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Title:
A SYSTEM FOR PRODUCING A BIOPHARMACEUTICAL PRODUCT
Document Type and Number:
WIPO Patent Application WO/2021/176106
Kind Code:
A1
Abstract:
A system for producing a biopharmaceutical product including continuous in-line monitoring of critical process parameters and biopharmaceutical product critical quality attributes in near real-time is described. The system comprises a bioreactor for containing a cell culture comprising cell culture medium and producer cells configured to produce the biopharmaceutical product, a bioreactor sampling module fluidically coupled to the bioreactor and configured to automatically remove a sample of cell culture from the bioreactor, and a cell removal module fluidically coupled to the autosampler and configured to receive an aliquot of the sample of cell culture from the autosampler and separate cells from the aliquot to provide a cell-free sample. A cell culture analysis module is fluidically coupled to the autosampler and configured to receive an aliquot of the sample of cell culture and analyse the sample to determine a critical process parameter from the cell culture sample selected from viable cell density, and qualitative and/or quantitative analysis of at least one metabolite. The system also comprises a liquid handling module fluidically coupled to the cell removal module and configured to receive a cell free sample from the cell removal module and optionally store the cell free sample, a liquid chromatography-mass spectrometry analysis module configured to receive the cell free sample from the liquid handling module and determine in near real-time a plurality of biological product critical quality attributes in the pre-concentrated cell free sample selected from titre, aggregation profile, charge variant analysis, and middle up mass analysis, and a sample navigation controller operatively coupled to the cell removal module, cell culture analysis module, liquid handling module and liquid chromatography-mass spectrometry module and configured to control the routing of sample through the system during in-line monitoring of critical process parameters and critical product attributes of the biological product.

Inventors:
BONES JONATHAN (IE)
DORIVAL GARCIA NOEMI (IE)
FLORIS PATRICK (IE)
Application Number:
PCT/EP2021/055821
Publication Date:
September 10, 2021
Filing Date:
March 08, 2021
Export Citation:
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Assignee:
NATIONAL INSTITUTE FOR BIOPROCESSING RES AND TRAINING (IE)
International Classes:
C12M1/34; C12M1/36; G06N3/02; G16B40/00; G16C20/10
Attorney, Agent or Firm:
PURDY, Hugh, Barry (IE)
Download PDF:
Claims:
CLAIMS:

1. A system for producing a biopharmaceutical product including continuous in-line monitoring of critical process parameters and biopharmaceutical product critical quality attributes in near real-time, comprising: a bioreactor (1) for containing a cell culture comprising cell culture medium and producer cells configured to produce the biopharmaceutical product; a bioreactor sampling module (2, 3) fluidically coupled to the bioreactor and configured to automatically remove a sample of cell culture from the bioreactor; a cell removal module (4) fluidically coupled to the bioreactor sampling module (2,

3) and configured to receive an aliquot of the sample of cell culture and separate cells from the aliquot to provide a cell-free sample; a cell culture analysis module (5) fluidically coupled to the bioreactor sampling module (2, 3) and configured to receive an aliquot of the sample of cell culture and analyse the sample to determine a critical process parameter from the cell culture sample optionally selected from viable cell density, and qualitative and/or quantitative analysis of at least one metabolite; a liquid handling module (6) fluidically coupled to the cell removal module (4) and configured to receive a cell free sample from the cell removal module and optionally store the cell free sample; a liquid chromatography-mass spectrometry analysis module (7) configured to receive the cell free sample from the liquid handling module (6) and determine in near real-time a plurality of biopharmaceutical product critical quality attributes in the pre-concentrated cell free sample optionally selected from titre, aggregation profile, charge variant analysis, hydrophobic interactions, hydrophilic interactions, and middle up mass analysis; a sample navigation controller (11) operatively coupled to the cell removal module, cell culture analysis module, liquid handling module and liquid chromatography- mass spectrometry module and configured to control the routing of sample through the system during in-line monitoring of critical process parameters and critical product attributes of the biopharmaceutical product; and a computer processor (8) operatively coupled to the cell culture analyser (5) and liquid chromatography-mass spectrometry analysis module (7) and configured to receive the determined critical process parameters from the cell culture analyser and biopharmaceutical product critical quality attributes from the liquid chromatography-mass spectrometry analysis module, in which the system is configured to determine at least one critical process parameter and a plurality of biopharmaceutical product critical quality attributes in tandem during the biopharmaceutical production process within 2-4 hours of withdrawal of removal of the sample of cell culture from the bioreactor.

2. A system according to Claim 1, in which the computer processor (8) is operatively coupled to the bioreactor (1) and comprises a computational model configured to compare the determined critical process parameters and biopharmaceutical product critical quality attributes with reference critical process parameters and biological product critical quality attributes and modify operational parameters of the bioreactor based on the comparison.

3. A system according to Claim 1 or 2, in which the liquid chromatography-mass spectrometry analysis module (7) comprises: a pre-concentration module (23A) configured to pre-concentrate the cell free sample; an autosampling module (26) configured to receive the pre-concentrated cell free sample, neutralise part of the pre-concentrated cell-free sample, and reduce part of the pre-concentrated cell free sample; a liquid chromatography analysis module (24A-24D, 28) configured to receive an aliquot of the pre-concentrated and neutralised cell free sample and an aliquot of the pre-concentrated reduced cell free sample from the autosampling module and pass the aliquots through one of a plurality of liquid chromatography columns; an optical detector module configured to receive the chromatographed samples from the liquid chromatography analysis module and perform optical analysis on the chromatographed samples; and a mass spectrometry module (28, 30) configured to receive the chromatographed reduced sample from the optical detector module and perform in near real-time middle-up analysis of the reduced cell free sample.

4. A system according to Claim 1 or 2, in which the liquid chromatography-mass spectrometry analysis module (7) comprises: a pre-concentration module (23A) configured to pre-concentrate the cell free sample; an autosampling module (26) configured to receive the pre-concentrated cell free sample, neutralise part of the pre-concentrated cell-free sample, and reduce part of the pre-concentrated cell free sample; a liquid chromatography analysis module (24A-24D, 28) configured to receive an aliquot of the pre-concentrated and neutralised cell free sample and an aliquot of the pre-concentrated reduced cell free sample from the autosampling module and pass the aliquots through one of a plurality of liquid chromatography columns; and a mass spectrometry module (28, 30) configured to receive the chromatographed samples from the liquid chromatography analysis module and perform mass spectrometry analysis of the chromatographed samples to determine one or more biopharmaceutical product critical quality attributes based on the mass spectrometry analysis.

5. A system according to Claim 3 or 4, in which the liquid chromatography-mass spectrometry analysis module (24A-24D, 28) is configured to determine in near real-time a plurality of biological product critical quality attributes including titre, aggregation profile, charge variant analysis, sequence, post-translational modification profile, and glycan profile. 6. A system according to Claim 3, 4 or 5, in which the liquid chromatography analysis module (24A-24D) comprises a reverse phase chromatography column and one or more of an affinity chromatography column, a size exclusion chromatography column and a cation exchange chromatography column, and is configured to pass the reduced sample through the reverse phase chromatography column and a neutralised sample through one or more of the affinity chromatography column, size exclusion chromatography column and cation exchange chromatography column.

7. A system according to any of Clams 3 to 6, including a first pump module (20A) fluidically coupled to the liquid handling module (6) and configured to inject cell-free sample and a first mobile phase into the preconcentration module (23), and a second pump (20B) configured to inject pre-concentrated and neutralised or reduced cell free sample from the autosampler (26) and a selected second mobile phase into the liquid chromatography analysis module.

8. A system according to any of Claims 3 to 7, including a third pump module configured to continuously inject mass spectrometry mobile phase into the mass spectrometer during in line monitoring of critical process parameters and biological product critical quality attributes.

9. A system according to any of Claims 3 to 8, including an optical detector module and divert valve configured to (a) receive chromatographed neutralised sample from the liquid chromatography module, perform optical analysis on the chromatographed neutralised sample, and directed the sample to waste, or (b) receive chromatographed reduced sample from the liquid chromatography module, perform optical analysis of the reduced sample, and direct the reduced sample to the mass spectrometer.

10. A system according to any of Claims 3 to 9, including a first valving system upstream of the liquid chromatography-mass spectrometry analysis module and fluidically connected to the first and second pumps and autosampler, and configured to route the sample through the liquid chromatography-mass spectrometry analysis module.

11. A system according to any of Claims 3 to 10, including a second valving system fluidically connected to the liquid chromatography module and optical detector module and configured to route chromatographed sample from the liquid chromatography module to the optical detector module.

12. A system according to any of Claims 3 to 11 , in which the liquid chromatography analysis module comprises an affinity chromatography column fluidically coupled to a optical detector module for determining titre, a size exclusion chromatography column fluidically coupled to a optical detector module for determining aggregation profile, and a cation or anion exchange chromatography column fluidically coupled to an optical detector module for determining charge variant profile.

13. A system according to any of Claims 3 to 12, in which the pre-concentration module comprises a perfusion chromatography column.

14. A system according to any preceding Claim, configured to determine a plurality of critical process parameters from the cell culture sample and a plurality of biological product critical quality attributes in tandem in near-real time.

15. A method of producing a biological product including continuous in-line monitoring of critical process parameters and critical product attributes of the biological product in near real-time, comprising: culturing a cell culture comprising producer cells and cell culture medium in a bioreactor during a culturing period, in which the producer cells produce a biological product during the culturing period; periodically removing, in line, a sample of cell culture from the reactor during the culture period using an autosampler fluidically coupled to the reactor; transferring, in line, a first aliquot of the sample of cell culture from the autosampler to a cell removal device fluidically coupled to the autosampler and removing cells from the first aliquot of the sample of cell culture in the cell removal device to provide a cell-free sample; transferring, in line, a second aliquot of the sample of cell culture from the autosampler to a cell culture analysis module fluidically coupled to the autosampler, and analysing the second aliquot of the sample of cell culture to determine a critical process parameter from the cell culture sample selected from viable cell density, and qualitative and/or quantitative analysis of at least one metabolite; transferring, in line, an aliquot of the cell free sample to a liquid chromatography- mass spectrometry module and processing the sample to determine a biological product critical quality attribute selected from titre, aggregation profile, charge variant analysis, hydrophobic analysis, hydrophilic analysis, and middle up mass analysis; storing, by a computer processor operatively coupled to the cell culture analysis module and the liquid chromatography-mass spectrometry module, the determined critical process parameters and biological product critical quality attributes; and displaying, by a graphical display, the determined critical process parameters and biological product critical quality attributes, in which the critical process parameter and the plurality of biopharmaceutical product critical quality attributes are determined in tandem during the biopharmaceutical production process within 2-4 hours of withdrawal of removal of the sample of cell culture from the bioreactor.

16. A method according to Claim 15, including the steps of: comparing, by a computational model, the determined critical process parameters and biological product critical quality attributes with reference critical process parameters and biological product critical quality attribute; and modifying the bioreactor conditions based on the comparison.

17. A method according to Claim 16, including a step of determining a plurality of critical process parameter from the cell culture sample and a plurality of biological product critical quality attributes in tandem within 2-4 hours of withdrawal of removal of the sample of cell culture from the bioreactor. 18. A method according to Claim 16 or 17, in which the step of processing the sample by the liquid chromatography-mass spectrometry module includes the steps of: pre-treating the sample to concentrate the sample; neutralising part of the concentrated sample to provide a concentrated neutralised sample; reducing part of the concentrated sample to provide a concentrated reduced sample; assaying aliquots of the concentrated neutralised sample by liquid chromatography to determine a plurality of biopharmaceutical product critical quality attributes selected from titre, aggregation profile, hydrophilic interactions, hydrophobic interactions, and charge variant analysis; assaying the reduced sample by liquid chromatography and middle-up mass spectrometry analysis to determine a biological product parameter selected from amino acid sequence, post-translational modification, higher order structure, and glycan parameters of the biological product.

19. A method according to any of Claims 16 to 18, in which the step of assaying the concentrated neutralised sample by liquid chromatography comprises determining biological product critical quality attributes including titre, aggregation profile, and charge variant analysis.

20. A method according to any of Claims 18 or 19, in which the step of assaying the reduced sample by mass spectrometry comprises performing an amino acid sequence, post-translational modification, and glycan analysis of the biological product.

21. A method according to any of Claims 16 to 20, including a step of injecting by a first pump the cell-free sample and a first mobile phase into a first liquid chromatography module for concentration of the cell free sample, and injecting by a second pump pre concentrated and neutralised or reduced cell free sample and a selected second mobile phase into a second liquid chromatography module. 22. A method according to any of Claims 16 to 21, including a step of injecting by a third pump mass spectrometry mobile phase continuously into the mass spectrometer during in line monitoring of critical process parameters and biological product critical quality attributes.

23. A method according to any of Claims 16 to 22, in which the steps of determining a critical quality attribute of the biological product comprises analysing the concentrated and neutralised sample by liquid chromatography followed by optical analysis or analysing the reduced sample by optical analysis followed by mass spectrometry analysis.

24. A method according to any of Claims 16 to 23, in which titre of the concentrated neutralised sample is determined by affinity chromatography.

25. A method according to any of Claims 16 to 24, in which aggregation profile of the concentrated neutralised sample is determined by size exclusion chromatography.

26. A method according to any of Claims 16 to 25, in which charge variant profile of the concentrated neutralised sample is determined by cation exchange chromatography.

27. A method according to any of Claims 16 to 26, in which aggregation profile of the concentrated neutralised sample is determined by size exclusion chromatography.

28. A method according to any of Claims 16 to 23, in which cell free sample is concentrated by perfusion chromatography.

29. A method according to any of Claims 16 to 28, in which at least three of titre, aggregation profile, charge variant analysis, hydrophobic analysis, hydrophilic analysis, and middle up mass analysis are determined in tandem within 2-4 hours of removal of the sample of cell culture from the bioreactor.

30. A method according to any of Claims 16 to 29, in which at least four of titre, aggregation profile, charge variant analysis, hydrophobic analysis, hydrophilic analysis, and middle up mass analysis are determined in tandem within 2-4 hours of removal of the sample of cell culture from the bioreactor. 31. A method according to any of Claims 16 to 28, in which at least five of titre, aggregation profile, charge variant analysis, hydrophobic analysis, hydrophilic analysis, and middle up mass analysis are determined in tandem within 2-4 hours of removal of the sample of cell culture from the bioreactor.

32. A method according to any of Claims 16 to 28, in which all of titre, aggregation profile, charge variant analysis, hydrophobic analysis, hydrophilic analysis, and middle up mass analysis are determined in tandem within 2-4 hours of removal of the sample of cell culture from the bioreactor.

33. A method according to any of Claims16 to 32, including steps of determining critical process parameters and biological product critical product attributes at a plurality of time points during the culturing cycle.

Description:
TITLE

A system for producing a biopharmaceutical product

Field of the Invention

The present invention relates to a system for producing a biopharmaceutical product. Also contemplated are methods of producing a biopharmaceutical product.

Background to the Invention

A major problem encountered within the bioprocessing industry is the time taken to generate analytical data on process parameters and product quality attributes post sample collection. This situation arises due to the need for offline sample processing and then sample analysis using single attribute monitoring methods in the laboratory setting. The time between sampling and result acquisition range from hours to days to weeks. During this time, the process progresses or indeed has completed without clear knowledge or understanding of the levels of process parameters or product quality attribute levels and, therefore, the opportunity to intervene to resolve a problem or enhance performance has been lost. This lack of knowledge can cause issues with batch attrition due to out of specification results. Statistical analysis of the data relates only at the time of sampling and offers no predictive ability for future performance.

Current processes involve sample collection, transfer for purification or sample processing followed by laboratory analysis using different analytical instrumentation depending on the test requested. One or more of these operations are carried out manually requiring highly trained personnel. In some instances spectroscopic monitoring of bioprocesses has been performed, however, the data generated is limited as it relies significant post acquisition processing to generate chemometric features that behave as the analytical signal and only ever provides a “fingerprint” rather than the detailed molecular level information provided by mass spectrometry. The major problems encountered include:

1. disconnection of equipment and systems that then results in sample disconnection, i.e. samples collected from different time points in the process do not provide a clear overview of the state of the process and the product being produced.

2. No insight into future process performance, current technology platforms only allow retrospective data on the process due to the analysis not being performed in near real time.

It is an object of the invention to overcome at least one of the above-referenced problems.

Summary of the Invention

The technical objectives are met by the provision of a system fluidically coupled to a bioreactor and configured for aseptic in-line sampling of cell culture from the bioreactor and automated, in-line and near real-time processing of the sample including cell culture analysis to determine one or more critical process parameters (CPP) from the cell culture sample, cell separation to provide a cell free sample, and automated and in-line analysis of the cell free sample by liquid phase separation-mass spectrometry to provide in near real time one or more critical quality attributes (CQA) of the biological product selected from titre, aggregation profile, charge variant analysis, sequence, post translational modification and glycan analyses. The system is configured to determine CPP’s and CQA’s within a few hours of the sample being taken (e.g. within 2-4 hours), allowing the process conditions to be modified quickly to address any problems revealed by the CPP and CQA data during the same production run, and to modify subsequent production processes to avoid problems. The system is a fully automated in-line system, meaning that human intervention is not required.

The system generally comprises a bioreactor sampling module configured to periodically remove a sample of cell culture periodically during a production cycle, a cell removal system fluidically coupled to the bioreactor sampling module and configured to separate cells from the cell culture sample and provide a cell free sample, a cell culture analysis module fluidically coupled to the autosampler and configured to determine one or more critical process parameters (CPP) of the cell culture, a liquid handler fluidically coupled to the cell removal system for receipt of a cell free sample, and a liquid chromatography-mass spectrometry (LC-MS) analysis module fluidically coupled to the liquid handler and configured to receive the cell free sample and analyse the sample to determine one or more CQA’s of the biological product. The system generally includes a computer processor operatively coupled to the cell sample analyser and LC-MS analysis module for receipt of sample-specific CPP and CQA inputs, and optionally a display module for graphical display of the data. In one embodiment, the computer processor comprises a computational model configured to receive the CQA and/or CPP data, compare the data with reference CPP and/or CQA data, and provide an output based on the comparison. In one embodiment, the processor is operatively coupled to the bioreactor (i.e. via a bioreactor control module) and configured to modify the operational parameters of the bioreactor based on the outputs of the computational model.

The LC-MS analysis module generally comprises a -concentration module, a CQA analysis module including an optical detector (for example a UV or fluorescence-based optical detector), an MS module, one or more liquid chromatography columns, and an autosampler configured to prepare and route samples through the LC-MS system. The autosampler is generally fluidically coupled to the pre-concentration module for receipt of pre-concentrated cell free sample, and configured to neutralise the sample and transfer aliquots of the preconcentrated and neutralised sample to the one or more liquid chromatography columns and an aliquot of the preconcentrated and neutralised sample to the MS module.

In a first aspect, the invention provides a system (typically a system for producing a biopharmaceutical product including continuous in-line monitoring of critical process parameters and biopharmaceutical product critical quality attributes in near real-time), comprising: a bioreactor for containing a cell culture comprising cell culture medium and producer cells configured to produce the biological product; a bioreactor sampling module fluidically coupled to the bioreactor and configured to automatically remove a sample of cell culture from the bioreactor; a cell removal module fluidically coupled to the autosampler and configured to receive an aliquot of the sample of cell culture from the autosampler and separate cells from the aliquot to provide a cell-free sample; a cell culture analysis module fluidically coupled to the autosampler and configured to receive an aliquot of the sample of cell culture and analyse the sample to determine a critical process parameter (CPP) from the cell culture sample a liquid handling module fluidically coupled to the cell removal module and configured to receive a cell free sample from the cell removal module and optionally store the cell free sample; a liquid phase separation-mass spectrometry (typically liquid chromatography-mass spectrometry) analysis module configured to receive the cell free sample from the liquid handling module and determine in near real-time one or more biopharmaceutical product critical quality attributes (CQA) in the cell free sample optionally, a sample navigation controller operatively coupled to the cell removal module, cell culture analysis module, liquid handling module and liquid chromatography-mass spectrometry module and configured to control the routing of sample through the system during in-line monitoring of critical process parameters and critical product attributes of the biological product; and optionally, a computer processor operatively coupled to the cell culture analyser and liquid chromatography-mass spectrometry analysis module and configured to receive the determined critical process parameters from the cell culture analyser and biological product critical quality attributes from the liquid chromatography- mass spectrometry analysis module.

In one embodiment, the cell culture analysis module is configured to determine a CPP selected from viable cell density, and qualitative and/or quantitative analysis of at least one metabolite.

In one embodiment, the liquid chromatography-mass spectrometry analysis module is configured to determine one or more biopharmaceutical product CQA’s selected from titre, aggregation profile, charge variant analysis, hydrophobic interactions, hydrophilic interactions, and middle up mass analysis. In any embodiment, the computer processor is operatively coupled to the bioreactor and comprises a computational model configured to compare the determined critical process parameters and biological product critical quality attributes with reference critical process parameters and biological product critical quality attributes, and modify bioreactor conditions based on the comparison.

In any embodiment, the liquid chromatography-mass spectrometry analysis module comprises: a pre-concentration module configured to pre-concentrate the cell free sample; an autosampling module configured to receive the pre-concentrated cell free sample, neutralise the pre-concentrated cell-free sample, and reduce part of the pre-concentrated neutralised cell free sample; a liquid chromatography analysis module configured to receive an aliquot of the pre-concentrated and neutralised cell free sample and an aliquot of the pre- concentrated reduced cell free sample from the autosampling module and pass the aliquots through one of a plurality of liquid chromatography columns; an optical detector configured to receive the chromatographed samples from the liquid chromatography analysis module and perform optical analysis on the chromatographed sample; and a mass spectrometry module configured to receive the reduced cell free sample from the optical detector and perform in near real-time middle-up analysis of the reduced cell free sample.

In one embodiment, the mass spectrometry module is configured to receive the chromatographed samples from the liquid chromatography analysis module, wherein one or more CQA’s are determined from mass spectrometry data.

In any embodiment, the liquid chromatography-mass spectrometry analysis module is configured to determine in near real-time a plurality of biological product critical quality attributes, for example at least 4, 5 or 6 critical quality attributes selected from titre, aggregation profile, charge variant analysis, hydrophobic interactions, hydrophilic interactions, sequence, post-translational modification profile, higher order structure, and glycan profile.

In any embodiment, the liquid chromatography analysis module comprises a reverse phase chromatography column and one or more of an affinity chromatography column, a size exclusion chromatography column and an ion exchange chromatography column, and is configured to pass the reduced sample through the reverse phase chromatography column and a neutralised sample through one or more of the affinity chromatography column, size exclusion chromatography column and cation exchange chromatography column.

In any embodiment, the liquid chromatography analysis module is configured to pass aliquots of the neutralised sample through the affinity chromatography column, size exclusion chromatography column and cation (or anion) exchange chromatography column.

In any embodiment, the system comprises a first pump module fluidically coupled to the liquid handling module and configured to inject cell-free sample and a first mobile phase into the preconcentration module.

In any embodiment, the system comprises a second pump configured to inject pre concentrated and neutralised cell free sample from the autosampler and a second mobile phase into the liquid chromatography analysis module. In an embodiment in which the CQA’s are determined by liquid chromatography followed by mass spectrometry analysis, the mobile phase is a mass spectrometry compatible mobile phase.

In any embodiment, the system comprises a third pump module configured to continuously inject mass spectrometry mobile phase into the mass spectrometer during in-line monitoring of critical process parameters and biological product critical quality attributes.

In another embodiment, the system comprises a single pump system configured to inject cell-free sample and a first mobile phase into the preconcentration module, inject pre concentrated and neutralised cell free sample from the autosampler and a second mobile phase into the liquid chromatography analysis module, and continuously inject mass spectrometry mobile phase into the mass spectrometer during in-line monitoring of critical process parameters and biological product critical quality attributes.

In any embodiment, the system comprises an optical detector and divert valve configured to (a) receive chromatographed sample from the liquid chromatography module, perform optical analysis on the chromatographed sample, and directed the sample to waste, or (b) receive reduced sample from the autosampler, perform optical analysis of the reduced sample, and direct the reduced sample to the mass spectrometer.

In any embodiment, the system comprises a first valving system upstream of the liquid chromatography-mass spectrometry analysis module and fluidically connected to the first and second pumps and autosampler, and configured to route the sample through the liquid chromatography-mass spectrometry analysis module.

In any embodiment, the system comprises a second valving system fluidically connected to the liquid chromatography module and UV analyser module and configured to route chromatographed sample from the liquid chromatography module to the UV analysis module.

In any embodiment, the valving system is a multiple port multiple position switching valve.

In any embodiment, the liquid chromatography analysis module comprises an affinity chromatography column fluidically coupled to a UV detector for determining titre. In one embodiment, the affinity chromatography column is a MabPac Protein A column. Other equivalent affinity-type separation systems may be employed.

In any embodiment, the liquid chromatography analysis module comprises a size exclusion chromatography column fluidically coupled to a UV detector for determining aggregation profile. In one embodiment, the size exclusion chromatography column is a MabPac SEC-1 column. Other equivalent separation systems may be employed, for example liquid phase separation systems based on analyte size or hydrodynamic volumes.

In any embodiment, the liquid chromatography analysis module comprises a cation exchange chromatography column fluidically coupled to a UV detector for determining charge variant profile. In one embodiment, the cation exchange chromatography column is a MabPac SCX-10 column. Other equivalent separation systems may be employed, for example separation based on differences in surface charge arising from the presence of absence of posttranslational modifications or other alterations to the primary sequence. In another embodiment, this column may be an anion exchange column should the isoelectric point of the molecules under study require or be more suited to separation on such a stationary phase.

In any embodiment, the system comprises the pre-concentration module comprises a perfusion chromatography column. In one embodiment, the perfusion chromatography column is a POROS A20 column. Other separation technologies may be employed, for example r any equivalent chemistry for the separation or preconcentration of analyte based on affinity or another appropriate chemical, physical or biochemical interaction.

In any embodiment, the system is configured to determine a critical process parameter from the cell culture sample and a plurality of biological product critical quality attributes in tandem in near-real time.

In another aspect, the invention provides a method of producing a biological product including continuous in-line monitoring of critical process parameters and critical product attributes of the biological product in near real-time, comprising: culturing a cell culture comprising producer cells and cell culture medium in a bioreactor during a culturing period, in which the producer cells produce a biological product during the culturing period; periodically removing, in line, a sample of cell culture from the reactor during the culture period using an autosampler fluidically coupled to the reactor; transferring, in line, a first aliquot of the sample of cell culture from the autosampler to a cell removal device fluidically coupled to the autosampler and removing cells from the first aliquot of the sample of cell culture in the cell removal device to provide a cell-free sample; transferring, in line, a second aliquot of the sample of cell culture from the autosampler to a cell culture analysis module fluidically coupled to the autosampler, and analysing the second aliquot of the sample of cell culture to determine a critical process parameter from the cell culture sample; transferring, in line, an aliquot of the cell free sample to a liquid phase separation- mass spectrometry (generally a liquid chromatography-mass spectrometry) module and processing the sample to determine a biological product critical quality attribute; optionally, storing, by a computer processor operatively coupled to the cell culture analysis module and the liquid chromatography-mass spectrometry module, the determined critical process parameters and biological product critical quality attributes; and optionally, displaying, by a graphical display, the determined critical process parameters and biological product critical quality attributes.

In one embodiment, the method comprises determining a CPP selected from viable cell density, and qualitative and/or quantitative analysis of at least one metabolite.

In one embodiment, the method comprises determining a biopharmaceutical product CQA selected from titre, aggregation profile, charge variant analysis, hydrophobic interactions, hydrophilic interactions, and middle up mass analysis.

In any embodiment, the method comprises the steps of: comparing, by a computational model, the determined critical process parameters and biological product critical quality attributes with reference critical process parameters and biological product critical quality attribute; and modifying the bioreactor conditions based on the comparison.

In any embodiment, the step of processing the sample by the liquid chromatography-mass spectrometry module includes the steps of: pre-treating the sample to concentrate the sample; neutralising the concentrated sample to provide a concentrated neutralised sample; treating part of the concentrated neutralised sample to provide a concentrated reduced (or hydrolysed or enzymatically transformed) sample; assaying aliquots of the concentrated neutralised sample by liquid chromatography (and optionally mass spectrometry) to determine a biological product critical quality attributes selected from titre, aggregation profile, and charge variant analysis; assaying the reduced sample by liquid chromatography and middle-up mass spectrometry analysis to determine a biological product parameter selected from amino acid sequence, post-translational modification, and glycan parameters of the biological product.

In any embodiment, the method comprises a step of assaying the concentrated neutralised sample by liquid chromatography to determine biological product critical quality attributes including titre, aggregation profile, and charge variant analysis.

In any embodiment, the method comprises a step of assaying the reduced sample by mass spectrometry to perform an amino acid sequence, post-translational modification, higher order structure, and glycan analysis of the biological product.

In any embodiment, the method comprises passing the reduced sample through a reverse phase chromatography column and a neutralised sample through one or more, and preferably all, of the affinity chromatography column, size exclusion chromatography column and cation exchange chromatography column.

In any embodiment, the method comprises a step of injecting (typically by a first pump) the cell-free sample and a first mobile phase into a first liquid chromatography module for concentration of the cell free sample, and injecting (typically by a second pump) pre concentrated and neutralised cell free sample a second mobile phase into a second liquid chromatography module. In any embodiment, the method comprises a step of injecting (typically by a third pump) mass spectrometry mobile phase continuously into the mass spectrometer during in-line monitoring of critical process parameters and biological product critical quality attributes.

In any embodiment, the steps of determining a critical quality attribute of the biological product comprises analysing the concentrated and neutralised sample by liquid chromatography followed by optical analysis or analysing the reduced sample by optical analysis followed by mass spectrometry analysis.

In any embodiment, the titre of the concentrated neutralised sample is determined by affinity chromatography.

In any embodiment, the aggregation profile of the concentrated neutralised sample is determined by size exclusion chromatography.

In any embodiment, the charge variant profile of the concentrated neutralised sample is determined by cation or ion exchange chromatography.

In any embodiment, the aggregation profile of the concentrated neutralised sample is determined by size exclusion chromatography.

In any embodiment, the cell free sample is concentrated by perfusion chromatography

In any embodiment, the method comprises a step of determining a plurality of critical process parameter from the cell culture sample and a plurality of biological product critical quality attributes in tandem in near-real time.

In any embodiment, the method comprises a step of determining critical process parameters and biological product critical product attributes at a plurality of time points during the culturing cycle.

There is also provided a computer program comprising program instructions for causing a computer program to carry out the above method which may be embodied on a record medium, carrier signal or read-only memory. Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

Figure 1 is an illustration of a system for producing a biopharmaceutical product according to the invention.

Figure 2 is an illustration of a liquid chromatography-mass spectrometry analysis module forming part of the system of Figure 1.

Figure 3 is another illustration of a liquid chromatography-mass spectrometry analysis module forming part of the system of Figure 1.

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and general preferences

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

As used herein, the terms “biopharmaceutical product” or “biological product” are used interchangeably and refer to a product produced by a cell in culture in a bioreactor. Examples of cells (i.e. producer cells) include but are not limited to prokaryotic cells such as bacteria and eukaryotic cells such as Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) cells. Examples of biopharmaceutical products include antibodies (i.e. monoclonal antibodies), proteins (i.e. Fc-fusion proteins), blood factors, thrombolytic agents, hormones, interferons, haematopoietic growth factors, and nucleic acid-based biopharmaceutical products (such as gene therapy drugs).

As used herein, the term “critical process parameter” or “CPP” should be understood to refer to viable cell density, or a qualitative and/or quantitative analysis of at least one metabolite.

As used herein, the term “critical quality attribute” or “CQA” as applied to the biopharmaceutical product refers to one ore more of titre, aggregation profile, charge variant analysis, hydrophobic interactions, hydrophilic interactions, and middle-up mass analysis (including one or more of amino acid sequence, post -translational modification, higher order analysis, and glycan analysis). Generally, the method of the invention comprises determining a plurality of CQA’s for the biopharmaceutical product, optionally including titre, aggregation profile, charge variant analysis, hydrophobic interactions, hydrophilic interactions, and middle-up mass analysis. Determination of the or each CQA usually employs pre-treatment of the sample by a liquid chromatography separation technique, and then optical (i.e. UV) analysis of the chromatographed sample. In another embodiment, one or more CQA’s may be determined by liquid chromatography followed by mass spectrometry analysis of the chromatographed sample, where CQA’s are determined from the mass spectrometry data. Middle-up mass analysis generally employs mass spectrometry. Other CQA’s are determined using liquid phase separation systems, for example liquid chromatography columns or electrophoresis (capillary electrophoresis or microchip electrophoresis).

As used herein, the term “automated” as applied to the system of the invention means that the routing of sample through the system is controlled by the system and does not require human intervention. Thus, the system generally comprises a sample navigation controller operatively coupled to most or all of the autosampler, cell removal module, liquid handler, and LC-MS analysis module. In one embodiment, the term includes the analysis of CPP and CQA data by computer processor, and optional control of bioreactor operation based on the analysis of the data.

As used herein, the term “in-line” as applied to the system of the invention means that the parts of the system are fluidically connected as described herein allowing the end-to-end “hands-free” routing of sample through the various parts of the system from the bioreactor to the LC-MS system, and optionally the collection and optional analysis of CPP and CQA by a computer processor.

As used herein, the term “near real-time” as applied to the determination of a CPP or CQA of a biological product during a biopharmaceutical production process means that time taken from withdrawal of a sample from a bioreactor to the determination of one or more CPP and/or CQA’s takes less than 12 hours, and typically less than 6, 5, 4, 3.5 or 3 hours. In one embodiment, the term means within about 2-4 or 2-3 hours of withdrawal of sample from the bioreactor.

As used here, the term “bioreactor sampling module” refers to a device fluidically coupled to the bioreactor and configured to aseptically remove a sample of cell culture from the bioreactor upon actuation. It may comprise a sample pilot unit operatively connected to a modified dip tube positioned within the bioreactor vessel’

As used here, the term “cell removal module” refers to a unit configured to process a sample of cell culture to provide a cell free sample. In one embodiment, the module is configured to pass the cell culture through a TFF membrane

As used here, the term “cell culture analysis module” refers to a device configured to analyse a sample of cell culture for online metabolite or viable cell density (VCD) analysis. In one embodiment, the device is an automated cell culture analyse such as a Nova Flex II analyser or other equivalent device.

As used here, the term “liquid handling module” refers to an automated robotic liquid handling device fluidically connected to the LC-MS analysis module and configured to receive cell free sample routed from the cell removal module and store or aliquot the sample into multiple samples. One example is a Gilson GX-271 liquid handler.

As used here, the term “liquid chromatography-mass spectrometry analysis module” or “LC-MS analysis module” refers to an analysis module comprising one or more liquid chromatography columns, a UV analyser, a mass spectrometry analyser, and typically an autosampler configured for preparation of sample for analysis and optionally aliquoting of samples. It will be appreciated that the liquid chromatography column(s) may be replaced with a different liquid phase separation system for example an electrophoresis-based separation system (i.e. capillary electrophoresis or microchip electrophoresis), in which case the LC-MS module may be a LPS-MS module (liquid phase separation-mass spectrometry module). In one embodiment, the LC-MS analysis module is configured to route sample through a pre-concentration LC module (i.e. a Protein A column) to an autosampler, neutralise the pre-concentrated sample, and route aliquots of the pre concentrated and neutralised sample to the LC analysis columns, generally in a sequential fashion. The autosampler is also configured to reduce an aliquot of the pre-concentrated and neutralised sample, for downstream middle-up analysis by the mass spectrometer. The LC-MS analysis module also typically comprises a plurality of pumps and switching valves. In any embodiment, the module comprises a first pump module fluidically coupled to the liquid handling module and configured to inject cell-free sample and a first mobile phase into the preconcentration module. In any embodiment, the module comprises a second pump configured to inject pre-concentrated and neutralised cell free sample from the autosampler and a second mobile phase into the liquid chromatography analysis module. In any embodiment, the system comprises a third pump module configured to continuously inject mass spectrometry mobile phase into the mass spectrometer during in line monitoring of critical process parameters and biological product critical quality attributes.

As used herein, the term “sample navigation controller” refers to system operatively coupled to the cell removal module, cell culture analysis module, liquid handling module and liquid chromatography-mass spectrometry module and configured to control the parts of the system of the invention including the periodic withdrawal of cell culture fluid from the bioreactor, routing of cell culture to the cell culture analyser or the cell removal system, and the downstream management of the analytical cascade in the LC-MS system during in-line monitoring of critical process parameters and critical product attributes of the biopharmaceutical product. In one embodiment, a MAST system (Lonza) is employed as the sample navigation controller.

As used here, the term “autosampler” refers to a liquid handling device forming part of the LC-MS analysis module and configured to collect samples, prepare samples for analysis (for example sample neutralisation and sample reduction) and prepare aliquots of any sample. Many autosamplers for liquids consist of a carousel and the sampling apparatus. The carousel holds the samples, and revolves around its centre so that samples change their horizontal position. There may be several concentric rings holding samples in a carousel. The sampling apparatus can be fixed horizontally, only moving up and down to allow the carousel to move, or it can also move horizontally, depending on the design of the system. The sampling apparatus in most of such autosamplers consist of a needle connected to a remote pumping syringe via tubing.

As used herein, the term “sample concentration” or “sample pre-concentration” refers to treatment of the cell free sample to increase the concentration of the biopharmaceutical product in the sample. This may be achieved by passing the cell free sample through a perfusion chromatography column, for example a POROS A20 column. The mobile phase employed may be a mixture of (a) 1x PBS (pH 7.5) and (b) 1x PBS 30 mM HCL (pH 1.9). The mobile phase and cell free sample from the liquid handler are generally injected into the chromatography column using a first pump module, for example a UltiMate LPG- 3400RS quarternary pump. In one embodiment, the biopharmaceutical product in the cell free sample is concentrated by at least 50% and up to 500%, and preferably about 250%.

As used herein, the term “sample neutralisation” should be understood to mean the automated addition of liquid to change the chemical properties of the collected sample. In one embodiment this might include the addition of a basic or alkaline solution to an acidic eluent to raise the pH of the collected sample towards neutrality or another pH value as required by subsequent analytical steps. In another embodiment this might include the reverse situation. In a further embodiment, this might include the addition of water or other hypotonic solutions to adjuct chemical parameters such as osmolality or conductivity of the solution to facilitate downstream analysis. In other embodiments, this may also include the addition of specific chemical or biochemical entities to the collected sample to enable transformation of the physical, chemical or biochemical properties of the analytes to facilitate downstream analysis.

As used herein, the term “optical detector” or “optical detector module” refers to a UV detector or a fluorescence-based detector.

The embodiments in the invention described with reference to the drawings comprise a computer apparatus and/or processes performed in a computer apparatus. However, the invention also extends to computer programs, particularly computer programs stored on or in a carrier adapted to bring the invention into practice. The program may be in the form of source code, object code, or a code intermediate source and object code, such as in partially compiled form or in any other form suitable for use in the implementation of the method according to the invention. The carrier may comprise a storage medium such as ROM, e.g. CD ROM, or magnetic recording medium, e.g. a floppy disk or hard disk. The carrier may be an electrical or optical signal which may be transmitted via an electrical or an optical cable or by radio or other means.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

The proposed fully automated ‘end to end’ multi-attribute analytical platform provides an integrated scalable analytical solution to address the unique problem of fully automated Critical Quality Attributes (CQAs) sampling and analysis for which no market solution currently exists. The platform is based on state of the art LC-MS instrumentation, in particular liquid chromatography instrumentation with fractionating autosampler capabilities that allows for liquid handling and sample preparation within the instruments fluidics combined with high resolution intact mass profiling using world leading mass spectrometers. The combined technical solution proposed vastly surpasses current state of art and will enable Biopharma Industries to increase process understanding in a rapid fashion, improve the level of product quality and related data available during a process campaign and enable the development of more advanced process control strategies resulting in enhanced process robustness and performance. Considering that the majority of Irish biopharmaceutical manufacturing sites produce monoclonal antibodies (mAbs) and Fc fusion proteins (FcFPs), the proposed platform has been specifically formulated for monitoring bioproduction process of these biotherapeutics.

The system consists in an automated on-line multi-attribute analytical platform for monitoring therapeutic protein CQAs and associated critical process parameters (CPPs) in near real time using high resolution LC-MS in tandem with appropriate additional nutrient and metabolite analysers in the process development and/or production environment.

Using an automated bioreactor sampling system, the aseptic sampling of media is optimised, as well as a further generation of free cell samples and hyphenation the high resolution LC- MS system is achieved, to enable product-level information and in depth fully automated CQAs, which includes the determination of: (i) Product titre; (ii) Aggregate profile; (iii) Charge variant analysis; (iv) Middle-up mass analysis for sequence verification, and post- translational modification (PTM) identification and glycan analysis.

This will in one embodiment be achieved by using the fractionating autosampler capabilities within the LC to redirect and manipulate the sampled product on to various separation chemistries prior to high resolution accurate mass profiling using high resolution Orbitrap mass spectrometry. In parallel, an aliquot of sample stream will be directed to a commercial nutrient and metabolite analyser for profiling. Bioinformatics and associated software development will enable the generation of a simplified visualisation interface for interpretation of the multi-attribute analytical data. Data aggregation and visualisation will be executed in manner which meets the needs of process development scientist as well as process operators.

Referring to the drawings, and initially to Figure 1, a system according to one embodiment of the invention is illustrated. The system comprises a bioreactor 1 containing a cell culture (cell culture medium plus producer cell) configured to produce a biopharmaceutical product, a bioreactor sampling module 2 comprises a sampling dip tube 3 configured to aseptically withdraw a sample of cell culture, a cell removal system 4 fluidically coupled to the sampling module 2 and configured to receive an aliquot of the cell culture and treat the cell culture to remove cells and provide a cell free sample, and a cell culture analysis module 5 fluidically coupled to the sampling module 2 and configured to receive a sample of the cell culture and analyse the cell culture for a critical process parameter selected from viable cell density and qualitative and/or quantitative metabolite analysis.

The system also includes a liquid handling module 6 fluidically coupled to the cell removal module 4 and configured to receive and store cell free samples from the cell removal module 4, a liquid chromatography-mass spectrometry (LC-MS) analysis module 7 configured to determine one or more critical process parameters (CPP’s) and/or critical quality attributes (CQA’s) of the cell free sample, and a computer processor 8 operatively coupled to the LC- MS analysis module 7 and cell culture analysis module 5 and configured to receive sample- specific CPP and CQA data, and display the CPP and CQA data on a screen. In one embodiment, the computer processor 8 comprises a computational model configured to compare the CPP and/or CQA data with reference data, and provide an output based on the comparison. The computer processor 8 may be operatively coupled to a bioreactor control module 10 configured to modify the bioreactor conditions in response to the computational model output.

The system also includes a sample navigation controller 11 operatively coupled to the bioreactor sampling module 2, cell removal system 4, liquid handling module 6 and LC-MS analysis module 8 and configured to control the parts of the system of the invention including the periodic withdrawal of cell culture fluid from the bioreactor, routing of cell culture to the cell culture analyser or the cell removal system, and the downstream management of the analytical cascade in the LC-MS system during in-line monitoring of critical process parameters and critical product attributes of the biopharmaceutical product. In one embodiment, a MAST system (Lonza) is employed as the sample navigation controller.

Referring to Figure 2, the LC-MS analysis system is described in more detail, and comprises a first pump/mobile phase module 20A configured to inject a first mobile phase and cell free sample from the liquid handling module 6 through a first switching valve 22A that routes the cell free sample into a pre-concentration column 23A of the liquid chromatography instrument where the biopharmaceutical product in the sample is concentrated before being routed by a second switching valve 22B to an autosampler 26. In the autosampler, the concentrated sample is neutralised with basic buffer and aliquots of the concentrated and neutralised sample are generated and optionally stored prior to further analysis. Aliquots are then individually combined with second mobile phase and injected via a second pump/mobile phase module 20B through the switching valve 22A where an aliquot of the concentrated neutralised sample is routed through each of the three liquid chromatography analytical columns 24A-24C and a UV analyser 28 via a switching valve 22B, for determination of titre, aggregation profile and charge variant analysis (as described in further detail below).

In the autosampler 26, an aliquot of the pre-concentrated sample is reduced with guanidine HCI and 125 mM TCEP and incubated for 1 hour at 40°C. The reduced sample is injected via second pump/mobile phase module 20B and switching valves 22A and 22B through reverse phase chromatography column 24D and UV analyser 28. Switch valve 29 downstream of UV analyser 28 is configured to direct chromatographed samples from LC columns 24A-24C to waste 31 and chromatographed samples from LC column 24D to a mass spectrometer 30 for middle-up analysis. Third pump/mobile phase module 20 is fluidically connected to the mass spectrometer 30 and configured to continuously inject MS mobile phase into the mass spectrometer 30 during an analytical cascade. For ease of explanation, the transfer of neutralised sample and reduced sample from autosampler 26 to switching valve 22A is illustrated with different lines, but it will be appreciated that the same conduit may be used and the system will be flushed after neutralised sample has been analysed in columns 24A-24C and prior to reverse phase analysis of reduced sample in column 24D.

The details of analytical cascade controlling software and functions, mobile phases pumped by each of the three pump/mobile phase modules, and switching valve positions are described in more details below.

Automated bioreactor sampling system.

A platform for automated and aseptic bioreactor sampling should be selected. The Modular Automated Sampling Technology (MAST™) from Lonza is one of the options. The MAST connect Master Controller Software allows to harness the full potential of the MAST system through 2 controllers: Master Controller and Analytical Navigator.

The Master Controller is the brain of the MAST system and is used to control all connected devices and modules, as the sample pilot (SP) module, used to safely collect samples maintaining the bioreactor sterility. The Master Controller operates the system and maintains critical operational interlocks. The Master Controller communicates with the MASTconnect software allowing the user to operate the MAST system.

The Analytical Navigator controller is responsible of directing the samples to the 2 different analytical destinations, either the metabolite analyser (where BioProfile® FLEX2™ from Nova® Biomedical is one of the options) or the Cell Removal System (CRS) module. The Analytical Navigator controller facilitates communications and data integrity when samples arrive to the different destinations.

The CRS module will generate cell-free samples from culture media coming from the bioreactor, using tangential flow filtration (TFF), isolating cells from the whole sample in the retentate, producing a cell-free permeate sample that will be directed afterwards to the Gilson® GX-271 liquid handler, where the sample will be collected and further injected to the LC-MS system for analysis.

Automated analytical platform scouting.

Figure 3 shows the full setup for the analytical platform, indicating a detailed description of (i) the required modules and instruments, as well as their configuration; (ii) analytical columns; and (iii) mobile phases.

The generic full setup for the analytical platform contains:

. 3 analytical pumps (indicated as A, B, and C), whose functions are defined later.

. 5 analytical columns, distributed in

. 2 column compartments (TCC).

. 2 7-ports 6-position switching valves.

. 1 UV detector.

. 1 autosampler/fraction collector.

. 1 Mass Spectrometer detector.

Chromeleon™ 7.2.9, working with MAST as a service in the background controls the analytical cascade in the LC-MS that is launched from the MAST master controller software using Chromeleon eWorkflows.

The analytical cascade consists of a sequence of analysis for the CQAs assessment of the sampled product at different intervals over the culture time:

. Step 1. Antibody purification and collection.

. Step 2. Antibody neutralization and Titre analysis. . Step 3. Aggregation analysis.

. Step 4. Charge variant analysis.

. Step 5. System flushing for preparation for reverse phase chromatography analysis.

. Step 6. Antibody reduction step and Middle up analysis.

. Step 7. Final system flushing for preparation for the next analytical cascade.

Table 1 shows the sample block for the first eWorkflow sequence. It will be required as many eWorkflows as many samples are analysed. All these eWorkflows differ in the vial position where the antibody will be collected, and the well plate position where the antibody reduction step is performed. Both positions are defined according to the user’s convenience.

Table 1. Sample block for the first eWorkflow sequence

Considerations for instrument methods. Table 2 presents all analytical parameters that should be specified in the instrument methods for the analysis of the mentioned CQAs. The included parameters values are presented as example, which can be modified/customised by the user according to their specific analytical conditions and needs. Table 2. Analytical features of instrument methods.

Total (min) 206.1

(*) Equilibration time corresponds to the incubation time for the antibody reduction Total (h) 3.4 Although most of the settings are clearly defined, there are others that need to be explained:

Analytical pumps: The analytical platform requires 3 pump systems, indicated as A, B, and C in Figure 1. Pump A is dedicated to the injection of cell-free samples from the Gilson Liquid Handler to the LC-MS system, and works with mobile phases for the step 1 of the cascade. Pump B is used for the rest of the cascade (steps 2 to 7), working with 3 different combinations of mobile phases for the required analyses. Pump C is dedicated to pump a mixture of methanol:water (1 :1) to the MS detector while the system is running UV-based methods.

Divert valve position at MS: after the UV detector, the chromatographic fluid arrives to a 2- position divert valve before the MS detector. As most of the analyses require only UV detection, once the chromatographic fluid arrives to the divert valve, is directed to waste (which is “position 2” in Table 2). The middle-up analysis is the only one that requires MS detection, for this reason once the chromatographic fluid arrives to the divert valve, is directed to the MS (which is “position 1” in Table 2).

MS flushing method: As the chromatographic fluid is diverted to waste before arriving the MS detector for the UV-based analyses, the MS will be “acquiring” data without any sample entering the MS, which may damage some components of the HESI source. To solve this problem, the system was configured to continuously pump a mixture of methanol: water (1:1) through the MS while the system is running the UV-based analyses. This is shown in Table 2 as “On - going to MS" for UV detection-based methods, which is also configured when the divert valve is in “position 2”. For the middle-up analysis, as the chromatographic fluid is directed to the MS, the MS flushing solvent is diverted to waste, which explains the description “On - going to waste", that is also configured when the divert valve is in “position 1”, as it is also shown in Table 2.

Equilibration time: This is the time dedicated to equilibrate the system to the conditions for the upcoming analysis: mobile phases, flow rates, column temperature. The time indicated in Table 2 was calculated based mainly on the experience, taking into consideration also the type of mobile phases and flow rates.

Injection preparation time: This parameter considers the time required for the injection process before the actual gradient separation takes place (which is indicated as run time). As some methods of the analytical cascade require sample preparation steps, all injection methods are defined by the user, being adjusted according to the user needs. User-Defined Program (UDP): Besides full and partial injection modes, Chromeleon software offers UDPs to define the characteristics of the injection process according to the user needs. This is particularly important when the sequence includes different types of analyses. As well, UDPs are useful to configure the sample preparation steps that are required specifically in 2 stages of the analytical cascade: (i) antibody neutralisation with basic buffer after its collection under acid conditions; (ii) antibody reduction step and incubation for the middle-up analysis, which consists in previous antibody denaturation with 4 M Guanidine. HCI, further reduction with 125 mM TCEP and incubation during 1 h at 40°C. All these distinctive steps for the antibody reduction are generated as part of the UDP. Both sample preparation steps take place in the autosampler.

Autosampler racks distribution for the analytical cascade: As different types of analyses will take place, including sample preparation steps, it is critical to organise properly where all these specific events will occur.

The UltiMate™ 3000 autosampler has 3 vials racks called as R, B and G. Each rack has a specific purpose in the analytical cascade.

Rack R contains a vial with water in position RA1 that will trigger Step 1 : the initial injection to the LC system of 2 ml_ cell-free culture sample from the CRS step collected in a vial in the Gilson rack (see Figure 1). This step will produce the further collection of the antibody in the autosampler.

Rack B will be used for the antibody collection, and will contain empty vials to this end. Chromeleon has been configured to collect the antibody from the first sample in position BA1. For this reason, the further injections for titre, aggregation and charge variant analysis (Steps 2, 3 and 4), show BA1 as the vial to be injected (see Table 1). The next samples will be collected in positions BA2, BA3, BA4, etc. and consequently, a different eWorkflow should be created for all of them showing the corresponding new vial positions.

Rack G contains a 96-positions well plate, and will be used for the sample preparation for middle-up analysis (Step 6), consisting in the reduction of the antibody. As it is shown in the line 6 of the eWorkflow from Table 1 , Chromeleon has been configured to perform the reduction step for the first sample in position GA1, which means that the next samples will be reduced in positions GA2, GA3, GA4, etc. and consequently, a different eWorkflow should be created for all of them showing the corresponding new positions.

System flushing steps: Steps 5 and 7 correspond to “flush” injections that have the purpose of conditioning the system to required chromatographic settings. “Flush 1” method (Step 5) will prepare the system for the middle-up analysis (reverse phase chromatography), so that all salt residues from previous mobile phases should be removed from the system. Consequently, “Flush 1” consists in pumping water with 0.1% formic acid (mobile phase line A3) through the LC system. “Flush 1” injection has also the purpose of increasing temperature in the autosampler from 15°C (temperature for most methods) to 40°C, which is needed for the reduction step; and to set the column compartment TCC2 to 80°C that is the required temperature for the reverse phase column (TCC2 is off for the other methods).

“Flush 2” is the last method of the eWorkflow, which will condition the system at the required initial conditions to start the analysis of the next sample. This includes to bring back the autosampler at 15°C, and to turn off the temperature control in TCC2. This injection also consists in pumping water with 0.1% formic acid (mobile phase line A3) through the LC system to remove acetonitrile residues that may be incompatible with the upcoming analysis for the next sample.

Considerations for processing methods and protein deconvolution features for PTMs analysis.

Each instrument method includes a processing method that integrates the resulting peaks from each analysis:

Titre determination: the area measured for the antibody peak is compared automatically with a calibration curve, producing the resulted titre of the antibody in the analysed sample.

Aggregation and Charge variant analysis: The corresponding measured areas for the aggregates and main peak (for aggregation analysis) and the acid, basic variants and main peak (for charge variant analysis) are used to calculate automatically relative areas with respect to the main peak for both cases. Middle-up analysis: Although measuring the areas for heavy and light chains produced from the antibody reduction step are not providing critical information, the processing method has to be created because the settings for the automatic protein deconvolution (which is needed for the determination of PTMs), are part of a processing method.

All relevant results from the analyses are included as part of a report, which will be created from report templates available in Chromeleon software, being adjusted and customised according to the user needs. The report will be generated automatically once the eWorkflow sequence finishes for each sample analysis. Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.