homology search) the genome database for matches to the one or more candidate sequences, producing one or more matched sequences for the one or more variable portions.

These and other features of the applicant's teachings are set forth herein.

Before one or more embodiments of the present teachings are described in detail, one skilled in the art will appreciate that the present teachings are not limited in their application to the details of construction, the arrangements of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings.

<FIG> is a block diagram that illustrates a computer system <NUM>, upon which embodiments of the present teachings may be implemented. Computer system <NUM> includes a bus <NUM> or other communication mechanism for communicating information, and a processor <NUM> coupled with bus <NUM> for processing information. Computer system <NUM> also includes a memory <NUM>, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus <NUM> for storing instructions to be executed by processor <NUM>. Memory <NUM> also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor <NUM>.

Computer system <NUM> may be coupled via bus <NUM> to a display <NUM>, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. Another type of user input device is cursor control <NUM>, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor <NUM> and for controlling cursor movement on display <NUM>. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

A computer system <NUM> can perform the present teachings. Consistent with certain implementations of the present teachings, results are provided by computer system <NUM> in response to processor <NUM> executing one or more sequences of one or more instructions contained in memory <NUM>. Such instructions may be read into memory <NUM> from another computer-readable medium, such as storage device <NUM>. Execution of the sequences of instructions contained in memory <NUM> causes processor <NUM> to perform the process described herein. Alternatively, hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

In various embodiments, computer system <NUM> can be connected to one or more other computer systems, like computer system <NUM>, across a network to form a networked system. The network can include a private network or a public network such as the Internet. In the networked system, one or more computer systems can store and serve the data to other computer systems. The one or more computer systems that store and serve the data can be referred to as servers or the cloud, in a cloud computing scenario. The one or more computer systems can include one or more web servers, for example. The other computer systems that send and receive data to and from the servers or the cloud can be referred to as client or cloud devices, for example.

The term "computer-readable medium" as used herein refers to any media that participates in providing instructions to processor <NUM> for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Volatile media includes dynamic memory, such as memory <NUM>. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus <NUM>.

Common forms of computer-readable media or computer program products include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disc (DVD), a Bluray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor <NUM> for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. An infra-red detector coupled to bus <NUM> can receive the data carried in the infra-red signal and place the data on bus <NUM>. Bus <NUM> carries the data to memory <NUM>, from which processor <NUM> retrieves and executes the instructions. The instructions received by memory <NUM> may optionally be stored on storage device <NUM> either before or after execution by processor <NUM>.

The following descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software, but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.

As described above, top-down and middle-down analysis of monoclonal antibodies (mAbs) has only be demonstrated when the amino acid sequence of a mAb is known. Determining the sequence of an unknown mAb is a much more difficult problem than identifying or confirming the sequence of a known mAb. It is a much more difficult problem, because, as described above, the variable portion of a mAb can have billions of different possible sequences.

As a result, there is a need for systems and methods to determine the sequence of an unknown mAb. More specifically, there is a need for systems and methods to determine the sequence of the variable portion of an unknown mAbs.

In various embodiments, systems and methods are provided to sequence the variable portion of an unknown mAb. Top-down or middle-down LC-MS/MS is used. Top-down LC-MS/MS is applied to an intact mAb. Middle-down LC-MS/MS is applied to reduced mAb subunits.

Theoretical product ion peaks for one or more constant portions of the known mAb class are calculated. These calculated theoretical product ion peaks are removed or subtracted from the one or more product ion spectra produced by the top-down or middle-down LC-MS/MS. De novo sequencing is applied to the one or more difference product ion spectra, producing one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunits. Finally, a genome database is searched (homology search) for matches to the one or more candidate sequences, producing one or more matched sequences for the one or more variable portions of the unknown mAb. In various embodiments, traditional top-down or middle-down MS/MS is performed again to validate the one or more matched sequences.

<FIG> is an exemplary schematic diagram <NUM> showing a system for sequencing one or more variable portions of an unknown mAb, in accordance with various embodiments. The system of <FIG> includes genome database <NUM>, separation device <NUM>, ion source device <NUM>, mass spectrometer <NUM>, and processor <NUM>.

Processor <NUM> instructs separation device <NUM> to separate an unknown intact mAb or reduced mAb subunits of a known mAb class from a sample. In various embodiments, processor <NUM> is used to control or provide instructions to separation device <NUM>, ion source device <NUM>, and mass spectrometer <NUM>, to search genome database <NUM>, and to analyze data collected. Processor <NUM> controls or provides instructions by, for example, controlling one or more voltage, current, or pressure sources (not shown). Processor <NUM> can be a separate device as shown in <FIG> or can be a processor or controller of one or more devices of mass spectrometer <NUM>. Processor <NUM> can be, but is not limited to, a controller, a computer, a microprocessor, the computer system of <FIG>, or any device capable of sending and receiving control signals and data and analyzing data.

In various embodiments, processor <NUM> instructs separation device <NUM> to separate an intact unknown mAb in a top-down method as shown in step <NUM>. An intact unknown mAb in solution <NUM> is supplied to separation device <NUM> in this top-down method, and intact unknown mAb <NUM> is desalted and separated by separation device <NUM>.

In various alternative embodiments, processor <NUM> instructs separation device <NUM> to separate a digested unknown mAb in a middle-down method as shown in step <NUM>. An intact unknown mAb in digest solution <NUM> is digested by applying IdeS to solution <NUM>. The digested unknown mAb is then supplied to separation device <NUM> in this middle-down method, and digested unknown mAb <NUM> is desalted by separation device <NUM>.

In various embodiments, dithiothreitol (DTT) is used to reduce the digested unknown mAb. For example, DTT is injected into separation device <NUM> for online reduction. Separation device <NUM> is separating the reduced IdeS digest of the unknown mAb.

Separation device <NUM> can be, but is not limited to, a liquid chromatography (LC) device or a capillary electrophoresis device. In a preferred embodiment, separation device <NUM> is an LC column that separates and desalts the unknown intact mAb or reduced mAb subunits over time.

Processor <NUM> instructs ion source device <NUM> to ionize unknown intact mAb <NUM> or reduced mAb subunits <NUM>. Ion source device <NUM> can be an electrospray ion source (ESI) device. Ion source device <NUM> is shown as part of mass spectrometer <NUM> in <FIG>.

Mass spectrometer <NUM> includes, among other devices, dissociation device <NUM> and mass analyzer <NUM>. Processor <NUM> instructs mass spectrometer <NUM> to fragment the ionized unknown intact mAb or reduced mAb subunits using dissociation device <NUM> and mass analyze resulting product ions using mass analyzer <NUM>, producing one or more product ion spectra <NUM>. Mass analyzer <NUM> can include, but is not limited to, a time-of-flight (TOF) mass analyzer, a quadrupole, an ion trap, a linear ion trap, an orbitrap, a magnetic sector mass analyzer, a hybrid quadrupole time-of-flight (Q-TOF) mass analyzer, or a Fourier transform ion cyclotron resonance mass analyzer. In a preferred embodiment, mass analyzer <NUM> is a TOF mass analyzer.

Dissociation device <NUM> fragments the ionized unknown intact mAb or reduced mAb subunits using ExD, IRMPD, CID, or UVPD, for example. In a preferred embodiment, dissociation device <NUM> is an ECD device.

<FIG> is a schematic diagram <NUM> of an ECD device, in accordance with various embodiments. The ECD device includes electron emitter or filament <NUM> and electron gate <NUM>. Electrons are emitted perpendicular to the flow of ions <NUM> and parallel to the direction of magnetic field <NUM>.

Returning to <FIG>, mass spectrometers that include a dissociation device, typically include another dissociation device, like Q2 dissociation device <NUM> for CID. Q2 dissociation device <NUM> is used to fragment compounds other than proteins or peptides, for example. During the analysis of proteins or peptides, Q2 dissociation device <NUM> acts as an ion guide and simply transmits product ions from dissociation device <NUM> to mass analyzer <NUM>.

<FIG> is a cutaway three-dimensional perspective view <NUM> of an ECD device and a CID collision cell, in accordance with various embodiments. <FIG> shows that fragmentation of ions selectively can be performed at location <NUM> in ECD device <NUM> or at location <NUM> in CID collision cell <NUM>.

Returning to <FIG>, in various embodiments, processor <NUM> instructs mass spectrometer <NUM> to use an electron energy between <NUM> and <NUM> eV to fragment the ionized unknown intact mAb or reduced mAb subunits using ECD dissociation device <NUM>. Mass spectrometer <NUM> simultaneously injects electrons and ions of the unknown intact mAb or reduced mAb subunits into ECD dissociation device <NUM>.

In various embodiments, ECD dissociation device <NUM> is a quadrupole, hexapole, or octupole dissociation device.

Processor <NUM> calculates theoretical product ion peaks <NUM> for one or more constant portions of the known mAb class. mAb classes can include, but are not limited to, IgG, IgE, IgD, IgM, and IgA.

Processor <NUM> removes or subtracts calculated theoretical product ion peaks <NUM> from one or more product ion spectra <NUM>, producing one or more difference product ion spectra <NUM>.

Processor <NUM> applies de novo sequencing to one or more difference product ion spectra <NUM>, producing one or more candidate sequences <NUM> for one or more variable portions of the unknown intact mAb or reduced mAb subunits. Processor <NUM> searches (homology search) genome database <NUM> for matches to one or more candidate sequences <NUM>, producing one or more matched sequences <NUM> for the one or more variable portions.

In de novo sequencing, one or more amino acid sequences are assigned to the product ions in a product ion spectrum, for example. Note that de novo sequencing is different from the database search that is performed by the First Fornelli Paper, the Second Fornelli Paper, and the Cotham Paper to find a sequence. There is no database search in order to determine the candidate sequence in de novo sequencing.

In various embodiments, processor <NUM> removes calculated theoretical product ion peaks <NUM> by removing theoretical product ion peaks of C-terminal product ions. Processor <NUM> applies de novo sequencing to remaining N-terminal product ions of one or more difference product ion spectra <NUM> to produce one or more candidate sequences <NUM>.

As described below, the system of <FIG> was found to produce a <NUM>-<NUM>% sequence coverage of the unknown mAb. In various embodiments, processor <NUM> determines the sequence coverage and confirms the one or more matched sequences by further comparing the one or matched sequences to the one or more product ion spectra of the unknown intact mAb or reduced mAb subunits.

In various embodiments, processor <NUM> calculates a peak list from the one or more product ion spectra <NUM>. For example, processor <NUM> can calculate the peak list from one or more product ion spectra <NUM> that are converted to singly charged product ion peaks. Processor <NUM> then removes the calculated theoretical product ion peaks <NUM> from the one or more product ion spectra by removing theoretical product ion peaks <NUM> from the peak list, producing a difference peak list. Processor <NUM> applies de novo sequencing to one or more difference product ion spectra <NUM> by applying de novo sequencing to the difference peak list.

<FIG> is an exemplary product ion spectrum <NUM> produced by fragmenting a non-reduced intact mAb using ECD dissociation, in accordance with various embodiments. In ECD product ion spectrum <NUM>, singly charged product ions appear between about <NUM> and <NUM>/z. Multiply charged precursor ions appear between about <NUM> and <NUM>/z.

<FIG> is an exemplary table <NUM> showing a difference product ion peak list produced by removing or subtracting theoretical product ion peaks for constant portions of an mAbs class from a calculated list of single charge converted product ion peaks and showing a list of de novo sequences that correspond to the difference product ion peak list, in accordance with various embodiments. In table <NUM>, the product ion peaks of list <NUM> of theoretical product ion peaks for constant portions of the known mAb class are compared to the product ion peaks of list <NUM> of singly charged products ions. Matching peaks are removed from list <NUM>, producing difference peak list <NUM>. De novo sequencing is applied to the product ion peaks of difference peak list <NUM>, producing list <NUM> of de novo candidate sequences of the variable portion of the unknown mAbs.

<FIG> is a flowchart showing a method <NUM> for sequencing one or more variable portions of an unknown mAb, in accordance with various embodiments.

In step <NUM> of method <NUM>, a separation device is instructed to separate an unknown intact mAb or reduced mAb subunits of a known mAb class from a sample using a processor.

In step <NUM>, an ion source device is instructed to ionize the unknown intact mAb or reduced mAb subunits using the processor.

In step <NUM>, a mass spectrometer is instructed to fragment the ionized unknown intact mAb or reduced mAb subunits using a dissociation device of the mass spectrometer and to mass analyze resulting product ions using a mass analyzer of the mass spectrometer using the processor, producing one or more product ion spectra.

In step <NUM>, theoretical product ion peaks are calculated for one or more constant portions of the known mAb class using the processor.

In step <NUM>, the calculated theoretical product ion peaks are removed from the one or more product ion spectra using the processor, producing one or more difference product ion spectra.

In step <NUM>, de novo sequencing is applied to the one or more difference product ion spectra using the processor, producing one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunits.

In step <NUM>, a genome database is searched for matches to the one or more candidate sequences using the processor, producing one or more matched sequences for the one or more variable portions.

In various embodiments, in additional step <NUM>, the one or matched sequences are compared to the one or more product ion spectra of the unknown intact mAb or reduced mAb subunits to confirm the one or more matched sequences and to determine the coverage of the one or matched sequences. For example, step <NUM> is performed to determine the <NUM>-<NUM>% sequence coverage of one or more matched sequences <NUM> of <FIG>.

In various embodiments, computer program products include a tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method for sequencing one or more variable portions of an unknown mAb. This method is performed by a system that includes one or more distinct software modules.

<FIG> is a schematic diagram of a system <NUM> that includes one or more distinct software modules that perform a method for sequencing one or more variable portions of an unknown mAb, in accordance with various embodiments. System <NUM> includes control module <NUM> and analysis module <NUM>.

Control module <NUM> instructs a separation device to separate an unknown intact mAb or reduced mAb subunits of a known mAb class from a sample. Control module <NUM> instructs an ion source device to ionize the unknown intact mAb or reduced mAb subunits. Control module <NUM> instructs a mass spectrometer to fragment the ionized unknown intact mAb or reduced mAb subunits using a dissociation device of the mass spectrometer. Control module <NUM> also instructs the mass spectrometer to mass analyze resulting product ions using a mass analyzer of the mass spectrometer, producing one or more product ion spectra.

Analysis module <NUM> calculates theoretical product ion peaks for one or more constant portions of the known mAb class. Analysis module <NUM> removes the calculated theoretical product ion peaks from the one or more product ion spectra, producing one or more difference product ion spectra. Analysis module <NUM> applies de novo sequencing to the one or more difference product ion spectra, producing one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunits. Finally, analysis module <NUM> searches a genome database for matches to the one or more candidate sequences, producing one or more matched sequences for the one or more variable portions.

In various embodiments, analysis module <NUM> further compares the one or matched sequences to the one or more product ion spectra of the unknown intact mAb or reduced mAb subunits to confirm the one or more matched sequences and to determine the coverage of the one or matched sequences.

ECD provides unique features, such as top-down sequencing, de novo sequencing, glycosylation analysis, and informative disulfide bond cleavage, and is an ideal tool to analyze intact antibodies. A small and high throughput ECD device based on an RF ion trap was used in a number of experiments. This technology was applied to an intact mAb in this work. It was also applied to a mAb subunits that were reduced using a novel online disulfide bond reduction technique.

The ECD cell was installed between Q1 and Q2 in a quadrupole-TOF system. A simultaneous trapping ECD mode was used for high throughput analysis, which is a simultaneous injection of the electron beam and precursor ions into the ECD device. Typical electron beam irradiation time was <NUM>, and the electron beam intensity was tuned to obtain appropriate dissociation efficiency. The mass resolution of the TOF was <NUM>,<NUM>-<NUM>,<NUM>, which resolved isotope patterns of fragments up to Z~<NUM>+. A desalting LC column (Waters) was used for desalting, online reduction, and LC separation. Humanized monoclonal IgG (NIST-mAb) was obtained from NIST for demonstration purposes.

To obtain the best sequence coverage in top-down analysis using the ECD-TOF system, (<NUM>) a lower charged precursor may be selected to obtain lower fragment charge state distribution, (<NUM>) ECD may be performed with electron energy of <NUM>-3eV, and (<NUM>) precursor consumption of <NUM>~<NUM>% may be used to detect large fragments in highly charged states. Using a longer electron irradiation (or stronger electron beam) was found to induce secondary dissociation of primary ECD fragments, which removes the large fragments and produces internal fragments, which are not informative for sequencing.

Intact NIST-mAb was analyzed by the LC-ECD-TOF mass spectrometer. De novo sequencing on the intact ECD spectrum obtained by a single LC run indicated three sequences, and two of them were matched to N terminal partial sequences of the variable parts in a light chain and a heavy chain found in the human genome. The intact ECD spectrum was further analyzed in top-down manner using the suggested full sequences (the full sequence is provided by NIST), where the data covered the variable parts of the light chain and the heavy chain of the mAb. ECD at <NUM> eV did not cleave the disulfide-bonded rings in the protein.

Claim 1:
A system (<NUM>) for sequencing one or more variable portions of an unknown monoclonal antibody, mAb, comprising:
a genome database (<NUM>) for homology search;
a separation device (<NUM>),
an ion source device (<NUM>);
a mass spectrometer (<NUM>) that includes a dissociation device (<NUM>) and a mass analyzer (<NUM>); and
a processor (<NUM>) configured
to instruct the separation device (<NUM>) to separate an unknown intact mAb or reduced mAb subunits of a known mAb class from a sample (<NUM>),
to instruct the ion source device (<NUM>) to ionize the unknown intact mAb or reduced mAb subunits (<NUM>),
to instruct the mass spectrometer (<NUM>) to fragment the ionized unknown intact mAb or reduced mAb subunits using the dissociation device (<NUM>) and to mass analyze resulting product ions using the mass analyzer (<NUM>), producing one or more product ion spectra (<NUM>, <NUM>),
to calculate theoretical product ion peaks (<NUM>) for one or more constant portions of the known mAb class (<NUM>),
to remove the calculated theoretical product ion peaks (<NUM>) from the one or more product ion spectra (<NUM>), producing one or more difference product ion spectra (<NUM>, <NUM>),
to apply de novo sequencing to the one or more difference product ion spectra (<NUM>), by assigning one or more amino acid sequences to the product ions in the one or more difference product ion spectra to produce one or more candidate sequences (<NUM>) for one or more variable portions of the unknown intact mAb or reduced mAb subunits (<NUM>), and
to search the genome database (<NUM>) for matches to the one or more candidate sequences (<NUM>), producing one or more matched sequences (<NUM>) for the one or more variable portions (<NUM>).