HPLC-based detection of aggregates in protein samples

Abstract

The present application provides a rapid and simple method for measuring residual pDADMAC in samples containing recombinant proteins of interest, based on the use of reversed-phase hydrophobic interaction HPLC in combination with charged aerosol detection.

Description

[Technical field]

[0001] The present invention relates to the field of recombinant protein production and the recovery and purification of recombinant proteins from host cell supernatants. The present invention utilizes a method to measure the levels of poly(diallyldimethylammonium chloride) pDADMAC in a sample and ensure its removal during protein purification. [Background technology]

[0002] In the therapeutic field, the use of biological entities such as proteins and antibodies, and especially antibody-derived molecules, is constantly growing in presence and importance, and with it the need for parallel and controlled production methods. The commercialization of therapeutic proteins requires the production of large amounts of therapeutic proteins, and much effort has been put into improving the host cells expressing the desired proteins and their processing, resulting in an increase in the titer of the product. As a result, higher amounts of biomass and debris are also observed at the cell culture level, which must be removed as part of the protein purification process.

[0003] One approach to remove biomass to obtain clarified cell culture fluid and aid in purifying the protein of interest from said fluid includes the use of flocculants such as cationic polymers that are used to encapsulate mammalian cells and enzymes and to flocculate microbial cell cultures.Typically, the flocculants are removed from the cell culture fluid after settling and then further processed by centrifugation and / or depth filtration to obtain the so-called clarified cell culture from which the protein of interest is purified.

[0004] Flocculants can be anionic, cationic or "multimodal". When the pH of a solution is below the pI of a particular protein, the protein carries a net positive charge. Under these conditions, cationic polyelectrolytes can precipitate impurities, leaving the protein of interest in solution. Conversely, anionic polyelectrolytes can precipitate the protein of interest to form a protein-polyelectrolyte precipitate, leaving impurities in solution.

[0005] Cationic polymers are particularly suitable for this application, given that cells and cell debris typically have an overall negative charge. This results in the formation of neutralized particles formed by ionic interactions that fall out of solution. Examples of cationic polymers used for flocculation of clarified cell culture fluid from cells producing recombinant proteins include poly(diallyldimethylammonium chloride) (pDADMAC), polyamines, polyamino acids, polyacrylamides and chitosan.

[0006] On the other hand, anionic flocculants have also been used in this context, in particular the anionic polyelectrolytes polyvinylsulfonic acid, polyacrylic acid and carboxymethyldextran sulfate, which have been evaluated for the concentration and selective precipitation of monoclonal antibodies from CHO cell harvests leaving impurities in the supernatant.

[0007] More recent approaches have attempted to use agglutinants with multimodal functionality that do not require customization and can be used with a wide range of antibodies.

[0008] Additional information on the use of flocculants to clarify cell culture supernatants can be found in Singh et al. 2016 Clarification technologies for monoclonal antibody manufacturing processes: current state and future perspectives Biotechnol Bioeng 113:698.

[0009] From the perspective of large-scale commercial manufacturing of therapeutic drugs, it is essential to ensure that such aggregates are removed to levels safe for human administration during the subsequent purification process, before the protein is prepared into an administrable form.

[0010] An example of a cationic polymer known to be useful as a flocculant in cell culture is poly(diallyldimethylammonium chloride) (pDADMAC) (see, e.g., Peck et al. 2015 Dosing considerations and impacts on the clarification of mammalian cell culture feed streams using poly-diallyldimethylammonium chloride flocculant in conjunction with Clarisolve® depth filters). To date, methods for measuring residual pDADMAC in pretreated cell culture feed streams are based on the use of surface plasmon resonance (see, e.g., Detection of residual pDADMAC using surface plasmon resonance spectroscopy, Merck KGaA). More recent methods are based on the use of evaporative light scattering detection (ELSD) (Khodadadian et al. 2019 Determination of residual poly diallyldimethylammonium chloride (pDADMAC) in monoclonal antibody formulations by size exclusion chromatography and evaporative light scattering detector) Biologicals 57:21. However, existing methods have limitations with respect to precision, sensitivity, dynamic range, and the nature of the calibration curve.

[0011] Therefore, there is a continuing need to provide a rapid and robust method for detecting the level of residual aggregating agent applicable to samples containing protein complexes, which would allow for the assessment of the clearance of this aggregating agent during the manufacturing process. Summary of the Invention [Means for solving the problem]

[0012] The present invention provides a rapid and simple method for measuring pDADMAC in a sample containing a recombinant protein of interest, which involves the use of reversed-phase hydrophobic interaction HPLC and detection of charged aerosols.

[0013] In a first aspect, the present invention provides a method for measuring pDADMAC in a sample containing a recombinant protein of interest, comprising the steps of: a) applying the sample to a reversed-phase hydrophobic interaction HPLC; b) eluting the bound pDADMAC from the column using a hydrophobic gradient elution; c) measuring the pDADMAC recovered in step b) using a charged aerosol detector, The sample applied in a) comprises 0.2-3% (v / v) of a CAD-compatible acid.

[0014] In a further aspect, the invention relates to a method of producing a protein of interest comprising the addition of pDADMAC during protein purification, the subsequent removal of said pDADMAC being measured according to a method of the invention for measuring pDADMAC. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a calibration curve obtained from measuring six concentrations of pDADMAC according to the method of the present invention. [Diagram 2]FIG. 1 shows the linearity of results obtained from plotting individual measured concentrations against the theoretical levels of pDADMAC spiked in drug substance samples (five levels evaluated from 1.0 to 10.0 μg pDADMAC / mL). [Diagram 3] FIG. 1 shows an overlay of chromatographic profiles of drug substance analyzed "as is" (unspiked) and spiked with 1.0 μg pDADMAC / mL. [Figure 4] FIG. 1 shows validation of pDADMAC recovery at a spike level of 1.0 μg / mL in two different drug substance samples. [Diagram 5] FIG. 1 shows a summary of accuracy and precision results obtained with two drug substance samples spiked at three concentration levels (1.0-2.0 and 5.0 μg pDADMAC / mL). [Figure 6] FIG. 1 shows a summary of accuracy and precision results for additional engineered antibody types Fab-scFv (termed bispecific) and Fab-2x dsscFv (termed trivalent). [Figure 7] FIG. 1 shows an overlay of chromatographic profiles of drug substance analyzed "as is" (unspiked) and spiked with 1.0 μg pDADMAC / mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention solves the above-stated need by providing a new method for detecting pDADMAC in complex protein mixtures, which is fast and simple and allows for the assessment of the clearance of this aggregating agent during protein production. In particular, the method allows for the measurement of pDADMAC in real time during various purification steps.

[0017] In a first aspect, the present invention provides a method for measuring pDADMAC in a sample containing a protein of interest, comprising the steps of: a) applying the sample to a reversed-phase hydrophobic interaction HPLC; b) eluting the bound pDADMAC from the column using a hydrophobic gradient elution; c) measuring the pDADMAC recovered in step b) using a charged aerosol detector, The sample applied in a) comprises 0.2-3% of a CAD-compatible acid.

[0018] The Charged Aerosol Detector (CAD) is designed to be used for HPLC separations performed with volatile aqueous / organic mobile phases.

[0019] Hydrophobic gradient elution involves a change in the hydrophobicity of the mobile phase between the beginning (e.g., 90% aqueous) and end (e.g., 90% organic; methanol / acetonitrile) of the separation.

[0020] This method involves the use of CAD equipment, which requires an evaporation process for detection, and therefore requires a volatile mobile phase to which compatible additives can be added. Also, the additives contained in the sample injected in the chromatographic step as well as the mobile phase entering the CAD must be compatible. The method of the present invention uses a "CAD-compatible acid". As used herein, this term refers to any acid suitable for use in CAD. Typically, acidic additives used for CAD detection include TFA (trifluoroacetic acid), acetic acid, formic acid, or heptafluorobutyric acid.

[0021] As an example, a hydrophobic gradient can be obtained by using mobile phase A=0.1% TFA (trifluoroacetic acid) / HO, mobile phase B=0.1% TFA / CHCN and mobile phase C=0.1% TFA / MeOH. This example should not be considered limiting, considering that the skilled person knows different volatile mobile phases available for such gradients, such as mobile phase bases based on the use of ethanol or isopropanol as organic solvents, to which a CAD-compatible acid modifier will be added.

[0022] The CAD device allows the detection of all non-volatile and many semi-volatile analytes in the eluent. The compound of interest (pDADMAC polymer) does not need to have a chromophore as in UV detection, nor does it need to be ionized as in mass spectrometry. In CAD, the detector measures the charge imparted to the dry particles formed from the ultrafine aerosol of the analyte. The charge measured is proportional to the amount of analyte in the sample. The operation of the CAD detector is fairly simple, requiring only the setting of a few controllable parameters, and provides increased sensitivity compared to ELSD (Evaporative Light Scattering Detector) detection.

[0023] pDADMAC or poly(diallyldimethylammonium chloride) is a water-soluble cationic polymer that is highly effective as a flocculant in that it rapidly aggregates negatively charged cells and cell debris into larger particles via an ionic interaction mechanism. pDADMAC is available in a variety of polymer sizes, from less than 10 KDa to 10,000 KDa, but for flocculating mammalian cell cultures, a medium average molecular weight size (e.g., about 500 KDa) is often used and can be obtained by known providers such as Merck.

[0024] In a particular embodiment, the hydrophobic interaction HPLC (high pressure liquid chromatography) is a PLRP-S HPLC, i.e., Polymer Laboratories Reversed Phase-Styrene (divinylbenzene), supplied by Agilent or Phenomenex. The PLRP-S HPLC column is packed with a macroporous, essentially uniformly hydrophobic stationary phase with no residual surface functional groups (i.e., no typical silica problems of acidic silanol groups), allowing optimal separation power. Those skilled in the art will appreciate that the appropriate pore size is selected depending on the molecules to be separated. The 1000 Å pore size of the packing material is particularly suitable for the separation of high molecular weight species present in the samples of the invention (including pDADMAC polymers). The PLRP-S material is mechanically stable to the pressures obtained at optimized analytical conditions. Furthermore, separations can be performed at pH 1-14 using the PLRP-S HPLC column without any of the constraints associated with silica packing. Thus, in a particular embodiment of the invention, the PLRP-S column has a pore size of 1000 Å.

[0025] In another embodiment of the method of the invention, the sample comprises 0.1% to 3% of a CAD-compatible acid, such as 0.1% to 2% of a CAD-compatible acid, or 0.1% to 1.5% of a CAD-compatible acid, or 0.5% to 1.5% of a CAD-compatible acid. In a further embodiment of the invention, the sample comprises 1% of a CAD-compatible acid.

[0026] In one embodiment, the sample applied in step a) has a pH of 2-4.

[0027] Possible CAD-compatible acids in the context of the present invention include, but are not limited to, trifluoroacetic acid (TFA), heptafluorobutyric acid, acetic acid, or formic acid. In a particular embodiment of the method of the present invention, the CAD-compatible acid is trifluoroacetic acid.

[0028] The exact proportion and nature of the CAD-compatible acid will be selected by the skilled artisan, taking into account the specificity of the sample, and in particular the properties of the recombinant protein of interest that is the subject of purification.

[0029] In certain embodiments of the invention, the CAD-compatible acid is added to the sample prior to step a) of the above method, such as by addition from a stock solution of 15% (vol / vol) CAD-compatible acid.

[0030] Addition of acid to the sample may affect the solubility of recombinant proteins also contained in the sample, and in some cases may precipitate them. This precipitate may also contain some residual pDADMAC, which would result in an underestimation of the amount of pDADMAC present in the sample. To avoid such a phenomenon, the skilled artisan will recognize the beneficial effect of diluting the sample to reduce the concentration of recombinant proteins.

[0031] Similarly, if it is desired to adjust the protein concentration, the sample may be diluted using an appropriate buffer, such as, for example, by adding more buffer already present in the sample.

[0032] In one embodiment of the method of the invention, the sample applied in step a) comprises a recombinant protein concentration of less than 200 mg / ml, such as less than 100 mg / ml, for example less than 60 mg / ml. In a further particular embodiment, the sample comprises a recombinant protein concentration of between 20 and 60 mg / ml.

[0033] Similarly, other sample preparation techniques, such as filtration steps to remove smaller molecular weight molecules, may be desirable.

[0034] In one embodiment of the method of the invention, the sample applied in step a) has been filtered through a filter with a pore size of 80 kDa or less, 60 kDa or less, 50 kDa or less, in a further particular embodiment the sample is filtered through a filter with a pore size of 30 kDa.

[0035] Similarly, acid treatment of the sample may be desirable so that some or all of the proteins present in the sample are digested.In another embodiment, the method comprises acid treatment of the sample before applying the sample in step a).In a further embodiment of the method, the sample is subjected to an acid treatment step and a filtration step before applying the sample in step a).

[0036] In certain embodiments, the methods of the invention further comprise the step of determining the concentration of pDADMAC present in the sample by reference to a control.

[0037] Typically, a standard curve is generated using known concentrations of pDADMAC added to a formulation buffer and treated according to the methods of the invention.

[0038] When the method of the present invention is used in a quantitative assay, the pDADMAC charge measured in a sample containing an unknown amount of polymer is interpolated with the calibration curve to obtain the pDADMAC concentration of the sample. However, the method of the present invention can also be used in "limit test" assays, i.e. tests aimed at determining that the presence of pDADMAC is below a certain limit of interest. In these "limit tests", each sample is analyzed "as is" and spiked with an amount of pDADMAC that corresponds to the limit of interest. For example, if the limit of interest is 1 μg / ml pDADMAC, the sample is taken, split into two aliquots, one resulting aliquot is spiked with 1 μg / ml pDADMAC, the concentration of pDADMAC is measured by interpolation with the calibration curve, and then the remaining aliquot is directly measured by the method of the present invention, and the absence of pDADMAC is verified by interpolation with the calibration curve.

[0039] A sample containing the recombinant protein of interest may be obtained directly from a step in the purification process or may be obtained at the end of the process or from a step within the purification process, allowing analysis of the progressive clearance of pDADMAC.

[0040] In one embodiment, the sample applied in step a) is a sample obtained directly from a step of purification of said recombinant protein to which a CAD-compatible acid has been added.

[0041] In the therapeutic field, the proteins of interest may be selected, for example, from antibodies, cytokines, growth factors, hormones and other regulatory peptides and proteins. Such proteins may be extracted from tissues and secretions, but due to the development of biotechnology and the increasing need for larger quantities of these molecules and greater standardization of their preparation, they are often produced as recombinant proteins.

[0042] In a particular embodiment of the present invention, the recombinant protein of interest is an antibody, an antibody fragment or a derivative thereof.

[0043] Recombinant proteins of interest, such as antibodies, to be produced for large-scale commercial purposes can be produced by culturing eukaryotic host cells transfected with one or more expression vectors encoding the recombinant antibody, preferably mammalian cells, more preferably Chinese Hamster Ovary (CHO) cells.

[0044] Mammalian cells can be cultured in any medium that supports the growth and expression of the antibody, preferably the medium is a chemically defined medium that does not contain animal serum and animal derived products such as peptones. There are different cell culture media available to those skilled in the art that contain different combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or equivalent energy sources present at appropriate concentrations to allow cell growth and protein production. Additional cell culture medium components can be included in the cell culture medium at appropriate concentrations at different times during the cell culture cycle that would be known to those skilled in the art.

[0045] Mammalian cell culture can be carried out in any suitable vessel, such as shake flasks or bioreactors, which may or may not be operated in fed-batch mode, depending on the scale of production required. These bioreactors may be either stirred tanks or airlift reactors. A variety of large-scale bioreactors are available, with volumes of more than 1,000 L to 50,000 L, preferably 5,000 L to 20,000 L, or 10,000 L. Alternatively, small-scale bioreactors, such as 2 L to 100 L, can be used to produce antibodies according to the methods of the invention.

[0046] The antibody or antigen-binding fragment thereof that can be produced according to the method of the present invention is typically found in the supernatant of mammalian host cell culture, typically CHO cell culture.In the case of CHO culture process, the target protein, such as antibody or antigen-binding fragment thereof, is secreted into the supernatant, and the supernatant is typically collected by centrifugation according to the method known in the art.For the avoidance of doubt, supernatant refers to the liquid that is above the sedimented cells resulting from centrifugation of cell culture.

[0047] Flocculation is the process by which particles in suspension form aggregates or clusters of larger size. During this process, particles come out of suspension in the form of flocs by the addition of a flocculant. The flocculant can be anionic or cationic polymers. There are natural flocculants as well as synthetic flocculants that can be manufactured to have specific molecular weights and distributions.

[0048] Cationic polymers interact with negatively charged particles such as organic matter. In cell culture supernatants, cationic polymers interact with negatively charged particles, such as living and non-living cells, cell metabolic products including nucleic acids, proteins and liposomes, and cell debris. The aggregation of cationic polymers with negatively charged compounds found in cell culture supernatants leads to the formation of larger particles that then fall out of solution.

[0049] The addition of cationic polymers such as poly(diallyldimethylammonium chloride) (pDADMAC) to mammalian cell culture medium that contains or has contained cells expressing recombinant proteins aggregates negatively charged particles including cells (viable and non-viable), cell metabolites and cell debris. These large aggregated particles can then be removed by centrifugation or by gravity settling, producing a feedstream that has a significantly higher collection filter train throughput compared to typical centrifuged harvest feedstreams.

[0050] The supernatant collected after this filter train is typically called clarified cell culture fluid and is then further processed in several steps, typically including two or three chromatography steps and an ultrafiltration / diafiltration step, to obtain the purified protein of interest.

[0051] During this further or downstream purification process, residual flocculating agents such as pDADMAC, as well as other impurities including host cell proteins, protein aggregates and degradation products, are also removed.

[0052] In this context, it may be important to measure the final concentration of pDADMAC in the purified protein sample (typically referred to as the drug substance) to ensure sufficient clearance, but also to make this measurement during the purification steps to improve control over the process.

[0053] Thus, in certain embodiments of the process of the invention, the sample is obtained from the final purified protein of interest, and in alternative embodiments of the method of the invention, the sample is obtained from a step in the purification stream of said protein.

[0054] In a second aspect, the invention relates to a method for producing a protein of interest, the method comprising the step of adding pDADMAC during protein purification, and the removal of said pDADMAC is measured according to the method described in the previous paragraph.

[0055] The term "antibody" or "antibodies" as used herein refers to monoclonal or polyclonal antibodies. The term "antibody" or "antibodies" as used herein includes, but is not limited to, recombinant antibodies produced by recombinant techniques known in the art. "Antibody" or "antibodies" includes antibodies of any species, particularly mammalian species, such as IgD, IgG1, IgG 2a , IgG 2bHuman antibodies of any isotype including IgE, IgG3, IgG4, and dimers of this basic structure including IgGA1, IgGA2, or antibodies produced as pentamers such as IgM and modified variants thereof, non-human primate antibodies, e.g. from chimpanzee, baboon, rhesus or cynomolgus monkey, rodent antibodies, e.g. from mouse or rat, rabbit, goat or horse antibodies, as well as camelid antibodies (e.g. from camel or llama such as Nanobodies™) and derivatives thereof, or from avian species such as chicken antibodies, or from fish species such as shark antibodies. The term "antibody" or "antibodies" also refers to "chimeric" antibodies in which a first portion of at least one heavy and / or light chain antibody sequence is derived from a first species and a second portion of the heavy and / or light chain antibody sequence is derived from a second species. Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences. "Humanized" antibodies are chimeric antibodies that contain sequences derived from a non-human antibody. In most cases, humanized antibodies are human antibodies (recipient antibodies) in which residues from the recipient's hypervariable regions are replaced with residues from the hypervariable regions [or complementarity determining regions (CDRs)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken, or non-human primate that have the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody outside the CDRs, i.e., in the framework regions (FRs), are further replaced by corresponding non-human residues. Additionally, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve the performance of the antibody. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to human disease treatment. Humanized antibodies and several different techniques for producing them are well known in the art. The term "antibody" or "antibodies" also refers to human antibodies, which can be produced as an alternative to humanization.For example, it is possible to generate transgenic animals (e.g., mice) that can produce a full repertoire of human antibodies upon immunization when endogenous mouse antibodies are not produced. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The introduction of human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies with specificity for a particular antigen when the transgenic animals carrying the human germline immunoglobulin genes are immunized with said antigen. Techniques for generating such transgenic animals and for isolating and producing human antibodies from such transgenic animals are known in the art. Alternatively, in transgenic animals, e.g., mice, only the immunoglobulin genes encoding the variable regions of mouse antibodies are replaced with the corresponding human variable immunoglobulin gene sequences. The mouse germline immunoglobulin genes encoding the antibody constant regions remain unchanged. In this way, the antibody effector functions in the immune system of the transgenic mouse, so that the development of B cells is essentially unchanged and can lead to improved antibody responses upon antigen exposure in vivo. Once the genes encoding a particular antibody of interest are isolated from such transgenic animals, the genes encoding the constant regions can be replaced with human constant region genes to obtain a fully human antibody. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies such as phage display or ribosome display technologies, using recombinant DNA libraries that are at least partially artificially or from donor immunoglobulin variable (V) domain gene repertoires. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells, which are ex vivo immunized with an antigen of interest and subsequently fused to generate hybridomas, which can then be screened for optimal human antibodies.The term "antibody" or "antibodies" as used herein also refers to non-glycosylated antibodies.

[0056] The term "antibody" or "antibodies" as used herein refers not only to uncleaved antibodies of any species, including human (e.g., IgG) and other mammalian species, but also to antibody fragments. Antibody fragments contain at least one heavy or light chain immunoglobulin domain as known in the art and bind to one or more antigens. Examples of antibody fragments according to the invention include Fab, Fab', F(ab')2, and Fv and scFv fragments, as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAbs), such as single domain antibodies (sdAbs), VFv fragments, and VIFv fragments. H H and V NAR Antibody fragments as defined above include those known in the art, including, but not limited to, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments or antibodies, including, but not limited to, Fab-Fv or Fab-Fv-Fv constructs.

[0057] In one embodiment, the antibody comprises: a) a polypeptide chain of formula (I): VH-CH1-X-V1, and b) a polypeptide chain of formula (II): VL-CL-Y-V2 a multispecific antibody molecule comprising or consisting of During the ceremony, VH represents the heavy chain variable domain; CH1 represents a domain of the heavy chain constant region, e.g., domain 1 thereof; X represents a bond or a linker; Y represents a bond or a linker; V1 represents a dsFv, sdAb, scFv or dsscFv, VL represents the light chain variable domain; CL represents a domain derived from the light chain constant region, such as Ckappa; V2 represents a dsFv, sdAb, scFv or dsscFv, wherein at least one of V1 or V2 is a dsFv or dsscFv described in WO 2015 / 197772, which is incorporated herein by reference.

[0058] As used herein, a "single-chain variable fragment" or "scFv" refers to a single-chain variable fragment comprising or consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL), stabilized by a peptide linker between the VH and VL variable domains. The VH and VL variable domains may be in any suitable orientation, for example, the C-terminus of the VH may be linked to the N-terminus of the VL, or the C-terminus of the VL may be linked to the N-terminus of the VH.

[0059] A "disulfide-stabilized single-chain variable fragment" or "dsscFv" refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains and which also contains an interdomain disulfide bond between the VH and VL.

[0060] A "disulfide-stabilized variable fragment" or "dsFv" refers to a single-chain variable fragment that does not contain a peptide linker between the VH and VL variable domains, but is instead stabilized by an interdomain disulfide bond between the VH and VL.

[0061] In one particular embodiment, the antibody is a multispecific antibody of the Fab-2x dsscFv type as described in WO 2015 / 197772, which is incorporated herein by reference.

[0062] In a further particular embodiment, the multispecific antibody molecule of the Fab-2x dsscFv type is a trivalent antibody, i.e. each Fv binds to a different epitope.

[0063] In a further particular embodiment, the multispecific antibody has the Fab-dsscFv-dsFv format as described in WO 2015 / 197772.

[0064] example The following examples were performed using a 1000Å 8μ particle (150×2.1 mm) PLRP-S column supplied by Agilent, and a 10% aqueous solution of pDADMAC supplied by Millipore (nr. 137069). The polycationic pDADMAC polymer was separated from other components of the prepared sample (protein, formulation excipients, ...) as detailed below.

[0065] Example 1: Creating a calibration curve Two concentrated solutions of 25 μg and 100 μg pDADMAC / mL were prepared in the appropriate protein formulation buffer using a 10% weight-to-volume standard of the polymer. These two solutions were further diluted with buffer to obtain a calibration curve with known theoretical concentrations of 0.5, 1.0, 2.0, 5.0, 8.0 and 10.0 μg pDADMAC / mL. Prior to HPLC analysis, each of the six calibration standards was acidified by adding 10 μL of 15% (v / v) TFA in water to 100 μL of the standard and injected in duplicate. Analysis was performed using a Corona™ Veo™ RS Charged Aerosol Detector, The gradient applied for elution was as follows: Solvent A: HO / TFA 0.1% Solvent B: CH3CN / TFA 0.1% Solvent C: MeOH / TFA 0.1%

[0066] [Table 1]

[0067] Calibration standards were injected before and after each series of samples was analyzed, and the areas of the 12 injections of the calibration standards were used to construct a quadratic (second order) regression line with a weighting of 1 / x. An example of the resulting calibration curve is shown in Figure 1. The coefficient of determination (r 2) was 1.00 (≧0.98, a typical acceptance criterion). The pDADMAC measurements obtained for each analytical sample were then interpolated to the calibration curve to determine the pDADMAC concentration.

[0068] Example 2: Linearity of results In one validation exercise, a drug substance (IgG4) sample containing 56.8 mg of antibody / mL of sample was initially spiked with pDADMAC at five concentration levels (1.0, 2.0, 5.0, 8.0 and 10.0 μg / mL), with triplicate preparations at each level. Prior to HPLC analysis, each sample was acidified by adding 10 μL of 15% aqueous TFA to 100 μL of standard and further diluted by adding 110 μL of the appropriate protein formulation buffer. This method was applied to analyze 15 preparations, and the resulting individual measured concentrations (in μg pDADMAC / mL of spiked sample) were plotted against the theoretical levels of added pDADMAC. As shown in Figure 2, the resulting coefficients of determination (r 2 ) was 1.00 (≥0.98, typical acceptance criterion). Thus, the method is linear in the range evaluated (1.0–10.0 μg pDADMAC per mL of drug substance sample).

[0069] Example 3: Quantitative measurement: Each sample was analyzed "as is" and spiked at 1.0 μg pDADMAC / mL sample (for each triplicate preparation). Each of the six preparations was subjected to an acidification and dilution step as follows: Add 10 μL of 15% TFA in water and 110 μL of the appropriate protein formulation buffer to 100 μL of sample prior to HPLC analysis.

[0070] An overlay of typical chromatographic profiles obtained for a drug substance sample (containing 97.8 mg IgG4 antibody / mL and derived from a manufacturing process applying pDADMAC aggregation) analyzed without and with 1.0 μg pDADMAC / mL is shown in Figure 3.

[0071] To avoid possible contamination or contamination of the detector, a switching valve on the CAD instrument was used to direct flow to the detector sprayer only for the time required to read the pDADMAC peak signal (i.e., approximately 1 min before and 1 min after the pDADMAC peak retention time). The profile showed a peak representing the pDADMAC polymer near 5.3 min. The specificity of the method was demonstrated as no interfering peak was detected at the retention time of the pDADMAC peak (5.3 min). Furthermore, no significant carryover of pDADMAC was observed between samples, as established by performing measurements of a calibration curve starting from the highest pDADMAC concentration and tapering off to the lowest concentration reference standard, and injecting one blank after the calibration curve.

[0072] A regression line was obtained using injections of six calibration standards taking into account theoretical pDADMAC concentrations. The absence of pDADMAC in the unspiked samples was verified, while the concentrations measured in the spiked samples were interpolated from the curve. Using the pDADMAC concentrations measured in the triplicate preparations, a spike level of 1.0 μg / mL is verified as follows: The percent recovery of measured and theoretical concentrations of pDADMAC should be between 60% and 140%. The %RSD (relative standard deviation) for the three pDADMAC measured concentrations must be ≤15%.

[0073] The pDADMAC recoveries at the 1.0 μg / mL spike level obtained with two different drug substance samples are shown in Figure 4.

[0074] Example 4: Accuracy, precision and limits of quantification: Validation of the pDADMAC assay was performed using two drug substance samples. The samples were obtained from two different manufacturing batches of the same antibody, one produced using an aggregating agent (hereafter referred to as the "aggregated" sample) and one produced without an aggregating agent (hereafter referred to as the "non-aggregated" sample). The non-aggregated sample contained 56.8 mg IgG4 antibody / mL, whereas the aggregated sample contained 97.8 mg IgG4 antibody / mL. Both samples were spiked with three concentration levels, 1.0, 2.0 and 5.0 μg pDADMAC / mL, and analyzed twice (on separate days) with the method of the present invention in triplicate preparations at each level. Each preparation underwent the same acidification and dilution steps as described in Examples 2 and 3 above. The following parameters were evaluated: Actual measured pDADMAC concentrations compared to the theoretical pDADMAC concentrations (average of triplicate preparations at each level within each run) Based on this, the accuracy (% recovery) was calculated. Precision-repeatability (%RSD, n=3) was calculated using each pDADMAC concentration (measured at each level within each run for each preparation). · Each pDADMAC concentration (measured for each preparation at each level across both occasions) was used to calculate the overall intermediary precision (%RSD, n=6).

[0075] As shown in Figure 5, such low levels of impurities met typical acceptance criteria for the three parameters at the three concentration levels evaluated. -Average (n=3) % recovery within 60%-140% for accuracy Precision - Mean repeatability (n=3) %RSD≦15% - Overall average (n=6) %RSD for intra-laboratory precision ≦20%

[0076] The limit of quantification of pDADMAC in the samples (calculated as the lowest concentration added to the sample at which three typical acceptance criteria were met) was 1.0 μg pDADMAC per mL of sample.

[0077] Example 5: Application of the method to additional antibody types: In further experiments, the method of the invention was validated for Fab-scFv formulated at 58.2 mg / mL, as well as for Fab-2x dsscFv described in WO 2015 / 197772 formulated at 80 mg / mL. The method was limit tested at the limit of 1.0 μg pDADMAC / mL of interest. In this case, pDADMAC concentrations (μg / mL) measured in spiked samples were quantified against a calibration curve (0.3-5.0 μg pDADMAC / mL) to calculate % recovery relative to the theoretical 1.0 μg / mL spiked concentration.

[0078] The contributions to method variability due to column, instrument and operator factors were assumed to be the same for both engineered antibody types. Therefore, four runs (six replicates each) were initially performed with Fab-scFv samples spiked with 1.0 μg pDADMAC per mL to assess accuracy, precision-repeatability and inter-laboratory precision. Additionally, a fifth experiment examined accuracy and precision-repeatability for a similarly spiked Fab-2x dsscFv Fab.

[0079] For both Fab-scFv and Fab-2x dsscFv samples, six replicates of addition at 1.0 μg pDADMAC / mL were subjected to an acidification step by addition of 10% aqueous TFA, followed by two-fold dilution in the appropriate buffer. The results are shown in FIG.

[0080] For both modified antibody formats, accuracy and precision-repeatability were evaluated and met typical acceptance criteria (% recovery within 60-140% and %RSD ≤ 15% n=6 preparations). Furthermore, for the Fab-scFv antibody format, intra-laboratory precision assessed on four occasions was within the acceptance criteria (overall RSD ≤ 20% for pDADMAC concentrations measured in 24 spiked samples).

[0081] Example 6: Application of the method with different sample preparations: An alternative sample preparation was evaluated as a limit test method (detection limit 1 μg / ml). For this determination, two samples were prepared. -One sample is neat (the drug substance obtained from the manufacturing process), - the other was a spiked sample with 1 μg pDADMAC per mL, Both are then incubated with strong acid (in this case 37% hydrochloric acid) at 80 °C for 3 h. After incubation, the samples are allowed to evaporate overnight until completely dry. They are then reconstituted in 0.5% aqueous TFA solution before being centrifuged in centrifugal filter units (30 kDa cutoff pore size) for purification and pDADMAC concentration. The sample containing the pDADMAC retained by the filter is reconstituted in 0.1% aqueous TFA solution.

[0082] The pDADMAC is then separated from the protein residues and excipients and impurities remaining on the PLRP-S column and measured according to the methods of the present invention using a CAD detector.

[0083] Exemplary results are shown in Figure 7. The method has been validated for several antibody types, evaluating specificity and detection limits with six replicates in each case. The length of incubation with hydrochloric acid was optimized in each case and determined to be within 3-6 h at 80 °C.

[0084] The addition of these sample preparation steps is particularly interesting for assessing pDADMAC clearance in in-process samples because matrix interferences are completely removed. The present disclosure includes the following preferred embodiments. (1) A method for measuring pDADMAC in a sample containing a recombinant protein of interest, comprising: a) subjecting the sample to reverse phase hydrophobic interaction HPLC; b) eluting the bound pDADMAC from the column using a hydrophobic gradient elution; c) measuring the pDADMAC recovered in step b) using a charged aerosol detector (CAD), The method of claim 1, wherein the sample applied in a) comprises 0.2-3% (v / v) of a CAD-compatible acid. (2) The method according to (1), wherein the sample applied in step a) comprises a protein concentration of less than 200 mg / ml, such as between 20 and 100 mg / ml, for example between 20 and 60 mg / ml. (3) The method according to (1) or (2), wherein the sample applied in step a) has a pH of 2 to 4. (4) The method according to any one of (1) to (3), wherein the sample applied in step a) contains 0.5 to 1.5% (v / v) of a CAD-compatible acid. (5) The method according to any one of (1) to (4), wherein the CAD-compatible acid is selected from trifluoroacetic acid, acetic acid, heptafluorobutyric acid, and formic acid. (6) The method according to any one of (1) to (5), wherein the reversed-phase hydrophobic interaction HPLC is PLRP-S HPLC. (7) The method according to any one of (1) to (6), further comprising the step of determining the concentration of pDADMAC present in the sample by reference to a control. (8) The method according to any one of (1) to (7), wherein the target recombinant protein is an antibody, an antibody fragment, or a derivative thereof. (9) The method according to any one of (1) to (8), wherein the sample applied in step a) is a sample obtained directly from a step of purifying the recombinant protein to which a CAD-compatible acid has been added. (10) A method for producing a protein of interest comprising adding pDADMAC during protein purification, wherein subsequent removal of the pDADMAC is measured according to a method according to any one of (1) to (9).

Claims

1. 1. A method for measuring pDADMAC in a sample containing a recombinant protein of interest, comprising: a) subjecting said sample to a reversed-phase hydrophobic interaction HPLC; b) eluting the bound pDADMAC from the column using hydrophobic gradient elution; c) measuring the pDADMAC recovered in step b) using a charged aerosol detector (CAD), A method, wherein the sample applied in a) comprises 0.2-3% (v / v) of a CAD-compatible acid.

2. 2. The method of claim 1, wherein the sample applied in step a) comprises a protein concentration of less than 200 mg / ml.

3. A method according to claim 1 or 2, wherein the sample applied in step a) comprises a protein concentration of 20 to 100 mg / ml.

4. A method according to claim 1 or 2, wherein the sample applied in step a) comprises a protein concentration of 20 to 60 mg / ml.

5. The method according to any one of claims 1 to 4, wherein the sample applied in step a) has a pH of from 2 to 4.

6. The method according to any one of claims 1 to 5, wherein the sample applied in step a) comprises 0.5 to 1.5% (v / v) of a CAD-compatible acid.

7. The method of any one of claims 1 to 6, wherein the CAD-compatible acid is selected from the group consisting of trifluoroacetic acid, acetic acid, heptafluorobutyric acid and formic acid.

8. The method according to any one of claims 1 to 7, wherein the reversed-phase hydrophobic interaction HPLC is a reversed-phase hydrophobic interaction HPLC having a column packed with poly(styrene / divinylbenzene).

9. 9. The method of any one of claims 1 to 8, wherein the method further comprises determining the concentration of pDADMAC present in the sample by reference to a control.

10. The method according to any one of claims 1 to 9, wherein the recombinant protein of interest is an antibody, an antibody fragment or a derivative thereof.

11. The method according to any one of claims 1 to 10, wherein the sample applied in step a) is a sample obtained directly from a step of purification of the recombinant protein to which a CAD-compatible acid has been added.

12. 12. A method of producing a protein of interest comprising adding pDADMAC during protein purification, wherein the subsequent removal of said pDADMAC is measured according to the method of any one of claims 1 to 11.

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