Systems and methods for top-down analysis of antibodies in mass spectrometry

Abstract

The separation device separates unknown complete mAb or reduced mAb subunits of known mAb classes from the sample. The ion source device ionizes mAb. The mass spectrometer uses an ECD device to fragment the ionized mAb and uses a mass analyzer to perform mass analysis on the resulting product ions, thereby generating one or more product ion spectra. The theoretical product ion peaks of one or more constant parts of the mAb class are calculated. The theoretical product ion peaks are removed from one or more product ion spectra to generate one or more differential product ion spectra. De novo sequencing is applied to one or more differential product ion spectra to generate one or more candidate sequences for one or more variable parts of mAb. A match with one or more candidate sequences is searched in a genomic database to generate one or more matching sequences for one or more variable parts.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 655,540, filed on April 10, 2018, and U.S. Provisional Patent Application Serial No. 62 / 813,877, filed on March 5, 2019, the contents of both of which are incorporated herein by reference in their entirety. Technical Field

[0003] The teaching of this paper relates to a mass spectrometry system and method for sequencing the variable portion of an unknown monoclonal antibody (mAb). More specifically, an electron capture dissociation (ECD) device is used to fragment the unknown mAb that is complete, digested or reduced, thereby producing a product mass spectrum. From the product mass spectrum, the theoretical product ion peak calculated for the constant portion of the mAb class is removed, thereby producing a difference product mass spectrum. De novo sequencing is applied to a difference product ion spectrum, thereby producing a candidate sequence for the variable portion of mAb. Use so-called homology search to search for the matching with one or more candidate sequences in a genomic database, thereby producing a matching sequence for a variable portion.

[0004] The systems and methods disclosed herein are also compatible with a processor, controller, microcontroller, or computer system such as Figure 1 computer system) together. Background Art

[0005] Monoclonal antibody recognition

[0006] mAbs are proteins made by immune cells. These antibodies are monoclonal because a given antibody will only bind to a single binding site or paratope. Generally speaking, antibodies 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 to also be removed or damaged.

[0007] mAb can be produced in large quantities in the laboratory for many different species. The mAbs produced in these laboratories have been shown to be highly effective and well-tolerated biopharmaceuticals for humans. They have been found to be useful for combating a variety of different types of cancers and autoimmune diseases, and are currently being tested in other diseases including Alzheimer's disease. According to Cotham et al., Anal. Chem., 2016, 88, 4004-4013 (hereinafter referred to as "Cotham paper"), more than 300 therapeutic mAb candidates and their derivatives were in clinical drug development as late as 2016.

[0008] Figure 2 An example illustration 200 of an IgG mAb is shown. Figure 2 A mAb includes two identical long heavy chains 210 and two identical shorter light chains 220. Each of the heavy chain 210 and the light chain 220 includes a constant portion and a variable portion. There are not many variations in the amino acid sequence of the constant or fixed portion of a mAb. For example, the IgG class of mAbs has four different subclasses. For each of these four different subclasses, the constant portion of an IgG mAb can vary. Therefore, for the entire IgG class, there are only four different variations of the constant portion.

[0009] In contrast, the variable parts of mAbs can vary significantly and give mAbs the unique ability to find and attach to billions of different types of foreign or diseased tissues. These variable parts include the binding site or paratope of the mAb. Although the human genome includes less than 20,000 genes, the body is able to produce antibodies with billions of different variable parts (or more specifically, billions of different paratopes). Dr. Susumu Tonegawa won the 1987 Nobel Prize in Physiology or Medicine for discovering that different parts of genes can be selected and combined to produce these billions of different variable parts.

[0010] Figure 2 Each heavy chain 210 of the IgG mAb includes constant portions 211, 212, and 213 and one variable portion 214. Each light chain 220 includes one constant portion 221 and one variable portion 222. The variable portion 214 of each heavy chain 210 includes a binding site 215, and the variable portion 222 of each light chain 220 includes a binding site 223.

[0011] The increased use of therapeutic mAbs has increased the need to experimentally identify their amino acid sequences. Mass spectrometry / mass spectrometry (MS / MS) is often used to determine the amino acid sequence of peptides and proteins. Recently, top-down and mid-down MS / MS methods have been used to identify the sequences of known therapeutic mAbs.

[0012] For example, Fornelli et al., Mol Cell Proteomics, Dec 2012; 11(12): 1758-67 (hereinafter referred to as the "first Fornelli paper") proposed a top-down MS / MS approach to sequence intact mAbs. This approach was chosen to characterize mAbs and their post-translational modifications (PTMs).

[0013] Figure 3 is a schematic diagram 300 showing the top-down MS / MS method used in the first Fornelli paper. Figure 3In step 310, a sample of many copies of a known intact mAb in solution is prepared. In step 320, liquid chromatography (LC) is performed on the solution to separate and desalt the mAb.

[0014] In step 330, MS / MS is performed on the separated mAb. For example, the separated mAb is ionized using an ion source device 331, and the mAb precursor ions are fragmented using electron transfer dissociation (ETD) in a linear ion trap (LTQ) 332. The first Fornelli paper describes that electron capture dissociation (ECD) and ETD are ion activation techniques that allow peptide fragmentation with reduced PTM losses. Finally, the precursor ions and product ions are detected using an orbital trap Fourier transform mass spectrometer (FT-MS) 333, thereby generating multiple product ion spectra 334.

[0015] In step 340, analysis of the fragmentation patterns in the multiple product ion spectra 334 is performed using top-down MS / MS analysis software. Searches are performed against a custom sequence database 341 containing known sequences of both the light and heavy chains of known mAbs. From these searches, the first Fornelli paper reported matching sequences 342 with approximately 33% coverage of the complete known mAbs.

[0016] Fornelli et al., Anal.Chem.2014, 86, 3005-3012 (hereinafter referred to as the "Second Fornelli Paper") proposed a mid-down MS / MS method to sequence mAbs. The second Fornelli paper describes that the success of mAbs as therapeutic agents has required that these mAbs be fully characterized structurally to ensure their safety, efficiency, batch-to-batch consistency and stability. Therefore, the 33% sequence coverage of the method of the first Fornelli paper must be improved. To this end, the second Fornelli paper proposes chemical reduction of mAbs to large fragments or subunits before MS / MS analysis. This use of reduced mAb subunits is called mid-down MS / MS analysis.

[0017] Figure 4 is a schematic diagram 400 showing the mid-down MS / MS method used in the second Fornelli paper. Figure 4 In step 410, a sample of many copies of a known intact mAb in solution is prepared. However, in step 420, an immunoglobulin G degrading enzyme Streptococcus pyogenes (IdeS) digestion of the mAb and reduction of the digested mAb using a disulfide bond reducing agent 421 are performed prior to LC. In step 430, LC is performed on the mAb subunits to separate and desalt the mAb subunits.

[0018] In step 440, MS / MS is performed on the separated mAb subunits. For example, the separated mAb subunits are ionized using an ion source device 441, and the mAb subunit precursor ions are fragmented using electron transfer dissociation (ETD) in a linear ion trap (LTQ) 442. Finally, the precursor ions and product ions are detected using an orbital trap FT-MS 443, thereby generating a plurality of product ion spectra 444.

[0019] In step 450, analysis of the fragmentation patterns in the multiple product ion spectra 444 is performed using MS analysis software. Searches are performed against a database 451 containing known sequences of both the light and heavy chains of known mAbs. From these searches, the second Fornelli paper reported matching sequences 452 with approximately 50% coverage of the complete known mAbs.

[0020] Like the second Fornelli paper, the Cotham paper describes that the therapeutic efficacy of mAbs is regulated by the structural integrity of the primary sequence and the presence and abundance of PTMs. Therefore, the Cotham paper found that in addition to PTM recognition and site localization, the characterization of the primary sequence of antibodies is also critical to ensuring mAb safety and efficacy. The Cotham paper reviewed the methods of the first and second Fornelli papers and proposed a middle-down method like the second Fornelli paper but using 193nm ultraviolet photodissociation (UVPD) instead of ETD fragmentation.

[0021] Figure 5 is a schematic diagram 500 showing the mid-down MS / MS method used in the Cotham paper. Figure 5 In step 510, a sample of many copies of a known intact mAb in solution is prepared. In step 520, IdeS digestion of the mAb and reduction and denaturation of the digested mAb are performed prior to LC. In step 530, LC is performed on mAb subunits.

[0022] In step 540, MS / MS is performed on the separated mAb subunits. For example, the separated mAb subunits are ionized using an ion source device 541, and the mAb subunit precursor ions are fragmented using UVPD in a higher energy collision dissociation (HCD) chamber 542. Finally, the precursor ions and product ions are detected using an orbital trap FT-MS 543, thereby generating a plurality of product ion spectra 544.

[0023] In step 550, the plurality of product ion spectra 544 are analyzed using MS analysis software. Figure 5 Using this approach, the Cotham paper reported approximately 60% total sequence coverage of known mAbs.

[0024] In step 550, analysis of the fragmentation pattern of a single spectrum 545 averaged from multiple product ion spectra 544 is performed using MS analysis software. Searches are performed against a database 551 containing known sequences of both the light and heavy chains of known mAbs. From these searches, the Cotham paper reports matching sequences 552 with approximately 60% coverage of the complete known mAbs.

[0025] The first Fornelli paper, the second Fornelli paper, and the Cotham paper all aim to identify known mAbs in sample solutions. As described in 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 efficacy as the innovator product.

[0026] 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 solution. This is a more difficult problem because, as mentioned above, the variable portion of a mAb can have billions of different possible sequences.

[0027] Therefore, there is a need for systems and methods for determining the sequence of an unknown mAb. More specifically, there is a need for systems and methods for determining the sequence of the variable portion of an unknown mAb.

[0028] Mass spectrometry background

[0029] Mass spectrometry (MS) is an analytical technique used to detect and quantify chemical compounds based on analysis of the m / z values ​​of ions formed from those compounds. MS involves ionization of one or more compounds of interest from a sample, generation of precursor ions, and mass analysis of the precursor ions.

[0030] 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 for the one or more compounds, fragmentation of the one or more precursor ions into product ions, and mass analysis of the product ions.

[0031] Both MS and MS / MS can provide qualitative and quantitative information. The measured precursor or product ion spectra can be used to identify molecules of interest. The intensities of the precursor ions and product ions can also be used to quantify the amount of the compound present in the sample.

[0032] Fragmentation technology background

[0033] Electron-based dissociation (ExD), ultraviolet light dissociation (UVPD), infrared light dissociation (IRMPD), and collision-induced dissociation (CID) are often used as fragmentation techniques for tandem mass spectrometry (MS / MS). ExD may include, but is not limited to, electron capture dissociation (ECD) or electron transfer dissociation (ETD). CID is the most common dissociation technique in tandem mass spectrometers.

[0034] As mentioned above, in top-down and middle-down proteomics, intact or digested proteins are ionized and subjected to tandem mass spectrometry. For example, ECD is a dissociation technique that preferentially dissociates peptides and protein backbones. Therefore, this technique is an ideal tool for analyzing peptide or protein sequences using top-down and middle-down proteomics approaches. Summary of the invention

[0035] Disclosed are systems, methods, and computer program products for sequencing one or more variable portions of an unknown mAb. The system includes a genomic database, a separation device, an ion source device, a mass spectrometer having one or more dissociation devices, and a processor.

[0036] The processor instructs the separation device to separate the unknown complete mAb or reduced mAb subunit of the known mAb class from the sample. The processor instructs the ion source device to ionize the unknown complete mAb or reduced mAb subunit. 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 use the dissociation device to fragment the ionized unknown complete mAb or reduced mAb subunit, and uses the mass analyzer to mass analyze the resulting product ions, thereby generating one or more product ion spectra.

[0037] The processor calculates the theoretical product ion peak of one or more constant parts of known mAb classes. The processor removes the calculated theoretical product ion peak from one or more product ion spectra, thereby producing one or more difference product ion spectra. The processor applies de novo sequencing to one or more difference product ion spectra, thereby producing one or more candidate sequences for one or more variable parts of unknown complete mAb or reduced mAb subunits. The processor searches (homology search) for the matching of one or more candidate sequences in a genome database, thereby producing one or more matching sequences for one or more variable parts.

[0038] These and other features of the applicant's teachings are set forth herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Those skilled in the art will appreciate that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0040] Figure 1 is a block diagram illustrating a computer system upon which embodiments of the present teachings may be implemented.

[0041] Figure 2 is an example illustration of an IgG monoclonal antibody (mAb).

[0042] Figure 3 is a schematic diagram showing the top-down MS / MS approach used in the first Fornelli paper.

[0043] Figure 4 is a schematic diagram showing the mid-down MS / MS approach used in the second Fornelli paper.

[0044] Figure 5 is a schematic diagram showing the mid-down MS / MS method used in the Cotham paper.

[0045] Figure 6 is an exemplary schematic diagram illustrating a system for sequencing one or more variable portions of an unknown mAb according to various embodiments.

[0046] Figure 7 is a schematic diagram of an electron capture dissociation (ECD) apparatus according to various embodiments.

[0047] Figure 8 is a cut-away three-dimensional perspective view of an ECD apparatus and a collision-induced dissociation (CID) collision cell according to various embodiments.

[0048] Fig. 9 is an exemplary product ion spectrum generated by fragmenting an unreduced intact mAb using ECD dissociation according to various embodiments.

[0049] Fig.10 is an exemplary table according to various embodiments, which shows a differential product ion peak list generated by removing or subtracting a theoretical product ion peak for a constant portion of a mAb class from a list of calculated singly charged transition product ion peaks, and shows a list of de novo sequences corresponding to the differential product ion peak list.

[0050] Fig.11 is a flow chart illustrating a method for sequencing one or more variable portions of an unknown mAb according to various embodiments.

[0051] Fig.12 is a schematic diagram of a system including one or more different software modules that execute a method for sequencing one or more variable portions of an unknown mAb according to various embodiments.

[0052] Before describing one or more embodiments of the present teaching in detail, those skilled in the art will recognize that the application of the present teaching is not limited to the details of the construction, the arrangement of components and the arrangement of steps set forth in the following detailed description or illustrated in the accompanying drawings. Moreover, it should be understood that the words and terms used herein are for descriptive purposes and should not be considered as limiting. DETAILED DESCRIPTION

[0053] Computer-implemented systems

[0054] Figure 1 1 is a block diagram illustrating a computer system 100 on which embodiments of the present teachings may be implemented. The computer system 100 includes a bus 102 or other communication mechanism for communicating information, and a processor 104 coupled to the bus 102 for processing information. The computer system 100 also includes a memory 106, which may be a random access memory (RAM) or other dynamic storage device, coupled to the bus 102 for storing instructions to be executed by the processor 104. The memory 106 may also be used to store temporary variables or other intermediate information during the execution of instructions to be executed by the processor 104. The computer system 100 also includes a read-only memory (ROM) 108 or other static storage device coupled to the bus 102 for storing static information and instructions for the processor 104. A storage device 110, such as a magnetic disk or optical disk, is provided and coupled to the bus 102 for storing information and instructions.

[0055] The computer system 100 may be coupled to a display 112, such as a cathode ray tube (CRT) or a liquid crystal display (LCD), via the bus 102 for displaying information to a computer user. An input device 114, including alphanumeric and other keys, is coupled to the bus 102 for communicating information and command selections to the processor 104. Another type of user input device is a cursor control 116, such as a mouse, trackball, or cursor direction keys, for communicating direction information and command selections to the processor 104 and for controlling cursor movement on the display 112. Such input devices typically have two degrees of freedom in two axes, namely, a first axis (i.e., x) and a second axis (i.e., y), which allows the device to specify a position in a plane.

[0056] The computer system 100 can perform the present teachings. Consistent with certain embodiments of the present teachings, results are provided by the computer system 100 in response to the processor 104 executing one or more sequences of one or more instructions contained in the memory 106. Such instructions can be read into the memory 106 from another computer-readable medium such as the storage device 110. The execution of the sequence of instructions contained in the memory 106 causes the processor 104 to perform the processing described herein. Alternatively, hard-wired circuits can be used instead of software instructions or in combination with software instructions to implement the present teachings. Therefore, the embodiments of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0057] In various embodiments, computer system 100 can be connected to one or more other computer systems (like computer system 100) across a network to form a networked system. The network can include a private network or a public network such as the Internet. In a networked system, one or more computer systems can store data and supply data to other computer systems. In a cloud computing scenario, one or more computer systems that store and supply data can be referred to as servers or clouds. For example, one or more computer systems can include one or more web servers. For example, other computer systems that send data to and receive data from a server or cloud can be referred to as clients or cloud devices.

[0058] As used herein, the term "computer-readable medium" refers to any medium that participates in providing instructions to processor 104 for execution. Such media can take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 110. Volatile media include dynamic memory, such as memory 106. Transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 102.

[0059] Common forms of computer readable media or computer program products include, for example, floppy disks, flexible disks, hard disks, magnetic tapes or any other magnetic media, CD-ROMs, Digital Video Disks (DVDs), Blu-ray Discs, any other optical media, thumb drives, memory cards, RAM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cassettes, or any other tangible medium from which a computer can read.

[0060] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 104 for execution. For example, the instructions may initially be carried on a disk of a remote computer. The remote computer may load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 100 may receive the data on the telephone line and use an infrared transmitter to convert the data into an infrared signal. An infrared detector coupled to bus 102 may receive the data carried in the infrared signal and place the data on bus 102. Bus 102 carries the data to memory 106, from which processor 104 retrieves and executes the instructions. The instructions received by memory 106 may optionally be stored on storage device 110 before or after execution by processor 104.

[0061] According to various embodiments, instructions configured to be executed by a processor to perform the method are stored on a computer readable medium. A computer readable medium may be a device that stores digital information. For example, a computer readable medium includes a compact disk read-only memory (CD-ROM) for storing software as known in the art. The computer readable medium is accessed by a processor adapted to execute the instructions configured to be executed.

[0062] For the purpose of illustration and description, the following description of various embodiments of this teaching has been given. It is not exhaustive and does not limit this teaching to the precise form disclosed. In view of the above teaching, modifications and variations are possible, or can be obtained from the practice of this teaching. In addition, the described embodiments include software, but this teaching can be implemented as a combination of hardware and software or individually implemented in hardware. This teaching can be implemented with both object-oriented and non-object-oriented programming systems.

[0063] MABS variable part determination

[0064] As mentioned above, top-down and middle-down analysis of monoclonal antibodies (mAbs) has only been demonstrated when the amino acid sequence of the 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. This is an even more difficult problem because, as mentioned above, the variable portion of a mAb can have billions of different possible sequences.

[0065] Therefore, there is a need for systems and methods for determining the sequence of an unknown mAb. More specifically, there is a need for systems and methods for determining the sequence of the variable portion of an unknown mAb.

[0066] In various embodiments, systems and methods are provided for sequencing the variable portion of an unknown mAb. Top-down or mid-down LC-MS / MS is used. Top-down LC-MS / MS is applied to intact mAbs. Mid-down LC-MS / MS is applied to reduced mAb subunits.

[0067] Theoretical product ion peaks of one or more constant parts of known mAb classes are calculated. These theoretical product ion peaks calculated are removed or subtracted from one or more product ion spectra produced by LC-MS / MS from top to bottom or from the middle to bottom. De novo sequencing is applied to one or more differential product ion spectra, thereby producing one or more candidate sequences for unknown complete mAb or reducing one or more variable parts of mAb subunit. Finally, in the genome database, search (homology search) for the matching with one or more candidate sequences, thereby producing one or more matching sequences for one or more variable parts of unknown mAb. In various embodiments, traditional top-down or from the middle to bottom MS / MS is performed again to verify these one or more matching sequences.

[0068] A system for sequencing the variable portion of unknown mAbs

[0069] Figure 6 is an exemplary schematic diagram 600 illustrating a system for sequencing one or more variable portions of an unknown mAb according to various embodiments. Figure 6 The system includes a genome database 610, a separation device 620, an ion source device 630, a mass spectrometer 640 and a processor 650.

[0070] Processor 650 instructs separation device 620 to separate unknown complete mAb or reduced mAb subunits of known mAb classes from the sample. In various embodiments, processor 650 is used to control separation device 620, ion source device 630 and mass spectrometer 640 or provide instructions to separation device 620, ion source device 630 and mass spectrometer 640 to search genome database 610 and analyze the collected data. Processor 650 controls or provides instructions by, for example, controlling one or more voltage, current or pressure sources (not shown). Processor 650 can be, for example, Figure 6 640, or may be a processor or controller of one or more devices of mass spectrometer 640. Processor 650 may be, but is not limited to, a controller, a computer, a microprocessor, Figure 1 A computer system or any device capable of sending and receiving control signals and data and analyzing data.

[0071] In various embodiments, in a top-down approach, the processor 650 instructs the separation device 620 to separate the intact unknown mAb, as shown in step 601. In this top-down approach, the intact unknown mAb in the solution 603 is supplied to the separation device 620, and the intact unknown mAb 621 is desalted and separated by the separation device 620.

[0072] In various alternative embodiments, as shown in step 602, in a middle-down approach, the processor 650 instructs the separation device 620 to separate the digested unknown mAb. The intact unknown mAb in the digestion solution 603 is digested by applying IdeS to the solution 603. Then, in this middle-down approach, the digested unknown mAb is supplied to the separation device 620, and the digested unknown mAb 622 is desalted by the separation device 620.

[0073] In various embodiments, dithiothreitol (DTT) is used to reduce the digested unknown mAb. For example, DTT is injected into the separation device 620 for online reduction. The separation device 620 separates the reduced IdeS digest of the unknown mAb.

[0074] The separation device 620 can be, but is not limited to, a liquid chromatography (LC) device or a capillary electrophoresis device. In a preferred embodiment, the separation device 620 is an LC column that separates and desalts unknown intact mAbs or reduced mAb subunits over time.

[0075] The processor 650 instructs the ion source device 630 to ionize the unknown intact mAb 621 or the reduced mAb subunit 622. The ion source device 630 may be an electrospray ion source (ESI) device. Figure 6 , ion source device 630 is shown as part of mass spectrometer 640.

[0076] Mass spectrometer 640 includes, among other devices, a dissociation device 641 and a mass analyzer 643. Processor 650 instructs mass spectrometer 640 to fragment the unknown intact mAb or reduced mAb subunits of ionization using dissociation device 641, and mass analyzes the resulting product ions using mass analyzer 643, thereby generating one or more product ion spectra 644. Mass analyzer 643 may include, but is not limited to, a time-of-flight (TOF) mass analyzer, a quadrupole, an ion trap, a linear ion trap, an orbital trap, 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 643 is a TOF mass analyzer.

[0077] The dissociation device 641 fragments the ionized unknown intact mAb or reduced mAb subunits, for example using ExD, IRMPD, CID or UVPD. In a preferred embodiment, the dissociation device 641 is an ECD device.

[0078] Figure 7 700 is a schematic diagram of an ECD device according to various embodiments. The ECD device includes an electron emitter or filament 710 and an electron gate 720. Electrons are emitted perpendicular to the flow of ions 730 and parallel to the direction of a magnetic field 740.

[0079] return Figure 6 , a mass spectrometer including a dissociation device typically includes another dissociation device, such as a Q2 dissociation device 642 for CID. The Q2 dissociation device 642 is used, for example, to fragment compounds other than proteins or peptides. During the analysis of proteins or peptides, the Q2 dissociation device 642 acts as an ion guide and simply transfers product ions from the dissociation device 641 to the mass analyzer 643.

[0080] Figure 8 is a cutaway three-dimensional perspective view 800 of an ECD apparatus and a CID collision cell according to various embodiments. Figure 8 It is shown that fragmentation of ions can be selectively performed at position 811 in the ECD device 814 or at position 812 in the CID collision cell 815 .

[0081] return Figure 6 In various embodiments, the processor 650 instructs the mass spectrometer 640 to fragment the ionized unknown intact mAb or reduced mAb subunits using an electron energy between 0 eV and 3 eV using the ECD dissociation device 641. The mass spectrometer 640 simultaneously injects electrons and ions of the unknown intact mAb or reduced mAb subunits into the ECD dissociation device 641.

[0082] In various embodiments, the ECD dissociation device 641 is a quadrupole, hexapole, or octopole dissociation device.

[0083] Processor 650 calculates theoretical product ion peaks 651 for one or more constant portions of known mAb classes. mAb classes may include, but are not limited to, IgG, IgE, IgD, IgM, and IgA.

[0084] Processor 650 removes or subtracts calculated theoretical product ion peaks 651 from one or more product ion spectra 644 , thereby generating one or more difference product ion spectra 652 .

[0085] The processor 650 applies de novo sequencing to the one or more difference product ion spectra 652, thereby generating one or more candidate sequences 653 for one or more variable portions of the unknown intact mAb or reduced mAb subunit. The processor 650 searches (homology search) for matches with the one or more candidate sequences 653 in the genome database 610, thereby generating one or more matching sequences 654 for the one or more variable portions.

[0086] In de novo sequencing, for example, one or more amino acid sequences are assigned to product ions in the product ion spectrum. Note that de novo sequencing is different from the database search performed by the first Fornelli paper, the second Fornelli paper, and the Cotham paper to find the sequence. There is no database search in de novo sequencing to determine the candidate sequence.

[0087] In various embodiments, processor 650 removes calculated theoretical product ion peak 651 by removing the theoretical product ion peak of the C-terminal product ion. Processor 650 applies de novo sequencing to the remaining N-terminal product ions of one or more differential product ion spectra 652 to generate one or more candidate sequences 653.

[0088] As described below, it was found Figure 6 The system produces 70%-80% sequence coverage of the unknown mAb. In various embodiments, processor 650 determines sequence coverage and confirms one or more matching sequences by further comparing one or more matching sequences to one or more product ion spectra of the unknown intact mAb or reduced mAb subunit.

[0089] In various embodiments, processor 650 calculates a peak list from one or more product ion spectra 644. For example, processor 650 can calculate a peak list from one or more product ion spectra 644 that are converted into singly charged product ion peaks. Processor 650 then removes the calculated theoretical product ion peaks 651 from the one or more product ion spectra by removing the theoretical product ion peaks 651 from the peak list, thereby generating a difference peak list. Processor 650 applies de novo sequencing to one or more difference product ion spectra 652 by applying de novo sequencing to the difference peak list.

[0090] Fig. 9 is an exemplary product ion spectrum 900 generated by fragmenting an unreduced intact mAb using ECD dissociation according to various embodiments. In the ECD product ion spectrum 900, singly charged product ions appear between about 500 and 1500 m / z. Multiply charged precursor ions appear between about 2100 and 4500 m / z.

[0091] Fig.101 is an exemplary table 1000 showing a differential product ion peak list generated by removing or subtracting theoretical product ion peaks of the constant portion of a mAb class from a list of calculated singly charged conversion product ion peaks, and showing a list of de novo sequences corresponding to the differential product ion peak list, according to various embodiments. In table 1000, product ion peaks of a list 1020 of theoretical product ion peaks of the constant portion of a known mAb class are compared to product ion peaks of a list 1010 of singly charged product ions. Matching peaks are removed from list 1010, thereby generating a differential peak list 1030. De novo sequencing is applied to the product ion peaks of the differential peak list 1030, thereby generating a list 1040 of de novo candidate sequences of the variable portion of an unknown mAb.

[0092] Method for sequencing the variable portion of an unknown mAb

[0093] Fig.11 is a flow chart illustrating a method 1100 for sequencing one or more variable portions of an unknown mAb, according to various embodiments.

[0094] In step 1110 of method 1100, a processor is used to instruct a separation device to separate unknown intact mAbs or reduced mAb subunits of a known mAb class from a sample.

[0095] In step 1120, a processor is used to instruct an ion source device to ionize an unknown intact mAb or reduced mAb subunit.

[0096] In step 1130, a processor is used to instruct a mass spectrometer to fragment the ionized unknown intact mAb or reduced mAb subunit using a dissociation device of the mass spectrometer, and mass analyze the resulting product ions using a mass analyzer of the mass spectrometer to generate one or more product ion spectra.

[0097] In step 1140, a processor is used to calculate theoretical product ion peaks for one or more constant moieties of known mAb classes.

[0098] In step 1150, the calculated theoretical product ion peaks are removed from the one or more product ion spectra using a processor, thereby generating one or more difference product ion spectra.

[0099] In step 1160, de novo sequencing is applied to the one or more differential product ion spectra using a processor to generate one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunit.

[0100] In step 1170, a processor is used to search a genomic database for matches to the one or more candidate sequences, thereby generating one or more matching sequences for the one or more variable portions.

[0101] In various embodiments, in an additional step 1180, the one or matching sequences are compared to one or more product ion spectra of an unknown intact mAb or reduced mAb subunit to confirm the one or more matching sequences and determine coverage of the one or matching sequences. For example, step 1180 is performed to determine Figure 6 One or more matching sequences 654 have 70%-80% sequence coverage.

[0102] Computer program product for sequencing the variable portion of an unknown mAb

[0103] In various embodiments, a computer program product includes a tangible computer-readable storage medium, the contents of which include a program having instructions that are executed on a processor to perform a method for sequencing one or more variable portions of an unknown mAb. This method is performed by a system including one or more different software modules.

[0104] Fig.12 12 is a schematic diagram of a system 1200 including one or more different software modules for executing a method for sequencing one or more variable portions of an unknown mAb according to various embodiments. The system 1200 includes a control module 1210 and an analysis module 1220.

[0105] The control module 1210 instructs the separation device to separate the unknown intact mAb or reduced mAb subunit of the known mAb class from the sample. The control module 1210 instructs the ion source device to ionize the unknown intact mAb or reduced mAb subunit. The control module 1210 instructs the mass spectrometer to fragment the ionized unknown intact mAb or reduced mAb subunit using the dissociation device of the mass spectrometer. The control module 1210 also instructs the mass spectrometer to perform mass analysis on the obtained product ions using the mass analyzer of the mass spectrometer, thereby generating one or more product ion spectra.

[0106] The analysis module 1220 calculates theoretical product ion peaks for one or more constant portions of a known mAb class. The analysis module 1220 removes the calculated theoretical product ion peaks from one or more product ion spectra, thereby generating one or more differential product ion spectra. The analysis module 1220 applies de novo sequencing to one or more differential product ion spectra, thereby generating one or more candidate sequences for one or more variable portions of an unknown complete mAb or reduced mAb subunit. Finally, the analysis module 1220 searches for matches with one or more candidate sequences in a genomic database, thereby generating one or more matching sequences for one or more variable portions.

[0107] In various embodiments, the analysis module 1220 also compares the one or matching sequence to one or more product ion spectra of unknown intact mAb or reduced mAb subunits to confirm the one or matching sequence and determine coverage of the one or matching sequence.

[0108] Experimental data

[0109] ECD offers unique features such as top-down sequencing, de novo sequencing, glycosylation analysis, and informative disulfide bond cleavage, and is an ideal tool for analyzing intact antibodies. Small and high-throughput ECD devices based on RF ion traps are used in many experiments. In this work this technique was applied to intact mAbs. It was also applied to mAb subunits reduced using a novel online disulfide bond reduction technique.

[0110] The ECD chamber was installed between Q1 and Q2 in the quadrupole TOF system. Simultaneous capture ECD mode was used for high throughput analysis, which is to inject the electron beam and precursor ions into the ECD device simultaneously. The typical electron beam exposure time was 10ms, and the electron beam intensity was tuned to obtain appropriate dissociation efficiency. The mass resolution of TOF is 35000-47000, which resolves the isotopic patterns of fragments up to Z~30+. A desalting LC column (Waters) was used for desalting, online reduction and LC separation. For demonstration, humanized monoclonal IgG (NIST-mAb) was obtained from NIST.

[0111] To obtain optimal sequence coverage in top-down analysis using an ECD-TOF system, (1) lower charged precursors can be selected to obtain a lower fragment charge state distribution, (2) ECD can be performed with electron energies of 0-3 eV, and (3) 30% to 50% precursor consumption can be used to detect large fragments in high charge states. It was found that the use of longer electron irradiation (or a stronger electron beam) induces secondary dissociation of primary ECD fragments, which removes large fragments and generates internal fragments, which are not informative for sequencing.

[0112] The complete NIST-mAb was analyzed by LC-ECD-TOF mass spectrometer. De novo sequencing of the complete ECD spectrum obtained by a single LC run indicated three sequences, and two of them matched the N-terminal partial sequence of the variable part in the light chain and heavy chain found in the human genome. The complete ECD spectrum was further analyzed in a top-down manner using the proposed complete sequence (complete sequence provided by NIST), wherein the data covered the variable part of the light chain and heavy chain of the mAb. The ECD of 3eV did not cleave the disulfide ring in the protein.

[0113] To obtain almost complete sequence coverage, the IdeS enzyme (Genovis) was applied. To reduce disulfide bonds online, DTT was injected into the intact mAb and the IdeS digest was captured on a desalting column for 1 minute. By applying reduction, 84.7% sequence coverage of the light chain, 78.3% sequence coverage of the variable part of the heavy chain (Fd') and 84.7% sequence coverage of the fixed part of the heavy chain (scFc) were obtained. In addition, ECD indicated the glycosylation sites in the scFc and their mass, and CID informed the composition of the glycans.

[0114] Although the present teaching is described in conjunction with various embodiments, it is not intended that the present teaching be limited to such embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teaching encompasses various alternatives, modifications and equivalents.

[0115] In addition, when describing various embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, insofar as the method or process does not rely on the specific order of the steps set forth herein, the method or process should not be limited to the specific sequence of steps described. As will be appreciated by those of ordinary skill in the art, other sequences of steps may be possible. Therefore, the specific order of the steps set forth in the specification should not be interpreted as a limitation on the claims. Furthermore, claims to the method and / or process should not be limited to performing their steps in the order written, and those skilled in the art can readily appreciate that these sequences can vary and still remain within the spirit and scope of the various embodiments.

Claims

1. A system for sequencing one or more variable portions of an unknown monoclonal antibody (mAb), comprising: genomic databases for homology searches; Separation equipment; Ion source equipment; a mass spectrometer comprising a dissociation device and a mass analyzer; as well as A processor, the processor: instructing the separation device to separate unknown intact mAbs or reduced mAb subunits of a known mAb class from the sample, instructing the ion source device to ionize the unknown intact mAb or reduced mAb subunit, instructing the mass spectrometer to fragment the ionized unknown intact mAb or reduced mAb subunit using the dissociation device and to mass analyze the resulting product ions using the mass analyzer to generate one or more product ion spectra, calculating theoretical product ion peaks of one or more constant moieties of said known mAb class, removing the calculated theoretical product ion peaks from the one or more product ion spectra to produce one or more difference product ion spectra, applying de novo sequencing to the one or more differential product ion spectra to generate one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunit, and The genomic database is searched for matches to the one or more candidate sequences, thereby generating one or more matching sequences for the one or more variable portions. 2 . The system of claim 1 , wherein the processor removes the calculated theoretical product ion peak by removing the theoretical product ion peak of the C-terminal product ion.

3. The system of claim 2, wherein the processor applies de novo sequencing to the remaining N-terminal product ions of the one or more differential product ion spectra to generate one or more candidate sequences.

4. The system of claim 1, wherein the processor calculates a peak list from the one or more product ion spectra.

5. The system of claim 4, wherein the processor calculates a peak list from the one or more product ion spectra converted into singly charged product ion peaks.

6. The system of claim 4, wherein the processor removes calculated theoretical product ion peaks from the one or more product ion spectra by removing the theoretical product ion peaks from the peak list to generate a difference peak list.

7. The system of claim 6, wherein the processor applies de novo sequencing to the one or more differential product ion spectra by applying de novo sequencing to the differential peak list.

8. The system of claim 1 further comprises applying an immunoglobulin G degrading enzyme digested with Streptococcus pyogenes IdeS to the intact unknown mAb to produce the reduced unknown mAb before the processor instructs the separation device to separate and desalt the reduced unknown mAb.

9. The system of claim 8, further comprising injecting dithiothreitol (DTT) into the solution having the intact unknown mAb and into the separation device when the separation device separates and desalts the reduced unknown mAb digested with IdeS.

10. The system of claim 1, wherein the dissociation device comprises an electron capture dissociation (ECD) device.

11. The system of claim 10, wherein the processor instructs the mass spectrometer to fragment the ionized unknown intact mAb or reduced mAb subunit using an electron energy between 0 eV and 3 eV with the ECD device.

12. The system of claim 10, wherein the mass spectrometer simultaneously injects ions and electrons of the unknown intact mAb or reduced mAb subunit into the ECD device.

13. The system of claim 10, wherein the ECD device comprises a quadrupole, hexapole, or octopole dissociation device.

14. A method for sequencing one or more variable portions of an unknown monoclonal antibody (mAb), comprising: separating unknown intact mAbs or reduced mAb subunits of known mAb classes from a sample using a processor-instructed separation device; instructing an ion source device to ionize the unknown intact mAb or reduced mAb subunit using the processor; instructing a mass spectrometer using the processor to fragment the ionized unknown intact mAb or reduced mAb subunit using a dissociation device of the mass spectrometer, and mass analyzing the resulting product ions using a mass analyzer of the mass spectrometer to generate one or more product ion spectra; calculating, using the processor, theoretical product ion peaks for one or more constant moieties of the known mAb class; removing, using the processor, the calculated theoretical product ion peaks from the one or more product ion spectra, thereby generating one or more difference product ion spectra; applying de novo sequencing to the one or more differential product ion spectra using the processor to generate one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunit; as well as Using the processor, a genomic database is searched for matches to the one or more candidate sequences, thereby generating one or more matching sequences for the one or more variable portions.

15. A computer program product comprising a non-transitory and tangible computer-readable storage medium, the contents of which include a program having instructions executed on a processor to perform a method for sequencing one or more variable portions of an unknown monoclonal antibody (mAb), the method comprising: Providing a system, wherein the system comprises one or more different software modules, and wherein the different software modules comprise a control module and an analysis module; Using the control module to instruct a separation device to separate unknown intact mAbs or reduced mAb subunits of a known mAb class from a sample; Using the control module to instruct an ion source device to ionize the unknown intact mAb or reduced mAb subunit; instructing a mass spectrometer using the control module to fragment the ionized unknown intact mAb or reduced mAb subunit using a dissociation device of the mass spectrometer, and mass analyzing the resulting product ions using a mass analyzer of the mass spectrometer to generate one or more product ion spectra; calculating, using the analysis module, theoretical product ion peaks of one or more constant portions of the known mAb class; removing the calculated theoretical product ion peaks from the one or more product ion spectra using the analysis module to generate one or more difference product ion spectra; applying de novo sequencing to the one or more differential product ion spectra using the analysis module to generate one or more candidate sequences for one or more variable portions of the unknown intact mAb or reduced mAb subunit; as well as The analysis module is used to search a genomic database for matches to the one or more candidate sequences, thereby generating one or more matching sequences for the one or more variable portions.

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