Methods and kits for detecting specific host cell proteins

Abstract

The present invention relates to a method for detecting a host cell protein (HCP) native to a host cell species, in a composition of proteins produced by said host cell species and comprising HCPs and at least one biological entity that is not native to the host cell species, wherein the method comprises a dual-recognition immunoassay. The invention also relates to methods for assessing quality and reproducibility of aspects of biological production processes, as well as kits and use of kits in such methods.

Description

[0001] New method

[0002] Field of the invention

[0003] The present invention relates to methods and products for use in development and quality control of biotechnological production processes. In particular, it relates to methods and process for measuring and monitoring impurities arising in biological production processes and for investigating the reproducibility of methods for purifying a product produced through such processes.

[0004] Background

[0005] A large and growing number of medicinal products are proteins or other biological entities produced in host cells. Proteins originating from the host cell (i.e. Host Cell Proteins, or HCPs) or from the production process must be separated from the biological entity of interest before making a medicinal preparation of the protein of interest. Known methods and means for measuring and monitoring the successful removal of HCPs include immunoassays and mass spectrometry-based methods.

[0006] Immunoassays for measuring HCPs in biopharmaceutical production are typically sandwich immunoassays with polyclonal antibodies raised against a mixture of HCPs. These methods result in a single HCP value for a given production lot or other investigated sample, i.e. they do not measure individual HCPs specifically.

[0007] Mass spectrometry-based methods may identify individual HCPs but may on the other hand be overwhelmed by peptides derived from the protein of interest. While product-derived peptides may be removed through e.g. HPLC, this requires a manipulation of the sample under investigation which may interfere with results. MS-based methods may also require significant sample preparation, including reduction, alkylation, and proteolytic digestion, followed by separation, which also constitute sample manipulation.

[0008] Other methods and systems for identifying and quantitating low-abundance host cell proteins (HCP), in particular using ProteoMiner™ beads (Bio-Rad Laboratories, Inc., Hercules, CA), to monitor and control impurities in biopharmaceutical products are described in WO2021202554. Multiplex detection of proteins in body fluid samples from human and non-human primates have been achieved through e.g. proximity extension assays (PEA) and proximity ligation assays (PLA). PEA and PLA are described in WO 01 / 61037. Multiplexed PLA is further described in i.a. Lundberg et al. Molecular & Cellular Proteomics

[0009] 10:10.1074 / mcp. M110.004978, 1-10, 2011. Multiplexed PEA is further described in e.g. Assarsson et al., PLoS 1, 2014, 9, 4, e95192; Wik et al., 2021, Mol Cell Proteomics 20, 100168 and Siegbahn A, et al., PLoS One. 2023 Nov 14;18(11): e0293465, all incorporated herein by reference in their entirety.

[0010] Nan Liu et aL, BioProcess International 10(2) February 2022 describes an automated solidphase proximity ligation assay (PLA) for Chinese Hamster Ovary (CHO) HCP detection. The polyclonal antibody used for HCP detection was raised against a mixture of CHO HCPs. The detection is unspecific and quantifies HCPs as a group of proteins without identifying the specific, individual HCPs in the HCP mixture. In addition, solid-phase detection is less advantageous for the purpose as it requires additional washing steps which may decrease the yield of the final recombinant biological product.

[0011] Summary

[0012] The present invention is as described herein and in the appended claims.

[0013] Brief description of the drawings

[0014] Figure 1 shows the typical steps in bioprocess development and manufacturing. The steps may vary depending on drug substance but often include cell culture (102), harvest (104), clarification / filtration (106), capture (108), viral inactivation (110), polish (112), filtration (114), concentration (116), formulation (118), and fill finish (120). In each of these steps, samples may be drawn for analysis of critical quality attributes (CQAs), both product and process related. The samples are optionally diluted or otherwise prepared, if required for proper analysis. According to the invention, the identity and level of each host cell protein, or leached affinity ligand, is measured by a dual-recognition immunoassay. The generated data is used to optimize and monitor the process and to ensure that the process and product meet the set requirements e.g. for regulatory approval. Definition of terms and abbreviations

[0015] All terms and abbreviations used in the present specification shall be construed to have the meaning normally given to them in the relevant art, unless another meaning is clearly intended. For the sake of clarity, a few terms and abbreviations are defined below.

[0016] The singular “a” and “an” shall be construed as including also the plural.

[0017] Compositions “comprising” one or more recited elements may also include other elements not specifically recited. The term "comprising” also encompasses the term “consisting of”.

[0018] “Dual-recognition immunoassay” is an immunoassay wherein two (or more) simultaneous and specific binding events are required for a signal to be generated. This differs from e.g. sandwich ELISAs, Sandwich ELISA generally include capture of the analyte by a capture antibody and subsequent addition of a second antibody (detection antibody) coupled to a signal-generating moiety. While this involves two binding events, these two events are not both required to generate a signal, as the signal is generated solely by the signal-generating moiety on the detection antibody.

[0019] An "immunoassay” is a type of assay for a specific analytes to be detected, that utilizes molecules binding specifically and preferentially to the analyte in question and wherein binding events generate or result in a signal that can be detected. While the prefix “immuno-” implies, and originates from, the original use of antibodies as the specific binding molecules, immunoassays may also use other types of specific binding molecules.

[0020] “Readout” is intended to refer to the process of quantifying the amount of reporter molecules with the respective unique identification sequences and correlating these amounts to the amounts of the respective analytes of interest in the analyzed sample. Accordingly, a readout can be seen as a step of detecting the signal in the assay, or more particularly the reporter molecules, in a quantitative manner.

[0021] Multiplexing of biological assays, such as proximity assays, means performing a plurality of assays in parallel and, preferably, in the same reaction container, e.g. a test tube or a well in a microtiter plate. Multiplexing thus has the potential to massively increase throughput of samples and reduce footprint of the necessary equipment.

[0022] The term “plurality” or “multiple” as used in the present invention means more than one (that 20 is to say, two or more), in line with its standard definition. A Host Cell Protein (HCP) is a native protein originating from a host cell species that has been engineered to produce a recombinant biological entity not native to the host cell species. In this regard, it is often referred to an HCP or HCPs from the host cell species in the general sense and as group of proteins having such an origin. However herein, whenever a “specific HCP” (sHCP) or “an individual HCP” is referred to, this identifies a specific, individual protein within the group of HCPs.

[0023] Detailed description

[0024] The present invention relates to improved methods for monitoring the presence of Host Cell Proteins (HCPs), complexes between a HCP and a biological entity and / or a leached affinity ligand at one or more stages of purification of compositions produced in host cells, such as by recombinant protein production.

[0025] In a first aspect, the invention relates to a method for detecting at least one specific host cell protein (sHCP) native to a host cell species, in a mixture of proteins produced by said host cell species, wherein said mixture comprises a plurality of specific HCPs (sHCPs) and at least one biological entity that is not native to the host cell species, wherein the method comprises a dual-recognition immunoassay. Herein, the terms “mixture” and “composition” may be used interchangeably.

[0026] A dual-recognition immunoassay is an immunoassay wherein two specific binding events are required for a signal to be generated. This is typically achieved by having at least two affinity binders that may bind to the target at the same time. One or both of the affinity binders are equipped with attachments that can generate a signal only when both affinity binders are bound to the target. In the context of the present invention, when a method comprises a dual-recognition immunoassay and an sHCP is the target of the dual-recognition immunoassay, this means that said sHCP is detected by a pair of at least two affinity binders that are specific for the individual HCP. In other words, the affinity binders will not bind to just any sHCP in a mixture comprising a plurality of different sHCPs but only to the sHCP to which the affinity binders are specific. Alternatively, one of the affinity binders in a pair bind specifically to the sHCP whereas another affinity binder in the pair specifically binds to a protein recombinantly produced in the host cell, so that the pair together may detect a complex formed of the sHCP and the recombinantly produced protein. The affinity binders are also referred to herein as protein-binding moieties, target-specific binding domains or similarly. One advantage of using a dual recognition immunoassay to detect HCPs, as compared to standard immunoassays as used for detecting HCPs, is that it is possible to simultaneously detect all relevant HCPs specifically and thus get a much more granular and detailed measurement of which HCPs are present in the product under investigation in each step of the production process. This facilitates improved quality control measures in the production process, as the efficacy and reproducibility of purification steps can be assessed for each individual HCP. The presence and concentration of some HCPs may also be more critical to the final product, which makes identification of the specific HCPs particularly important. In addition to a specific detection of the selected individual HCPs, the concentration or amount of the individual HCP may be desired. The present invention provides the ability to detect individual HCPs simultaneously and to determine their amount in a mixture. This enables understanding of underlying protein profiles aside from a composite, parts per million (ppm) relative the biological entity of interest, measurement. While this, in theory, also could be achieved with the use of advanced MS based methods, such methods may run into problems of saturation when a single protein, e.g. the protein of interest in production, is present at a much higher concentration than the HCPs. This could be solved by removal of the high abundant protein(s) prior to analysis, but such removal may also remove HCPs that it would be relevant to measure. Furthermore, specific calibrators, chemistries and analysis are required making the implementation of MS based methods increasingly complicated.

[0027] The invention is hence embodied in a method comprising a dual-recognition immunoassay for detection and quantitative measurement of at least one specific host cell protein (sHCP) native to a host cell species, in a mixture of proteins produced by said host cell species, wherein said mixture comprises a plurality of specific HCPs (sHCPs) and at least one biological entity that is not native to the host cell species.

[0028] In one embodiment, the host cell is modified to produce the biological entity. The modification may be a genetic modification, such as an introduction of recombinant genetic material for transient or permanent expression of the biological entity.

[0029] In one embodiment, the biological entity is produced for inclusion in a medicinal product for human or animal use. The biological entity may thus be a protein, such as a therapeutic antibody, hormone, enzyme, antigen, auto-antigen, allergen. It may also be a virus or viruslike particle.

[0030] In one embodiment, the sHCP is present in the composition at an amount of less than 1% by weight of the amount of the biological entity. In other embodiments, the sHCP is present in the composition at an amount of 1-100 parts per million (ppm) by weight of the amount of the biological entity.

[0031] The amount of sHCP present in the composition as an amount of the weight of the biological entity present in the composition is usually the total amount of sHCPs but individual amounts of sHCPs may also be determined in the context of the present invention. Importantly, the total amount of sHCP present in the composition is preferably less than 1% by weight of the amount of the biological entity.

[0032] In one embodiment, a plurality of specific host cell proteins (sHCPs) are detected, such as at least 5, at least 10, at least 20, at least 50, or at least 100 specific host cell proteins (sHCPs).

[0033] When a plurality of sHCPs is detected, this means that a plurality of different sHCPs is detected, i.e. the method is multiplex in the sense that the presence and concentration of a plurality of different sHCPs are determined simultaneously in a single assay. The same applies to all aspects and embodiments of the invention whenever a plurality of sHCPs is to be detected or determined for presence or concentration in a composition as referred to herein.

[0034] In one embodiment, the host cell is a mammalian cell, such as a Chinese Hamster Ovary cell, a HEK-293 cell, or a HeLa cell.

[0035] Further embodiments include other mammalian host cells, such as mouse myeloma (e.g. NS20) cells and Vero cells.

[0036] In other embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, a fungal cell or a transgenic cell. A transgenic host cell is a cell in which genetic modifications have been made to introduce genetic material from another organism or from modified versions from the same organism.

[0037] Examples of suitable bacterial host cells include Escherichia coli cells, Lactococcus lactis cells, Pseudomonas fluorescens cells and Staphylococcus aureus cells. Suitable yeast host cells include Pichia pastoris cells and Saccharomyces cerevisiae cells. Examples of suitable insect host cells include e.g. Sf9 insect cells. A fungal host cell that is particularly useful includes the Cl filamentous fungus Thermothelomyces heterothallica Furthermore, suitable transgenic host cells include cells from bacteria, plants, animals and yeast which have been genetically modified.

[0038] The above exemplified host cells apply to all aspects and embodiments of the present invention.

[0039] In one embodiment, the method further comprises determining an absolute and / or a relative concentration of the specific HCP, a complex between the biological entity and the sHCP and / or the affinity ligand in the composition.

[0040] In one embodiment, the dual-recognition immunoassay is a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay.

[0041] Under certain conditions, some Host Cell Proteins may bind tightly to a produced biological entity, such as a protein, that it is desired to purify. The “piggybacking" problem refers to the co-purification of unwanted impurities (host cell proteins, HCPs, or other biomolecules) that bind to the target protein — often through non-covalent or secondary interactions — and are therefore “carried along” through affinity or chromatographic purification steps. In essence, impurities “piggyback” on the product molecule, escaping removal despite high-resolution purification techniques such as Protein A affinity chromatography. For instance, certain CHO (Chinese Hamster Ovary) host proteins, such as lipoprotein lipase or complement component proteins, are known to tightly associate with monoclonal antibodies and resist removal during Protein A capture. Also, enzymatic impurities (e.g., cathepsins) can co-purify and degrade the antibody post-purification.

[0042] The present invention can be used to address the piggybacking problem by providing a dualrecognition immunoassay wherein one of the affinity binders specifically binds the piggybacking HCP and the other affinity binder specifically binds the product to be purified. The dual-recognition immunoassay is thus designed to detect the complex formed between the HCP and the product. As understood by the skilled person, the affinity binders need to be selected to bind epitopes that are freely available when the HCP / product complex is formed.

[0043] The above mentioned piggyback assay may be combined with an assay that measures the free form of the piggybacking HCP. This assay may use the affinity binder that binds the epitope freely available when the HCP is bound to the product, but the second affinity binder is selected to bind specifically to an epitope that is not freely available when bound to the product. In this way, it is possible to design two dual recognition assays that together detect both free and bound HCP. When evaluating different purification protocols, it is then possible to optimize for conditions that disfavour piggybacking to improve the overall purification process.

[0044] Thus, in one embodiment, the present invention relates to a method for detecting at least one specific host cell protein (sHCP) native to a host cell species, in a mixture of proteins produced by said host cell species, wherein said mixture comprises at least one biological entity that is not native to the host cell species, wherein the method comprises a dualrecognition immunoassay configured to detect a complex of the sHCP and the biological entity.

[0045] In a further aspect, the present invention relates to a method for detecting a leached affinity ligand, in a composition that has been subjected to affinity purification using the affinity ligand, wherein the method comprises detecting the leached affinity ligand using a dualrecognition immunoassay.

[0046] In this aspect, the composition may be a mixture of proteins produced by a host cell species as described above.

[0047] In a further aspect, the invention relates to a method for quality assessment of a purification step in a purification method for removal of at least one specific host cell protein (sHCP) from a composition comprising the at least one sHCP and a biological entity not native to the host cell, the method for quality assessment comprising

[0048] (i) Determining a concentration of the at least one sHCP prior to the purification step;

[0049] (ii) Determining a concentration of the at least one sHCP after the purification step; and

[0050] (iii) Assessing the quality of the purification step by comparing the concentrations of the at least one sHCP before and after the purification step.

[0051] It is understood that the concentration is determined in the composition comprising the at least one sHCP and a biological entity not native to the host cell.

[0052] In one embodiment, the concentrations are absolute or relative.

[0053] In one embodiment, an absolute concentration for the at least one specific host cell protein is determined for a final product obtained in the purification process. In one embodiment, the concentration determinations in steps (i) and (ii) are done for at least 5, such as at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100 specific host cell proteins (sHCPs). In other words, the concentration determinations may be done for a plurality of sHCPs. In one embodiment, the method comprises a dual-recognition immunoassay configured to detect a complex between the biological entity and a sHCP.

[0054] In one embodiment, the method further comprises detecting a leached affinity ligand using a dual-recognition immunoassay.

[0055] In one embodiment the host cell is a human cell, such as a HEK293 cell, and the specific host cell proteins (sHCPs) are selected from the host cell proteins listed in Table 1.

[0056] In one embodiment, the host cell is a Chinese Hamster Ovary cell, and the specific host cell proteins (sHCPs) are selected from the host cell proteins listed in Table 2.

[0057] In one embodiment, the concentration determinations in steps (i) and (ii) are done with a dual recognition immunoassay, such as Proximity Extension Assay or Proximity Ligation Assay.

[0058] It is to be understood that concentration determinations may be performed for the at least one sHCP, a complex between the biological entity and a sHCP and / or for a leached affinity ligand, respectively, before and after the purification step.

[0059] In one aspect, the invention relates to a method for assessment of reproducibility of a process for purification of a biological entity produced by expressing the biological entity in a host cell, wherein the biological entity is not native to the host cell species, to obtain a composition comprising a plurality of specific host cell proteins (sHCPs) and the biological entity, the purification process comprising subjecting the composition to a plurality of purification steps to remove the sHCPs and retain the biological entity, the method for reproducibility assessment comprising

[0060] (i) Determining, for at least two separate runs of the same purification process, a concentration for at least one or each of the specific host cell proteins at, at least two instances selected from prior to, in between, or after the purification steps to obtain sHCP concentration values at each such instance; and

[0061] (ii) Assessing the reproducibility of the purification process by comparing the sHCP concentration values obtained from the separate runs of the purification process. In one embodiment, the sHCP concentration values are absolute or relative concentration values.

[0062] In one embodiment, the sHCP concentration values are obtained for at least 5, such as at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100 sHCPs. In other words, the sHCP concentration values may be obtained for a plurality of sHCPs.

[0063] In one embodiment, the method comprises a dual-recognition immunoassay configured to detect a complex between the biological entity and a sHCP.

[0064] In one embodiment, the method further comprises detecting a leached affinity ligand using a dual-recognition immunoassay.

[0065] In one embodiment, the host cell is a human cell, such as a HEK293 cell, and the specific host cell proteins are selected from the host cell proteins listed in Table 1.

[0066] In one embodiment, the host cell is a Chinese Hamster Ovary cell, and the specific host cell proteins are selected from the host cell proteins listed in Table 2.

[0067] In one embodiment, the sHCP concentration values are obtained with a dual recognition immunoassay, such as a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay.

[0068] It is to be understood that concentration determinations for assessing the reproducibility of the purification process may be performed for the at least one sHCP, a complex between the biological entity and a sHCP and / or for a leached affinity ligand, respectively, before and after the purification step.

[0069] In one embodiment, the comparison is performed by forming univariate or multivariate ratios of direct measurements of concentrations, multivariate data analysis, using dimensionality reduction algorithms such as Principal Component Analysis or Uniform Manifold Approximation and Projection; or deep learning-based reduction methods such as autoencoders.

[0070] In one aspect, the present invention relates to a kit for detecting a specific Chinese Hamster Ovary (CHO) protein (sCHOP) native to a CHO cell line, in a mixture of proteins produced by said CHO cell line, said kit comprising a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the specific CHO protein, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding.

[0071] In one aspect, the present invention relates to a kit for detecting a leached proteinaceous affinity ligand used in an affinity purification of a protein composition, said kit comprising a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the affinity ligand, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding.

[0072] In one aspect, the present invention relates to a kit for detecting a complex formed between a protein recombinantly produced in a host cell and a Host Cell Protein, said kit comprising a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the complex, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding.

[0073] In one embodiment, the kit according to this aspect is a kit wherein one protein-binding moiety is capable of binding specifically to the recombinantly produced protein and one protein-binding moiety is capable of binding specifically to the Host Cell Protein.

[0074] In one embodiment of the kits according to the invention, the first and second reagent compositions are provided as a single reagent composition or as separate reagent compositions.

[0075] In one embodiment of the kits according to the invention, the matched protein-binding moieties are coupled to oligonucleotides capable of interacting and forming a reporter nucleic acid molecule when the protein-binding moieties simultaneously bind to the host cell protein and in presence of the second reagent composition.

[0076] In one embodiment of the kits according to the invention, the protein-binding moieties are selected from antibodies and fragments thereof, nucleic acid molecules such as aptamers, molecular imprinted polymers.

[0077] In one embodiment of the kits according to the inventions, the said first reagent composition comprises matched protein-binding moieties capable of simultaneously and specifically binding to a plurality of specific CHO proteins (sCHOPs), such as at least 5, at least 10, at least 20, at least 50, or at least 100 specific CHO proteins (sCHOPs). Hence, the first reagent composition may comprise a plurality of different matched protein-binding moieties capable of binding to a plurality of sCHOPs.

[0078] In one aspect, the present invention relates to a method for detecting a proteinaceous affinity ligand in a sample, said method comprising contacting the sample with a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the affinity ligand, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding; inducing the generation of signal, and measuring the signal thereby detecting the affinity ligand.

[0079] In one embodiment, the matched protein-binding moieties are coupled to oligonucleotides capable of interacting and forming a reporter nucleic acid molecule when the protein-binding moieties simultaneously bind to the host cell protein and in presence of the second reagent composition.

[0080] In one embodiment, the protein-binding moieties are selected from antibodies and fragments thereof, nucleic acid molecules such as aptamers, molecular imprinted polymers.

[0081] In one embodiment, said proteinaceous affinity ligand is selected from the group consisting of Protein A, Protein G, Protein L, and functional variants thereof.

[0082] In one aspect, the present invention relates to the use of a kit for performing a dual recognition immunoassay in any method according to the invention.

[0083] In one embodiment, the dual recognition immunoassay is a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay.

[0084] The target of the dual-recognition assay, in the context of the present invention, is generally a Host Cell Protein (HCP) that it is desirable to detect, more particularly a specific Host Cell Protein (sHCP) in a mixture of different sHCPs. HCPs are proteins that are products of genes in the native genome of the host cell. In other aspects, the target is a protein, expressed by the host cell, that is not an HCP and not the biological entity of interest. Such targets may be e.g. viral proteins resulting from virus transfection of the host cell to introduce genetic material for production of the biological entity of interest. The target may also be a complex between a Host Cell Protein, or other impurity, and the biological entity of interest. The target may also be the biological entity of interest or a variant thereof that is considered an impurity. If the biological entity of interest is a protein, the impurity variant may e.g. be a variant that carries undesired post-translational modifications, or a fragment of the desired biological entity of interest.

[0085] Concentrations of HCPs determined in the context of the present invention may be absolute or relative. Determination of absolute concentrations is useful for certain purposes, such as determining that any specific HCP is below a certain absolute threshold required by product quality criteria or set by regulatory authorities, guidelines or frameworks. Determinations of relative concentrations may be useful when monitoring trends or progress along a purification process or comparison of concentrations between different production batches of the biological entity. By using a combination of relative and absolute measurements a deeper understanding of the HCP profile dynamics can be achieved.

[0086] Table 1: Human derived HCPs. Adapted from Smith et al., European Journal of Pharmaceutics and Biopharmaceutics 189 (2023) 276-280.

[0087] Table 2: HCPs from Chinese Hamster Ovary cells. Adapted from Molden et aL, mAbs, 2021, VOL. 13, NO. 1, 61955811.

[0088] A specific example of the use of the invention is found within the development of efficient production of biological pharmaceuticals, from early process development to drug substance release. In each case the produced material needs to be analyzed for its host cell protein (HCP) profile and content (i.e. residual proteins which pose a risk of creating a immunogenic or undesired side reaction in the recipient of the product). While a number of dual-recognition immunoassays may be used in the present invention, as discussed above, presently preferred embodiments are PEA and PLA. A panel of PEA or PLA assays may be developed to monitor relevant HCPs in the specific product development effort. A methodology to develop and run a panel detecting 94 unique target proteins is provided in Assarsson et al. PLoS One, 2014, 9(4), e95192. Similarly, the development of a multiplexed PLA assay is described in Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp. M110.004978, 1-10, 2011. These protocols can be adapted to detect all or a subset of the HCPs in Table 1 or Table 2, or other HCPs that are considered relevant in the specific case. The protocols provide for relative concentration measurements. If absolute quantification is desired, standard curves may be generated for all assays included in the panel, using recombinant HCPs for all assays. A general protocol to follow is described in Siegbahn A, et aL, PLoS One. 2023 Nov 14;18(11): e0293465.

[0089] Figure 1 provides a typical flow chart for biological production of a protein or other biological entity of interest, such as a therapeutic antibody, peptide hormone etc. The production comprises cell culture (102), harvest (104), clarification / filtration (106), capture (108), viral inactivation (110), polish (112), filtration (114), concentration (116), formulation (118), and fill finish (120). Not all these steps need to be present in a process under development or manufacturing. In each of these steps, samples may be drawn for analysis of HCP content according to the present invention. The samples are optionally diluted or otherwise prepared, if required for proper analysis. According to the invention, the identity and level of each host cell protein is measured and the data is used to optimize and monitor the process and to ensure that the process and product meet the requirements for drug approval.

[0090] Development of a biological production process as set out in Figure 1 takes place in several steps, which are generally described below in connection with implementation of the present invention.

[0091] The development and scaling of a biological production process can be divided into different steps: i) Small volume scale using e.g micro plates, deep well plates, or small shake bottles, for isolation of a high producing CHO cell line featuring high titer of product molecule e.g. 5- 10 g / L . Alternatively, the production system could be Human HEK 293 cells for generation of high titers of Adenovirus or Adenovirus like particles. Samples, ideally small volumes, are drawn and analyzed in this step to note and understand the presence of high-risk HCPs. ii) Scaling the process is then done by moving to small bioreactors < liter) and ends at the small pilot scale (>liters) to better mimic production. In this step the analyzed HCP profile may be used for monitoring the effectiveness of the purification process method and to define and set quality parameters for large-scale production. Analysis of the HCP profile is repeatedly performed as a way to control and adjust the process. For chromatography purification steps parameters such as buffer composition (ionic strength, pH, etc), flow speed, temperature, protein to chromatography bead capacity ratio, may be adjusted. Samples are collected from multiple pilot runs (>10 to hundreds liter scale) to support GMP (good manufacturing procedure) in terms of robustness and performance. At this step the production process should be locked and the HCP profile, both relative as well as absolute levels of individual HCP levels, constitute part of the “critical to quality”(CTQ) attribute of the drug substance, iii) At full pilot scale (<100 liter) the HCP profile may be obtained from multiple batches or during extended time. If a continuous bioproduction process is used the HCP profile is used to follow production performance over time, iv) At the final production scale (> hundreds liter) HCP data obtained from one or more steps of the production process are used to monitor that the process is within tolerances, as quality control data, and to support drug substance release for drug product formulation.

[0092] For each sampling and HCP measurement during steps i-iv the workflow is very similar differing mainly in the initial dilution of the sample. This allows for robust and reproducible HCP results in all development and production steps.

[0093] An example of a HCP profile is a set of either relative (NPX) or absolute (g / L) concentration values, or both, for a number of HCPs present (or, in certain cases, not present). The HCPs may be one or more proteins selected from Table 1 (if the host cell is human) or Table 2 (if the host cell is a Chinese Hamster Ovary cell), or other host cell proteins deemed relevant to include for a particular production process. A HCP profile may include as little as a single protein, but in general includes a plurality, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 proteins or even more. The results from the dual recognition immunoassay provide HCP-profiles of a resolution and precision that is transferable from process development into production, bridging these two critical bioproduction process steps.

[0094] The HCP profiles obtained in various instances of the invention may be compared to each other to assess the quality or reproducibility of the production process, or to monitor that a certain purification step has been adequately transferred from one development stage to another. This can be done in several ways, such as by forming univariate or multivariate ratios of direct measurements of concentrations. Another option is multivariate data analysis (MVDA), using dimensionality reduction algorithms such as Principal Component Analysis (PCA, Greenacre, et al. (2022) Nature Reviews Methods Primers, 2(1), 100)), Uniform Manifold Approximation and Projection (UMAP, Mclnnes et al., (2018) Journal of Open Source Software, 3(29), 861) or deep learning-based reduction methods such as autoencoders (Wang et al., (2016) Neurocomputing, 184, 232-242), to identify HOP profiles distinct from other HOP profiles. HOP profiles distinct from profiles with desired outcome would in this setting reveal batches where one or several steps in processing had an undesired an effect. Statistical modelling can be used to build models for e.g., control or quality. Various embodiments of Artificial Intelligence and Machine Learning can be used to assess product quality and e.g., predict batches likely to fail quality control in later steps to save resources. Trend Analysis can be used to monitor that a process does not drift out of a specified tolerated range for HCP content and to identify deviations at an early stage. These techniques can also be used in process development and optimization, where new protocols can be compared with earlier protocols in an automatic manner to identify parameters critical for performance improvement.

[0095] The purification process as outlined in Fig 1 may include affinity purification, e.g. in the capture step (108). Affinity purification is a useful downstream process in monoclonal antibody (mAb) manufacturing. It enables highly selective capture and purification of antibodies from complex biological mixtures, typically achieving >95% purity in a single step. The most common strategy employs affinity chromatography, followed by polishing steps to remove impurities and aggregates. One common approach to affinity chromatography uses Protein A (from Staphylococcus aureus) as an affinity ligand, as Protein A specifically binds to the Fc region of IgG. Common resins for use in Protein A chromatography includes MabSelect SuRe (Cytiva), POROS A (Thermo Fisher Scientific), ProSep (Repligen). Alternative affinity ligands include Protein G, Protein L, and engineered or synthetic peptide ligands. Protein G provides broader subclass specificity for IgG, as compared with Protein A, and is useful for antibodies with weak Protein A binding. Protein L binds K light chains and may be used for Fab, scFv, and non-IgG formats.

[0096] Ligand leaching is a critical quality and regulatory concern in affinity chromatography, especially in Protein A-based purification of monoclonal antibodies (mAbs). It refers to the release of immobilized affinity ligands (such as Protein A, Protein G, or synthetic ligands) from the chromatography resin into the product stream during purification. Regulatory agencies such as the FDA and EMA require monitoring and control of leached ligands as a process-related impurity.

[0097] Thus, in some embodiments, the methods and kits as described herein may include assays, method steps, and reagents as applicable for detection of a leached affinity ligand in the composition being studied. Detection of the leached affinity ligand is generally done in the same way as the detection of a specific Host Cell Protein. That is, a dual-recognition immunoassay is provided that detects the affinity ligand used in an affinity chromatography method used to purify a protein composition, in analogous fashion as provided for detection of any specific Host Cell Protein.

[0098] In one aspect, the invention relates to a method for detecting a proteinaceous affinity ligand in a sample, said method comprising contacting the sample with a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the affinity ligand, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding; inducing the generation of signal, and measuring the signal thereby detecting the affinity ligand.

[0099] In one aspect, the present invention relates to a method for detecting a leached affinity ligand, in a composition that has been subjected to affinity purification using the affinity ligand, wherein the method comprises detecting the leached affinity ligand using a dualrecognition immunoassay.

[0100] In one embodiment, the method further comprises determining an absolute and / or a relative concentration of the leached affinity ligand in the composition.

[0101] In one embodiment, any dual-recognition immunoassay is a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay.

[0102] In one aspect, the present invention relates to a method for quality assessment of a purification step comprising affinity purification using an affinity ligand, in a purification method for removal of at least one specific host cell protein (sHCP) from a composition comprising the at least one sHCP and a biological entity, the method for quality assessment comprising

[0103] (i) Determining a concentration of the at least one sHCP prior to the purification step;

[0104] (ii) Determining a concentration of the at least one sHCP after the purification step;

[0105] (iii) Assessing the quality of the purification step by (iiia). comparing the concentrations of the at least one sHCP before and after the purification step; and

[0106] (iiib). Determining the concentration of leached affinity ligand after the purification step.

[0107] In one embodiment, the concentrations are absolute or relative.

[0108] In one embodiment, an absolute concentration for the affinity ligand is determined for a final product obtained in the purification process.

[0109] In one embodiment, the concentration determinations in steps (i) and (ii) are done with a dual recognition immunoassay, such as Proximity Extension Assay or Proximity Ligation Assay.

[0110] Dual recognition immunoassays

[0111] Dual recognition immunoassays use at least two detection probes (referred to herein as a “probe pair”, but an assay may also include three or more detection probes in a “probe pair”), each probe comprising one affinity binding moiety (“affinity binder”), wherein the probes within a pair are capable of simultaneous specific binding to a target. Together, the probe pair also comprise one or more moieties that serve to generate a detectable signal when both affinity binders simultaneously bind to the target.

[0112] Preferred dual recognition immunoassays for use in the present invention are multiplexed PLA or multiplexed PEA. PEA and PLA are described in WO 01 / 61037. PLA is also described in i.a. Lundberg et al., Molecular & Cellular Proteomics

[0113] 10:10.1074 / mcp.M110.004978, 1-10, 2011. PEA is further described in WO 03 / 044231, WO 2004 / 094456, WO 2005 / 123963, WO 2006 / 137932, WO 2013 / 113699, WO 2021 / 191442, WO 2021 / 191448, WO 2021 / 191449, WO 2022 / 191450, and WO 2022 / 112300; Assarsson et aL, PLoS 1, 2014, 9, 4, e95192; Lundberg et al. Nucleic Acids Research, 2011, Vol. 39, No. 15 el02; and Wik et aL, 2021, Mol Cell Proteomics 20, 100168, all incorporated herein by reference in their entirety.

[0114] Other dual-recognition immunoassays that may be used in connection with the present invention are methods such as described in WO2017205719, WO2019191838, W02020180741, and US20220390442, all incorporated herein by reference in their entirety.

[0115] As is known in the art, the target-specific binding domain of a detection probe may be any entity capable of binding specifically to a target analyte (or part thereof), and being coupled to a signal generating moiety. That the binding domain is "specific” to a certain target means, as is known to the skilled person, that it recognizes the target with low cross-reactivity (off- target binding) with other potentially present molecules, within the relevant application and experimental context. A framework for determining specificity for binders have been established by an International Working Group for Antibody Validation (Uhlen et al. Nat Methods, 2016 Oct; 13(10), 823-827, incorporated herein by reference). In the context of the present invention, it is usually referred to a protein-binding domain as the target-specific binding domain.

[0116] Typically, the target-specific binding domain may be a protein, for example, an antibody, or an antigen-binding part thereof, including, but not limited to, monoclonal, recombinant monoclonal, and polyclonal antibodies and antigen-binding antibody derivatives and fragments. However, the target-specific binding domain may be of any nature, including lectins, soluble cell surface receptors, combinatorially derived proteins from phage display or ribosome display, peptides, carbohydrates, molecularly imprinted polymers (MIPs), nucleic acids, such as an aptamer or a nucleic acid molecule comprising the complementary sequence for a target nucleic acid, or combinations thereof.

[0117] Reagents useful as target-specific binding domains are commercially available from a number of manufacturers that offer off-the-shelf reagents or develop new binding reagents for specific targets and specific needs. Such manufacturers include, among others, Thermo Fisher Scientific (Boston, MA, USA), Abeam (Cambridge, United Kingdom), Bio-Techne (Minneapolis, MN, USA), Proteogenix (Schiltigheim, France), Sino Biological (Beijing, China), Agrisera (Vannas, Sweden), Novaptech (Pessac, France), Aptamer Group (York, United Kingdom). Reagents useful as target-specific binding domains may also be developed independently of commercial suppliers, according to protocols well-known to the skilled person. Such protocols are e.g. described in “Monoclonal Antibody Production” (National Academy Press, Washington, DC, USA, 1999), Carey-Hanly et al. (ILAR Journal, Volume 37, Issue 3, 1995, Pages 93-118), llgu and Nilsen-Hamilton (Analyst. 2016 March 7; 141(5): 1551-1568). Reagents may also comprise antibody derivates or fragments, such as Fab, Fab', F(ab')2, Fv fragments; diabodies; single-domain antibodies (sdAb, Desmyter et al. (1996) Nat. Structure Biol. 3:803-811), nanobodies, single-chain Fv (scFv, Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883), divalent scFV (di-scFvs), tandem scFvs, triabodies, diabodies, single-chain diabodies (scDb), bi-specific T-cell engagers (BiTEs, Kufer et al. (2004) Trends Biotechnol. 22:238-244), and Dual Affinity Retargeting molecules (DARTs, diabodies additionally stabilized through a C-terminal disulfide bridge). The specificity of target-specific reagents with regard to the intended detection assay may be evaluated using the framework proposed by the International Working Group for Antibody Validation, cited above.

[0118] Further, the target-specific binding domain may bind to the target directly or indirectly. In other words, the detection probe may be a primary reagent which binds directly to the target, or a secondary reagent which binds indirectly, by virtue of binding to an intermediate molecule (a primary reagent) which is itself bound directly to the target.

[0119] In addition to the target-specific binding domain, a detection probe pair as used in the present invention also comprises a moiety capable of generating a detectable signal upon dual recognition. Currently envisioned dual-recognition assays use a nucleic acid moiety, also referred to herein as an oligonucleotide, comprising one or more identification sequences. The identification sequence may e.g. be a unique sequence (usually termed a “barcode sequence” or simply “barcode”) that is detected in a sequence-specific manner, for example, sequenced for identification in the readout step, or which provides a specific binding (hybridization) site for a probe or primer used in the detection, e.g., a unique primer binding site that can be used for readout using quantitative PCR (qPCR).

[0120] The oligonucleotide must be long enough to comprise the necessary functional elements used in the detection assay for which the detection probe is intended to be used. That is, at least a sequence capable of generating an identification sequence in the reporter molecule. This is typically 5-20 nucleotides, such as 5-10, 5-15, 10-15 or 15-20 nucleotides. The oligonucleotide may also contain sequences related to primer sites and / or sequencing adaptors for read-out, as known in the art. Generally, the oligonucleotide has a length in the range of 20-100 nucleotides but may be shorter or longer as required in the specific detection assay in which the detection probe is intended to be used.

[0121] Conjugation of a nucleic acid moiety to an antibody can be performed in several ways known to the skilled person, e.g. as reviewed by Dugal-Tessier et al. (J. Clin. Med.2021, 10, 838).

[0122] Commercial kits for preparing antibody-oligonucleotide conjugates are also readily available from a number of suppliers. The oligonucleotides may be coupled to the analyte binding domains by any means known in the art, and which may be desired or convenient and may be direct, or indirect, e.g. via a linking group. For example, the domains may be associated with one another by covalent linkage (e.g. chemical cross- linking) or by non-covalent association e.g. via streptavid in-biotin based coupling (biotin being provided on one domain, particularly the oligonucleotide domain, and streptavidin on the other). The oligonucleotide and analyte binding domain (i.e. , the target-specific binding domain), are joined together either directly through a bond or indirectly through a linking group. Where linking groups are employed, such groups may be chosen to provide for covalent attachment of the nucleic acid moiety and analyte binding domain through the linking group. The linking group, when present, is in many embodiments biologically inert. In representative embodiments, the linking group is generally at least about 50 Daltons, usually at least about 100 Daltons and may be as large as 1000 Daltons or larger, for example, up to 1000000 Daltons if the linking group contains a spacer, but generally will not exceed about 500 Daltons and usually will not exceed about 300 Daltons. Generally, such linkers will comprise a spacer group terminated at either end with a reactive functionality capable of covalently bonding to the nucleic acid domain or analyte binding domain. Spacer groups of interest may include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may possibly contain heteroatoms. Spacer groups may also be comprised of ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex. Specific spacer elements include: 1 ,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenediamine-N,N- diacetic acid, 1 ,1 '-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), 4,4'- ethylenedipiperidine.

[0123] Potential reactive functionalities include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals. Specific examples include primary and secondary amines, hydroxamic acids, N- hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinylsulfones, and maleimides.

[0124] Specific linker groups that may find use in the subject proximity probes include heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p- azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide, bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N- maleimidobutyryloxysuccinimide ester, N- hydroxy sulfosuccinimidyl-4- azidobenzoate, N-succinimidyl [4-azidophenyl]-l ,3'- dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4-[N- maleimidomethyl]cyclohexane-l -carboxylate, 3-(2-pyridyldithio)propionic acid N- hydroxysuccinimide ester (SPDP), 4-(Nmaleimidomethyl)-cyclohexane-l -carboxylic acid N-hydroxysuccinimide ester (SMCC), and the like.

[0125] The nucleic acid domain of the detection probes may be made up of ribonucleotides and / or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions. Thus, the nucleic acid domain may be DNA or RNA or a combination or any modification thereof e.g. PNA or other derivatives containing non-nucleotide backbones.

[0126] In one embodiment, the detection probes are manufactured by coupling a universal oligonucleotide to the analyte-specific binding domain and subsequently hybridizing a tag oligonucleotide to the universal oligonucleotide, wherein the tag oligonucleotide comprises a sequence capable of generating the identification sequence in a reporter molecule, a sequence complementary to the universal oligonucleotide to facilitate hybridization, and any other functional sequences necessary to perform the detection assay for which the detection probes are intended. Methods for manufacturing such detection probes are described i.a. in international patent publication W02017 / 068116.

[0127] Where proximity probes are used as the detection probes, each nucleic acid moiety of a proximity probe of the matched pair or more may comprise an identification sequence, or a partial identification sequence. The reporter molecule which is generated may comprise an identification sequence from each of the matched proximity probes. In other words, the identification sequence of the reporter molecule may be a combination, or composite, of the identification sequences of the individual nucleic acid moieties of matched proximity probes. The identification sequences of individual matched proximity probes may be the same or different. In an embodiment, each identification sequence in matched proximity probes is indicative of, or corresponds to, the analyte of interest. However, it is not required for a particular identification sequence of an individual proximity probe to be indicative of an analyte of interest - it is the identification sequence of the reporter nucleic acid molecule which is indicative of the analyte of interest. As indicated above, the reporter identification sequence may be a combination or composite. Alternatively, the identification sequence of the reporter nucleic acid molecule may be derived from the nucleic acid moiety of a single proximity probe (although it will be understood that interaction of the nucleic acid moieties of matched proximity probes will be required for the reporter nucleic acid molecule to form).

[0128] As is known in the art, different detection modalities for the readout are possible. These include sequencing. Thus, for example, an identification sequence may be a barcode which is sequenced. Any method of sequencing may be used, including sequencing-by-synthesis and sequencing-by-hybridization methods. Thus, depending on the nature of the ID sequence, any suitable method may be used to identify the ID sequence, and this may involve the use of hybridization probes and / or primers. For example, the detection (readout) step may involve amplifying the reporter nucleic acid using one or more primers, at least one of which binds to the ID sequence. Alternatively, the detection method may involve amplifying the reporter and detecting the amplicons by means of specific hybridization probes which bind to the ID sequence (or to a complement thereof). In sequencing-by- hybridization, barcodes can be decoded using labelled hybridization probes, including in combinatorial fashion.

[0129] Sequencing advantageously allows a high level of multiplexing and is a convenient method of detection. As noted above, any form of sequencing may be used, including any method of sequencing-by-synthesis, for example, pyrosequencing, reversible dye terminator sequencing and ion torrent sequencing. Particularly, high throughput methods of sequencing are used, and especially massively parallel DNA sequencing. Massively parallel DNA sequencing using the reversible dye terminator method may be performed, for instance, using an Illumina® NovaSeq™ system.

[0130] In another embodiment, the ID sequences are primer binding sites, e.g. for a PCR primer, although other amplification methods may be used.

[0131] In still further embodiments, the ID sequences may be restriction sites (i.e. a nucleotide sequence recognized by a restriction enzyme). In this embodiment, the nucleic acid domain of a proximity probe may comprise a different restriction site (such that it is recognized and cleaved by a different restriction enzyme). Different combinations of restriction enzymes may thus be applied to differentiate different reporter nucleic acids.

[0132] Amplification methods based on PCR are convenient and conveniently the readout may involve quantitative PCR (qPCR) or real-time PCR. The amplicons may be detected using any convenient protocol, including the use of dyes and stains, or labels, e.g. intercalating dyes, or labelled probes which bind to the amplicons. These include molecular beacons and such like, e.g. probes with FRET labels etc.

[0133] For instance, when readout is performed by qPCR, it is preferable to be able to provide a limited set of qPCR primers that work for all panels of assays, regardless of the assay content of the various panels. It is also preferable to keep the number of qPCR primers low to reduce cost, risk of mismatched binding and other biological artefacts. With the present invention, it is possible to select a set of detection probes where all detection probes generate reporter molecules with unique identification sequences, while at the same time all those identification sequences also correspond to a limited set of qPCR primer binding sites, so that a corresponding limited set of qPCR primers can be used for read-out of the panel. In this way, the same set of qPCR primers can be used for readout of any panel compiled from the library according to the invention.

[0134] Thus, in one embodiment, the unique identification sequences in the library can be made to correspond to a set of qPCR primers that can be used for readout of any panel compiled from the library according to the invention.

[0135] In one embodiment, the identification sequences are binding sites for qPCR primers.

[0136] In one embodiment, the identification sequences are barcode sequences.

[0137] All prior publications cited in the present specification are incorporated by reference in their entirety.

[0138] Itemized embodiments

[0139] Without limiting the scope of the invention as disclosed herein, the invention i.a. relates to the following.

[0140] 1. Method for detecting a host cell protein (HCP) native to a host cell species, in a composition of proteins produced by said host cell species and comprising HCPs and at least one biological entity that is not native to the host cell species, wherein the method comprises a dual-recognition immunoassay.

[0141] 2. The method of item 1, wherein the host cell is a mammalian cell, such as a Chinese Hamster Ovary cell, a HEK-293 cell, or a HeLa cell.

[0142] 3. A method for quality assessment of a purification step in a purification method for removal of at least one specific host cell protein (sHCP) from a composition comprising the at least one sHCP and a biological entity, the method for quality assessment comprising (i) Determining a concentration of the at least one sHCP prior to the purification step;

[0143] (ii) Determining a concentration of the at least one sHCP after the purification step; and

[0144] (iii) Assessing the quality of the purification step by comparing the concentrations of the at least one sHCP before and after the purification step. The method according to any one of items 1-3, wherein the host cell is a human cell, such as a HEK293 cell, and the specific host cell proteins are selected from the host cell proteins listed in Table 1. The method according to any one of items 1-4, wherein the host cell is a Chinese Hamster Ovary cell, and the specific host cell proteins are selected from the host cell proteins listed in Table 2. . The method according to any one of items 3-5, wherein the concentration determinations in steps (i) and (ii) are done with a dual recognition immunoassay. The method according to item 6, wherein the dual recognition immunoassay is a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay. Method for assessment of reproducibility of a process for purification of a biological entity produced by expressing the biological entity in a host cell, wherein the biological entity is not native to the host cell species, to obtain a composition comprising a plurality of specific host cell proteins (sHCPs) and the biological entity, the purification process comprising subjecting the composition to a plurality of purification steps to remove the sHCPs and retain the biological entity, the method for reproducibility assessment comprising

[0145] (i) Determining, for at least two separate runs of the same purification process, a concentration for each of the specific host cell proteins at at least two instances selected from prior to, in between, or after the purification steps to obtain sHCP concentration values at each such instance; and

[0146] (ii) Assessing the reproducibility of the purification process by comparing the sHCP concentration values obtained from the separate runs of the purification process. 9. Kit for detecting a specific Chinese Hamster Ovary (CHO) protein native to a CHO cell line, in a mixture of proteins produced by said CHO cell line, said kit comprising a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the specific CHO protein, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding.

[0147] 10. The kit according to item 9, wherein the matched protein-binding moieties are coupled to oligonucleotides capable of interacting and forming a reporter nucleic acid molecule when the protein-binding moieties simultaneously bind to the host cell protein and in presence of the second reagent composition.

[0148] 11. Use of a kit for performing a dual recognition immunoassay in a method according to any one of items 1-10.

[0149] 12. Use according to item 11, wherein the dual recognition immunoassay is a homogenous assay,

[0150] 13. Use according to item 12, wherein the homogenous assay is Proximity Extension Assay.

[0151] 14. Use according to item 12, wherein the homogenous assay is Proximity Ligation Assay.

[0152] Examples

[0153] The invention will be further described in the following illustrative example(s). The example(s) are merely for facilitating understanding of the invention and shall not be construed as limiting the scope of the invention, which is that of the appended claims.

[0154] Example 1

[0155] This example relates to implementation of the invention for monitoring of a process for recombinant production of a protein of interest in a HEK293 cell line.

[0156] A panel of PEA-based assays for the most relevant HCPs (selected from Table 1) are prepared as known in the art (Assarsson et al., PLoS 1, 2014, 9, 4, e95192 for qPCR readout; or Wik et al., 2021, Mol Cell Proteomics 20, 100168 and WO2021 / 191442 for NGS readout, proximity probe manufacturing as disclosed in W02017 / 068116, absolute quantification as disclosed in Siegbahn A, et al., PLoS One. 2023 Nov 14;18(11): e0293465 ). An assay for the protein of interest is also included in the panel.

[0157] A sample containing a few pL-mL is withdrawn from, or between, any or multiple bioprocess steps of the method as described in Figure 1. If the withdrawn sample is from the cell culture, a cell lysis step is first performed to release the HCPs from within the cells. Protease inhibitors are added to the withdrawn sample which then is centrifuged to remove cell debris and insoluble materials. This step results in a clarified supernatant that contains soluble proteins, including HCPs. After centrifugation, the supernatant is filtered through a 0.22-0.45 pm filter to remove any remaining particulates and ensure sterility. Protease inhibitors may also be included after the 0.22-0.45 pm filtering step. If the sample is stored for later simultaneous analysis of multiple samples in the process, the sample is aliquoted and stored at -80°C until further analysis.

[0158] Prior to analysis, a sample aliquot is diluted in buffer with appropriate pH and ionic strength to optimize protein solubility and stability and to be compatible with the PEA analysis. The dilution factor is dependent on the expected HCP level in the sample, i.e. the cell culture or harvest contain high amounts of HCPs and thus need appropriate dilution to be in the dynamic range of the PEA analysis. Samples further down in the process in Figure 1 require a smaller dilution factor as the HCP levels decrease throughout the process. Appropriate dilution buffers are described in the above cited prior art.

[0159] The HCPs are then analyzed using the PEA panel developed above. For absolute quantification, standard curves of the HCPs are also included in the PEA analysis, generally as described in Siegbahn et aL, 2023.

[0160] The relative or absolute abundances of each analyzed HCP before and after multiple runs of a purification step in the bioprocess is used for quality assessment of the respective step. Quality in this regard is the reproducible performance of the respective step to consistently achieve the expected purification.

[0161] The absolute abundance values obtained for each HCP is compared to the measured amount of protein of interest and given as a part per million (ppm) value (amount of HCP divided by the amount of protein of interest). This ratio is a key quality indicator reported in the process and product specifications submitted for regulatory approval.

Claims

1. CLAIMS1. A method for detecting at least one specific host cell protein (sHCP) native to a host cell species in a composition of proteins produced by said host cell species, wherein said composition comprises a plurality of specific HCPs and at least one biological entity that is not native to the host cell species and wherein the method comprises a dual-recognition immunoassay.

2. The method of claim 1, wherein the host cell is modified to produce the biological entity.

3. The method of any one of claims 1-2, wherein the biological entity is produced for inclusion in a medicinal product for human or animal use.

4. The method of any one of claims 1-3, wherein the sHCP is present in the composition at an amount of less than 1% by weight of the amount of the biological entity, such as between 1-100 ppm by weight of the amount of the biological entity.

5. The method of any one of claims 1-4, wherein a plurality of specific host cell proteins (sHCPs) are detected, such as at least 5, at least 10, at least 20, at least 50, or at least 100 specific host cell proteins (sHCPs).

6. The method of any one of claims 1-5, wherein the host cell is a mammalian cell, such as a Chinese Hamster Ovary cell, a HEK-293 cell, or a HeLa cell.

7. The method of any one of the preceding claims, wherein the dual-recognition immunoassay is configured to detect a complex between the biological entity and a sHCP.

8. The method of any one of the preceding claims, further comprising detecting a leached affinity ligand using a dual-recognition immunoassay.

9. The method of any one of claims 1-8, further comprising determining an absolute and / or a relative concentration of the specific HCP (sHCP), a complex between the biological entity and the sHCP and / or the affinity ligand in the composition.

10. The method of any one of claims 1-9, wherein any dual-recognition immunoassay is a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay.

11. A method for quality assessment of a purification step in a purification method for removal of at least one specific host cell protein (sHCP) from a composition comprising the at least one sHCP and a biological entity, the method for quality assessment comprising(i) Determining a concentration of the at least one sHCP prior to the purification step;(ii) Determining a concentration of the at least one sHCP after the purification step; and(iii) Assessing the quality of the purification step by comparing the concentrations of the at least one sHCP before and after the purification step.

12. The method according to claim 11, wherein the concentrations are absolute or relative.

13. The method according to claim 11, wherein an absolute concentration for the at least one specific host cell protein (sHCP) is determined for a final product obtained in the purification process.

14. The method according to any one of claims 11-13, wherein the concentration determinations in steps (i) and (ii) are done for at least 5, such as at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100 specific host cell proteins (sHCPs).

15. The method of any one of claims 11-14, wherein the method comprises a dualrecognition immunoassay configured to detect a complex between the biological entity and a sHCP.

16. The method of any one of claims 11-15, further comprising detecting a leached affinity ligand using a dual-recognition immunoassay.

17. The method according to any one of claims 1-16, wherein the host cell is a human cell, such as a HEK293 cell, and the specific host cell proteins (sHCPs) are selected from the host cell proteins listed in Table 1.

18. The method according to any one of claims 1-16, wherein the host cell is a Chinese Hamster Ovary cell, and the specific host cell proteins (sHCPs) are selected from the host cell proteins listed in Table 2.

19. The method according to any one of claims 11-18, wherein the concentration determinations in steps (i) and (ii) are done with a dual recognition immunoassay.

20. The method according to claim 19, wherein the dual recognition immunoassay is a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay.

21. A method for assessment of reproducibility of a process for purification of a biological entity produced by expressing the biological entity in a host cell, wherein the biological entity is not native to the host cell species, to obtain a composition comprising a plurality of specific host cell proteins (sHCPs) and the biological entity, the purification process comprising subjecting the composition to a plurality of purification steps to remove the sHCPs and retain the biological entity, the method for reproducibility assessment comprising(i) Determining, for at least two separate runs of the same purification process, a concentration for at least one or each of the specific host cell proteins at at least two instances selected from prior to, in between, or after the purification steps to obtain sHCP concentration values at each such instance; and(ii) Assessing the reproducibility of the purification process by comparing the sHCP concentration values obtained from the separate runs of the purification process.

22. The method according to claim 21, wherein the sHCP concentration values are absolute or relative concentration values.

23. The method according to any one of claims 21-22, wherein the sHCP concentration values are obtained for at least 5, such as at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100 sHCPs.

24. The method of any one of claims 21-23, wherein the method comprises a dualrecognition immunoassay configured to detect a complex between the biological entity and a sHCP.

25. The method of any one of claims 21-24, further comprising detecting a leached affinity ligand using a dual-recognition immunoassay.

26. The method of any one of claims 21-25, wherein the host cell is a human cell, such as a HEK293 cell, and the specific host cell proteins (sHCPs) are selected from the host cell proteins listed in Table 1.

27. The method according to any one of claims 21-26, wherein the host cell is a Chinese Hamster Ovary cell, and the specific host cell proteins (sHCPs) are selected from the host cell proteins listed in Table 2.

28. The method according to any one of claims 21-27, wherein the sHCP concentration values are obtained with a dual recognition immunoassay.

29. The method according to claim 28, wherein the dual recognition immunoassay is a homogenous assay, such as Proximity Extension Assay or Proximity Ligation Assay.

30. The method according to any one of claims 21-29, wherein the comparison is performed by forming univariate or multivariate ratios of direct measurements of concentrations, multivariate data analysis, using dimensionality reduction algorithms such as Principal Component Analysis or Uniform Manifold Approximation and Projection; or deep learning-based reduction methods such as autoencoders.

31. Kit for detecting a specific Chinese Hamster Ovary (CHO) protein native to a CHO cell line, in a mixture of proteins produced by said CHO cell line, said kit comprising a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the specific CHO protein, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding.

32. Kit for detecting a leached proteinaceous affinity ligand used in an affinity purification of a protein composition, said kit comprising a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the affinity ligand, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding.

33. Kit for detecting a complex formed between a protein recombinantly produced in a host cell and a Host Cell Protein, said kit comprising a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the complex, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneous binding.

34. The kit according to claim 33, wherein one protein-binding moiety is capable of binding specifically to the recombinantly produced protein and one protein-binding moiety is capable of binding specifically to the Host Cell Protein.

35. The kit according to any one of claims 31-34, wherein the first and second reagent compositions are provided as a single reagent composition or as separate reagent compositions.

36. The kit according to any one of claims 31-35, wherein the matched protein-binding moieties are coupled to oligonucleotides capable of interacting and forming a reporter nucleic acid molecule when the protein-binding moieties simultaneously bind to the host cell protein and in presence of the second reagent composition.

37. The kit according to any one of claims 31-36, wherein the protein-binding moieties are selected from antibodies and fragments thereof, nucleic acid molecules such as aptamers, molecular imprinted polymers.

38. The kit according to any one of claims 31-37, wherein said first reagent composition comprises matched protein-binding moieties capable of simultaneously and specifically binding to a plurality of specific CHO proteins, such as at least 5, at least 10, at least 20, at least 50, or at least 100 specific CHO proteins.

39. The kit according to any one of claims 32, 35-38, wherein said proteinaceous affinity ligand is selected from the group consisting of Protein A, Protein G, Protein L, and functional variants thereof.

40. A method for detecting a proteinaceous affinity ligand in a sample, said method comprising contacting the sample with a first reagent composition comprising two or more matched protein-binding moieties capable of simultaneously and specifically binding to the affinity ligand, and a second reagent composition comprising reagents capable of generating a signal dependent on an occurrence of such simultaneousbinding; inducing the generation of signal, and measuring the signal thereby detecting the affinity ligand.

41. The method according to claim 40, wherein the matched protein-binding moieties are coupled to oligonucleotides capable of interacting and forming a reporter nucleic acid molecule when the protein-binding moieties simultaneously bind to the host cell protein and in presence of the second reagent composition.

42. The method according to any one of claims 40-41, wherein the protein-binding moieties are selected from antibodies and fragments thereof, nucleic acid molecules such as aptamers, molecular imprinted polymers.

43. The method according to any one of claims 40-42, wherein said proteinaceous affinity ligand is selected from the group consisting of Protein A, Protein G, Protein L, and functional variants thereof.

44. Use of a kit for performing a dual recognition immunoassay in a method according to any one of claims 1-30, 40-43.

45. Use according to item 44, wherein the dual recognition immunoassay is a homogenous assays, as Proximity Extension Assay or Proximity Ligation Assay.

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