WO2019229058A1 - Method and system for providing antibody fragments suitable for online quality control - Google Patents

Abstract

The invention pertains to a method for providing fragments of mAh and mAh variants for analysis, the method comprises the steps of, in a serial order; (1) obtaining a sample comprising an antibody, (2) fragmenting the antibody in said sample to obtain F_c-fragments and F(ab')₂-fragments, (3) capturing the antibody fragments in two steps: (3.1) capturing the F(ab')₂-fragments, (3.2) capturing the F_c-fragments. The method further comprises a washing and elution step (4) subsequent to step 3, wherein the captured antibody fragment of choice for analysis is washed and eluted in the washing and elution step (4), thereby providing an analytical quality sample of said antibody fragment. Furthermore, the invention pertains to a system for providing fragments of mAh and mAh variants for analysis.

Description

METHOD AND SYSTEM FOR PROVIDING ANTIBODY FRAGMENTS SUITABLE

FOR ONLINE QUALITY CONTROL

Field of the Invention

This invention pertains in general to a method for carrying out a fragmentation reaction of monoclonal antibodies (mAbs) or mAb-variants to obtain a sample with analytical quality in a short time, a prerequisite for online quality control. Furthermore, it pertains to a system for carrying out said mAh or mAb-variant reaction to obtain a sample with analytical quality in a short time. Background of the Invention

Monoclonal antibodies (mAbs), with their inherently high selectivity and specificity constitute a large and growing portion of the high-value biotherapeutics market. The majority of marketed mAbs belong to the IgG class. IgGs consist of two heavy chains and two light chains linked by a total of approx. 16 inter- or intra- molecular disulfide bonds. The two heavy chains are linked by disulfide bonds and each heavy chain is disulfide bonded to a light chain. IgGs include antigen-binding (Fab) and crystallizable (F_c) regions: the two Fab parts are responsible for binding to the antigen, while the F_c unit binds to F_cy receptors, which regulate immune responses.

Glycosylation is a common post-translational modification for IgG antibodies produced by mammalian cells, which are frequently used for production. Glycosylation plays an important role for complement-dependent cytotoxicity (CDC) and antibody- dependent cell-mediated cytotoxicity (ADCC) functions through modulating the binding to the F_cy receptor. Particular glyco forms may be necessary to achieve therapeutic efficacy. These glycoforms may be targeted by glycosylation engineering, but may also be affected by cell culture conditions. The important role of glycosylation in the therapeutic effect of mAbs makes the control of the glycosylation profile a key aspect to consider in regulatory requirements and quality compliance. Variations in the N-linked glycan profile appearing during the manufacturing process can arise from various conditions (e.g. dissolved oxygen, shear stress, pH, glucose and amino acid concentration).

Given the inherent variability in the mAh production process, continuous monitoring of the quality attributes, such as protein glycosylation, is essential, since it would enable in-process corrections when undesired protein characteristics are detected. Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. EP 3 266 800 Al relates to a method for the purification of a human lgG-CHl domain comprising molecule using an antigen-binding protein that is capable of binding to an epitope that is comprised in the CH1 domain of each of human lgGl, human lgG2, human lgG3 and human lgG4. The application presents these affinity binders and their use in affinity purification studies, using commonly used proteases such as papain and pepsin.

The golden standard for characterization of mAbs from production in field of biotheraputics is the use of LC/MS after enzymatic fragmentation of the large antibody molecule down to smaller subunits (e.g. Fragment crystallizable (F_c), Fragment antigen- binding (F(ab')2), Fight chain (Fc), Fragment of heavy chain in F(ab')2 (i.e. Fd). The enzyme IdeS supplied today by Genovis under the tradename FabRICATOR is at present the most commonly used enzymatic tool when fragmenting the mAh prior to FC/MS- analysis. This is due to its ease of use and simple data interpretation. However, when using the IdeS enzyme, it is still necessary to perform the enzymatic

fragmentation step in offline mode on the laboratory bench. The procedure is relatively labor-intense and time consuming, and together with the FC/MS step, the analytical answer is in the best-case scenario retrieved 2-3 hours after the sample initially was taken from the mAh production line. A drawback is the presence of manual steps in the handling of the material, creating possible errors leading to less accurate analyses.

Thus, there is a need for new approaches for characterization of mAbs, with high enough purity and accuracy within a short enough time span, to enable online quality control.

Summary of the invention

Consequently, the present invention seeks to mitigate, alleviate, eliminate or circumvent one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination by providing a method for mAh and mAb-variant fragment analysis, comprising the steps of, in a serial order; (1) obtaining an antibody sample, (2) fragmenting the antibodies in said sample to obtain F_c-fragments and F(ab')2- fragments, (3) capturing the antibody fragments in two steps: (3.1) capturing the

F(ab')2-fragments, (3.2) capturing the F_c-fragments, the method further comprises a washing and elution step (4), wherein the captured antibody fragment of choice for analysis is washed and eluted, thereby providing an analytical quality sample of said antibody fragment of choice. The present invention is superior to methods and systems known in the art, as the method creates high accuracy, high reproducibility and a short time for an assay cycle.

Furthermore, a system for mAb-fragment analysis is provided, comprising at least one sample inlet, at least a fragmenting column, a first capturing column and a second capturing column arranged in a serial arrangement with a serial inlet in the beginning of the fragmenting column and a serial outlet from the end of the second capturing column, and at least one processed sample outlet, wherein each of the columns furthermore comprises an individual inlet and outlet, which can be either opened or closed, to enable individual washing of, and elution from, a specific column, whereby analytical quality fragments bound to the columns may be washed and eluted from any of said columns by means of a washing circuit and an elution circuit, respectively, and eluted analytical quality fragments are further conveyed, optionally after further processing, to the processed sample outlet.

The automated system gives rise to high accuracy, high reproducibility and short time for an assay cycle. Consumption of reagents is reduced, while accuracy and flexibility are far improved for the system as compared to manual handling.

Brief Description of the Drawings

These and other aspects, features and advantages of which the invention is capable of will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which

Fig. 1 shows a schematic view of the mAbLAB workflow reaction from the target mAh perspective. The different mAb-subunits produced using enzymatic digestion (F_c, F(ab')2) can be sequentially purified, here followed by a reduction step to generate Lc- and Fd-subunits,

Fig. 2 is a schematic view of the mAbLAB workflow including a desalting column. Sampling is here directly from an online connected bioreactor. The F(ab')2- fragments are captured in the first capturing step (step 3.1) allowing F_c-fragments successfully fragmented to continue to the remaining capturing step (3.2). The purified mAb-fragments of analytical quality are finally online -processed and analysed using a mass spectrometer,

Fig. 3. is a schematic view of the mAbLAB workflow including a desalting column. Sampling is here directly from an online connected bioreactor. The Fc- fragments are captured in the first capturing step (step 3.2) allowing F(ab')2-fragments successfully fragmented to continue to the remaining capturing step (3.1). The purified mAb-fragments of analytical quality are finally online -processed and analysed using a mass spectrometer,

Fig. 4 shows data from a comprehensive study performed using the mAbLAB workflow with focus on the repeatability of analysis of F_c-glycan profile of the therapeutically important mAb Trastuzumab. Mass spectrometric data from 18 consecutively processed samples are compiled,

Fig. 5 visualizes the results from a mAbLAB study that focused on the Fd-glycan profile of the therapeutically important mAb Cetuximab over a 48 h mammalian cell cultivation. High reproducibility throughout the course of the study can be seen from the deconvo luted mass spectrometric data presented,

Fig 6 shows a schematic overview of a system of the invention, with a fragmenting column, a first capturing column and a second capturing column arranged in a serial arrangement,

Fig 7 shows a schematic overview of a system of the invention, with a fragmenting column, a first capturing column and a second capturing column arranged in a serial arrangement, directly coupled to an antibody production reactor and a MS- instrument. In this configuration, the system further comprises a pre-column, a valve and a pump to enable controlled movement of solutions in the system, sealed bags containing solutions used during operations, a reduction unit, and a desalting column to facilitate MS-analysis quality sample of antibody fragments from the antibodies of the antibody production reactor for direct analysis in the MS-instrument, and

Fig. 8 visualizes a photograph of the complete mAbLAB system including an autosampler.

Description of embodiments

The following description focuses on an embodiment of the present invention applicable to a method for carrying out the mAb-reaction to obtain a sample with analytic quality in a sufficiently short time to enable online quality control.

The pharmaceutical industry faces a paradigm shift with more emphasis on personalized treatments, resulting in an increasing number of proteins as therapeutic candidates, including especially monoclonal antibodies (mAbs) entering various stages of development. For antibody manufacturing process development, maintaining desired quality attributes while reducing time to market, maintaining cost effectiveness, and providing manufacturing flexibility are key issues in today's competitive market, where an array of companies are working on therapies for similar targets and clinical indications. Current antibody therapies often require large doses over a long period of time, and hence the manufacturing capacity becomes an issue, where large quantities are required with cost and time efficiency to meet clinical requirements.

The industrial manufacturing of a monoclonal antibodies is an extremely complex and sensitive process. During the production, it is crucial to achieve sufficient knowledge regarding the design space and its impact on the drug substance

specifications. Analytical tools that can facilitate the procedure are highly sought in order to save valuable downstream resources.

Antibody manufacturing process parameters can be measured either on-line (or at- line by direct connection to calibrated analyzers) or off-line via process intervention by laboratory personnel. Common measurements include pH, temperature, cell counting and viability measurements using a hemocytometer or automated cell counters, packed cell volume, osmolality and certain metabolite concentrations. Accurate gas and liquid flow measurements are typically conducted using mass flow meters and magnetic flow meters.

The US Food and Drug Adminstration, (FDA) defines process validation as “establishing documented evidence that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes”. This reflects that the product quality can be substantially affected by the manufacturing process. To meet this regulatory requirement, the process needs to be fully characterized and validated.

The mAbLAB system represents a standalone analytical platform developed for the direct connection to a bioreactor with subsequent detection, such as high-resolution mass spectrometric detection, for real time in-production monitoring of monoclonal antibody quality. In the mAbLAB system, the sample is treated in a sequence of steps according to the method presented below. All this without any interference or introduced bias from laboratory personnel.

Upon sampling, cells are removed, the antibodies to be analyzed may be separated from the rest of the sample by selective adsorption to a selective adsorbent. At this step, it is also possible to remove any impurities before the antibodies are released and transported automatically to the cleavage site, which constitutes a small column of immobilized selective proteases, in most cases the IdeS enzyme. Via the selective enzymatic cleavage each antibody is split into one F(ab')2-unit and two F_c-fragments. As the sample is transported further in the flow-system, the sample passes over two columns both packed with different selective adsorbents, which captures either the F(ab')2 and/or F_c-fragments. Fragments are released from this column via sequential elution using a buffer with low pH (< 3.0). Depending on the priorities of the analysis, either the F(ab')2 or F_c-fragments can be captured in a subsequent step where they are polished by washing buffer before being released.

The fragment of interest may then be eluted and transferred to an autosampler for further treatment using various detection methods or fed through a desalting step before it is ready to be injected or directly infused to a mass spectrometer.

The antibodies may be from different antibody variants, such as chimeric antibodies antibodies fused with certain affinity tags or pharmaceuticals, antibody-drug conjugates (ADC) or antibody-toxin conjugates. This could also include antibodies e.g. lacking the light chains and just comprised of 2 heavy chains. Starting from such antibodies, the system will provide fragments of mAb variants.

In one embodiment, a method for providing fragments of mAb and mAb variants for analysis, the method comprising the steps of, in a serial order; (1) obtaining a sample comprising an antibody, (2) fragmenting the antibody in said sample to obtain Fc- fragments and F(ab')2-fragments, (3) capturing the antibody fragments in two steps: (3.1) capturing the F(ab')2-fragments, (3.2) capturing the Fc-fragments, the method further comprising a washing and elution step (4) subsequent to step 3, wherein the captured antibody fragment of choice for analysis is washed and eluted in the washing and elution step (4), thereby providing an analytical quality sample of said antibody fragment.

In one embodiment, the antibodies are retrieved as a sample from an antibody production reactor or other vessel. In one embodiment, steps (2) and (3) are arranged in serial in a closed system.

Cell culture process development often starts with cell line generation and selection, followed by process and media optimization in small scale systems, such as multi-well plates or shaker flasks. Once conditions are defined, the process is often transferred to a pilot scale to test scalability and produce material for preclinical toxicology studies, and then larger scale manufacturing for production of clinical material under current good manufacturing practices (cGMP) regulations. This means that there is both a requirement for small scale manufacturing and large-scale manufacturing processes, with a high degree of quality control. Due to the highly optimized method of the invention, sample volume can be very low, such as 1 to 200 pL, 5 to 100 pL, such as 10 to 50 pL, which can be used both for large and small/pilot scale manufacturing. Usually, the sample volume results in purified mAb-fragment concentrations in nano- to micromolar range, which is more than enough for sensitive detection methods such as mass spectrometry.

In one embodiment, the volume of the the sample comprising an antibody is between 1 to 200 pL, 5 to 100 pL, such as 10 to 50 pL.

However, if a culture or sample has a very low antibody concentration, the method may comprise an enrichment step. This is most easily facilitated by using a pre- column containing a matrix with immobilized antibody-binding entities such as protein A, protein G or protein L for enriching the mAh or mAb-variants before the

fragmentation of said sample (step (2) of the method of the invention). As such, a sample may have a large sample size, up to several milliliters, such as up to 10 ml. After the mAh or mAb-variants have bound to the pre-column, they can be washed and then eluted with a small volume, such as 1 to 200 pL, 5 to 100 pL, such as 10 to 50 pL, and fragmented in step (2) of the method.

In one embodiment, the method further comprises a mAh or mAb-variant enrichment step prior to step (1) or step (2).

By using such an enrichment step, samples with femtomolar concentration of the mAh or mAb-variants may still be analyzed using the method of the invention.

The complete analysis sequence is usually carried out within 20-30 minutes. The rapid sample processing by the invention also enables parallel online control of several different cultures, such as several bioreactors. This is highly desirable, since in large scale manufacturing plants, cell culture bioreactors with sizes up to several thousand litres may be utilized, making it very costly for situations where a cultivation does not produce the desired target molecules.

In one embodiment, the fragments of mAh and mAh variants for analysis are provided in 1 hour or less, such as 50 minutes or less, such as 45 minutes or less, such as 30 minutes or less.

The fragmentation reaction and purification take place within an extremely short time span, also results are highly accurate and reproducible, as can be seen in Figs. 4 and 5, where mass spectrometric data from 18 consecutively processed samples are compiled (Fig. 4) and from the deconvoluted mass spectrometric data of a 48 h mammalian cell cultivation (Fig. 5). Figures 4 and 5 clearly illustrate the superiority of the present invention over solutions known in the art. The short time for sample analysis is highly important, since if a deviation in e.g. the glycosylation pattern, or a truncated peptide sequence is starting to appear, then one needs to act rapidly to stop the process and harvest the product. Alternatively, changes might change a large proportion of the mAbs, and then the whole batch is rendered worthless and needs to be destroyed. The time saved by avoiding termination, or by terminating a failed batch at an early stage, such as described above, can be highly valuable.

It was found that the order in which the fragments are captured may be varied to enable ultimate purity of the target fragment. If the target fragment for analysis is a F(ab')2-fragment (or Fd-fragment and/or an Lc-fragment resulting therefrom), the F_c- fragments should preferably be captured first (step 3.2). Thereby, fragments still containing an Lc-fragment will bind, allowing F(ab')2-fragments that are successfully digested to completion to continue to the remaining capturing step (3.1). If the F(ab')2- fragments captured in step 3.1 are eluted first, a pure subunit mAh fraction is obtained.

In one embodiment, step 3.2 precedes step 3.1.

Similarly, if the target fragment for analysis is a F_c-fragment, the F(ab')2- fragment should preferably be captured first (step 3.1). Thereby, fragments still containing a F(ab')2-fragment will bind, allowing F_c-fragments successfully fragmented to continue to the remaining capturing step (3.2). If the F_c-fragments captured in step 3.2 are eluted first, a pure subunit mAb-fraction is obtained.

In one embodiment, step 3.1 precedes step 3.2.

This innovative“guard column” concept is depicted in Figs. 2 and 3. It makes sure that only the pure fraction of a mAh subunit is recovered. Hence, in a monitoring and control situation of a mAh production process, even with a slightly deteriorating IdeS enzymatic activity (more and more semi-cleaved mAh attained over time), it will still result in the recovery of a pure subunit mAh fraction for analysis.

The bound fragments of choice for analysis are washed and eluted in step (4) of the invention. The washing steps make sure the sample is of analytical purity at an appropriate concentration for the analysis method of choice. The analytical quality samples may be collected in a container or autosampler, or directly sent to a suitable fragment analyser, such as a HPLC system or a LC-MS system.

However, due to the small volume and request of high purity of the sample, the sample may be sent directly to a mass spectrometry detector. In order not to destroy the performance of the sensitive MS-instrument, one must either operate with salt-free samples, or with samples containing volatile salts. As such, in the mAbLAB system, the sample material may be treated in an online desalting column by initially trapping the sample components while the salt present is removed. Elution with volatile organic solvents makes the sample ready for MS analysis. The sample may for example be washed with running buffer (RB) and eluted with methanol/water (MeOHTFO) solution containing 0.1 % formic acid.

In one embodiment, the method further comprises a desalting step (5) subsequent to the step of washing and eluting the captured fragment, the desalting step (5) comprising (5.1) capturing a F_c-fragment and/or a F(ab')2-fragment, (5.2) washing the captured fragment, (5.3) eluting the fragment, thereby providing a MS analysis quality sample of said antibody fragment.

In one embodiment, the method further comprises an analysis step, wherein the provided fragments of mAb and mAb variants are analyzed using mass spectrometry.

This enables an extremely detailed and rapid analysis of the mAb-fragments or mAb-variant fragments. The system may be a closed loop from the point of sampling to the fragment analysis. Having such a closed loop system all the way from sampling to detection offers unprecedented reproducibility of results, which makes it possible to track minor changes in product quality of the cell culture.

Depending on the fragment of choice for analysis, it may be preferable to reduce the F(ab')2-fragments into Fd- fragments and Fc-fragments. It is preferred, if such a reduction can be done in the closed loop environment, for rapid and reproducible results. Regular reducing agents known in the art, such as Dithiothreitol (DTT) or Tris- 2-carboxyethylphosphine hydrochloride (TCEP) may be used, but other suitable reducing agents may also be used.

In one embodiment, the method further comprises a reduction step, wherein F(ab')2-fragments are reduced to Fd-fragments and Fc-fragments.

The F(ab')2-fragments may be reduced after being captured in step (3), however, the fragments may also be reduced after the elution of step (4), in a dissolved state.

In one embodiment, the F(ab')2-fragments are reduced while captured in step (3).

When reducing the F(ab')2-fragments while bound, one has to take into account that the reducing agent should not interfere with the column matrix. However, it also presents the opportunity to rinse away the Fc-fragments during washing before elution of the desired Fd-fragments.

In one embodiment, the F(ab')2-fragments are reduced after the washing and elution step (4). When reducing the F(ab')2-fragments in solution, one does not have to take into account any negative effects on column material with regard to sensitivity or affinity. This enables the use of a wider selection of reducing agents and possibly higher purity of the resulting fragments. However, upon reduction, the solution will comprise both the Fd- and Lc-fragments.

Today, the standard for preparing monoclonal antibodies (mAbs) for quality control analysis involves the use of the IdeS enzyme followed by selective affinity purification. IdeS is a unique enzyme that specifically digests the hinge region of IgG, producing a F(ab')2-fragment and two F_c-fragments. The enzyme is highly specific and no other substrate besides IgG is known. Digestion of IgG with IdeS rapidly generates a homogenous pool of F(ab')2 and F_c-fragments and there is no over-digestion or further degradation of the fragments typically associated with other proteolytic enzymes.

However, there are also other enzymes that might be used, such as papain, a cysteine protease which cleaves immunoglobulin G molecules in the hinge region which results in the generation of two F(ab)-domains and a F_c/2-domain; pepsin, which digests immunoglobulins below the hinge region of the heavy chains, resulting in a F(ab')2- fragment which is composed of two F(ab)-fragments still linked together by the remaining portion of their respective heavy chains at the hinge region; a range of microbial enzymes including IgdE, a cysteine protease that digests human IgGl at one specific site above the hinge; SpeB, a recombinantly produced cysteine protease that under reduced conditions digests in the hinge region of antibodies; and KGP, a protease that digests human IgGl between K223 and T224 producing a homogenous pool of F_ab and Fc-fragments can also be used.

In one embodiment, the fragmentation in step (2) is performed by a fragmenting enzyme, the fragmenting enzyme being a proteolytic enzyme selected from the group containing among others IdeS, IgdE, SpeB, KGP, papain, and pepsin or any

combination thereof.

The fragmenting enzyme is preferably of microbial origin, such as IdeS, IgdE, SpeBor KGP, but may also be of non-microbial origin such as papain, or pepsin.

Glycosylation is one of the most common post-translational modifications of proteins in eucaryotes. Glycans may be attached to asparagine residues (N-linked glycosylation) or to serine or threonine residues (O-linked glycosylation) on the core protein. Enzymatic removal of oligosaccharides is gentle and can provide complete sugar removal with no protein degradation, resulting in a pure sample. Proteins such as EndoS, an IgG-specific endoglycosidase acting on complex type N-glycans at the F_c- glycosylation site of IgG; EndoS2, an IgG-specific endoglycosidase that hydrolyzes all glycoforms present at the F_c-glycosylation site; sialidase, an hydrolase that catalyzes the cleavage of glucosidic linkages between a sialic acid residue and a hexose or hexosamine residue at the non-reducing terminal of oligosaccharides in glycoproteins, glycolipids, and proteoglycans; and PNGaseF, an amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides, may be used to degycosylate mAbs, mAb-variants or fragments thereof.

In one embodiment, the method further comprises a deglycosylation step, wherein an enzyme, preferably of microbial origin, catalyzing deglycosylation is used, preferably the enzyme being EndoS, EndoS2, sialidase, or PNGaseF, either separately or in combination.

As such, the analysis of mAh or mAb-fragment may include, but not be limited to glycoprofiling of the mAh or mAb-fragments, such as monosaccharide analysis, sialic acid analysis, intact released glycan analysis, site specific glycan analysis; analysis of other post-translational modifications (PTM), such as phosphorylation, ubiquitination, acetylation and methylation; and protein primary structure analysis, which refers to analysis if the sequence of amino acids in the polypeptide chain, including post- translational modifications which cannot be read from the gene.

In one embodiment, the fragments of mAh and mAh variants provided for analysis is used for glycoprofiling, post-translational modification (PTM) analysis, protein primary structure analysis.

The system operates with a continuous low flow (e.g. 5 to 400 mE per minute such as 10 to 100 mE per minute, such as 6 to 30 mE per minute) to enable the sample processing to take place in a serial manner. However, bound fragments may be rinsed in washings steps using a higher flow rate, to make the washing step both faster and more efficient, wherein the spent wash solution is directly discarded and not fed into the closed loop system.

In one embodiment, the method uses a continuous low flow during steps 1 to 3, such as 5 to 400 mE per minute, such as 10 to 100 mE per minute, such as 6 to 30 mE per minute.

The mAbFAB systems represent a standalone analytical platform developed for the direct connection to a bioreactor with subsequent processing of the sample before analysis, such as high-resolution mass spectrometric detection, for real time in- production monitoring of monoclonal antibody quality. The mAbFAB system is based on the flow-injection concept, but in this case with an integrated range of different unit operations. Thus, in essence, all the process steps are carried out in a closed system from the sample inlet (which may be directly connected to one or several reaction vessels) to the sample outlet (which may be directly connected to a mass spectrometer or other detector). This makes it possible to keep sterility and avoid contamination of irrelevant material during long term operations.

The system comprises a sample inlet, where the sample is loaded or automatically sampled from a vessel or bioreactor comprising mAh and mAb-variants or fragments thereof. It further comprises at least of a first, a second, and a third column arranged in a serial arrangement with the serial inlet in the beginning of the first column and the serial outlet from the end of the last column. In the described method, the first column is a fragmenting column, the second column a first capturing column, and the third column a second capturing column. By loading the columns with different materials, such as mAb-fragment affinity matrices or immobilized selective proteases, reactions or capture and purification of mAh fragments of choice may take place. Each of the columns furthermore comprises individual inlets and outlets that can either be opened or closed, to enable individual washing and elution of the specific column, whereby analysis quality fragments may be selectively eluted from either the first, second, or third column.

As the sample is transported further through the flow-system, the sample passes a selective adsorbent column, which captures the F(ab')2 and/or F_c-fragments. Fragments are released from this column via sequential elution using a buffer with low pH.

Depending on the priorities of the analysis, either F(ab')2 or F_c can be captured in a subsequent step where it is polished by washing buffer before being released.

The target fragment of choice may then be eluted and transferred to an

autosampler for loading into analytical systems based on different detection methods, or fed through a desalting step before it is ready to be injected or directly fed to a mass spectrometer.

In one embodiment, a system 100, 200 for providing fragments of mAbs and mAh variants for analysis according to the herein disclosed method comprises at least one sample inlet 11, at least a fragmenting column 12, a first capturing column 13 and a second capturing column 14 arranged in a serial arrangement with a serial inlet 15 in the beginning of the fragmenting column 12 and a serial outlet 16 from the end of the second capturing column 14, and at least one processed sample outlet 17. Each of the columns 12, 13, 14 furthermore comprises an individual inlet l8a, 18b, l8c and outlet l9a. l9b, l9c, which can be (individually) either opened or closed, to enable individual washing of, and elution from, a specific column 12, 13, 14. In this way, analytical quality fragments bound to the columns 12, 13, 14 may be washed and eluted from any of said columns 12, 13, 14 by means of a washing circuit 20a, 20b, 20c and an elution circuit (2 la, 2 lb, 2lc), respectively. Eluted analytical quality fragments are further conveyed, optionally after further processing, to the processed sample outlet (17).

A schematic overview of the system 100 can be seen in figure 6. The fully integrated system, the small sample volume, and the integrated handling with each step under well controlled conditions (controlled by a strict computer program operating high-precision valves and pumps) eliminates influences of the human factor. Tests have shown that ELISA, which is a much simpler process, can be done in a flow system with an accuracy of ±0.5% while when carried out manually it has ±10-15% variation in the assays. Thus, the integrated flow system offers far better condition for handling sensitive analyses, as compared to the manual mode. This is illustrated in Figure 4, where mass spectrometric data from 18 consecutively processed samples are compiled, showing very consistent results.

Besides the positive facts concerning accuracy and reproducibility, the small sample volume, the speed of analysis and the markedly improved hygienic conditions and reduced risks of infection of the reactor from where the sampling is done contribute to a substantially improved analytical outcome as compared to what the conventional procedures generate. The integrated washing steps when the analyte is captured on a selective adsorbent contribute to a far better condition of the sample as compared to what is achieved by the conventional processes.

In one embodiment, the fragmenting column 12 comprises immobilized enzymes catalytically active in fragmenting mAbs, the first capturing column 13, or the second capturing column 14, comprises an affinity matrix for capturing of the F_c-fragments, the other capturing column 13, 14 comprises an affinity matrix for capturing of the (Fab’)2 fragments. Thus, the first capturing column 13 may comprise an affinity matrix for capturing of F_c-fragments, and the second capturing column 14 may comprise an affinity matrix for capturing of (Fab’)2 fragments. Alternately, the second capturing column 14 may comprise an affinity matrix for capturing of F_c-fragments, and the first capturing column 13 may comprise an affinity matrix for capturing of (Fab’)2 fragments.

Fragmentation of the mAb or mAb-variants is preferably enzymatic, using a fragmenting enzyme selected from the group containing, among others, IdeS, papain, pepsin, IgdE, SpeB, and/or KGP. Also, deglycosylating enzymes such as EndoS, EndoS2, sialidase, PNGaseF may be used to degycosylate the mAbs or mAb-variants or fragments thereof.

In one embodiment, the mAb-active catalytic enzyme is selected from the group containing IdeS, papain, pepsin, IgdE, SpeB and KGP, either separately or in combination. In one embodiment, the enzyme used to degycosylate mAbs or mAb- fragments are selected from the group containing EndoS, EndoS2, sialidase and

PNGaseF, either separately or in combination.

In one embodiment, the immobilized enzymes (i.e. fragmenting enzymes) are kept under controlled temperature conditions in order to optimize catalytic efficiency as well as storage stability while in resting state. By cycling the temperature between these states (depending on system usage), optimal enzymatic performance can be obtained.

In the example of Figure 2, the mAb-active catalytic enzyme is IdeS, and the affinity matrix for capturing of the F_c-fragments is F_c-specific matrix, the affinity matrix for capturing of the F(ab')2-fragments is CHl-specifie matrix.

Thus, in one embodiment, the mAb-active catalytic enzyme is IdeS for antibody fragmentation, the affinity matrix for capturing of the F_c-fragments is F_c -specific e.g. matrix, the affinity matrix for capturing of the F(ab')2-fragment is CHl-specific matrix. In one further embodiment, the affinity matrices are CaptureSelect matrices.

The sample inlet 11 may be directly connected to one or several reaction vessels or antibody production reactors 23. This not only makes it possible to maintain sterility and avoid contamination of irrelevant material during long-term operations, but also removes the variability of manual human sampling. Since the automatic sampling procedure also will result in a highly repeatable sample volume, consistent sampling conditions as well as consistency with regard to sampling intervals and sample treatment, a manufacturing process may be monitored with much less margin of error, making it possible to, at a much earlier stage when compared to manual sampling, adjust or halt production if the manufacturing process does not go as planned. The variability and low error rate and variability makes it possible to adjust to even very small changes, which would be out of reach when utilizing manual handling.

In one embodiment, the system is provided with means for direct connection 22 to at least one antibody production reactor 23, whereby a mammalian cell culture in an antibody production reactor 23 may be automatically sampled and produced antibodies analyzed; the system preferably comprising at least one antibody production reactor 23. Conventional analysis methods involve steam sterilization of the sampling port prior to the manual, or occasionally, automated recovery of the sample from the bioreactor into a sterile vial (which typically is directly frozen in wait for analysis). Furthermore, both the risk of bioreactor infection during sampling and the fact that it will take several hours before the data results from the manually recovered samples will become accessible, results in that a very low sampling frequency typically is achieved. In fact, there is no or little chance that these analytical results can be used to intervene and possibly correct a bioprocess that is on its way of going wrong. The only possible incentive in this scenario (using existing manual methods) would be to terminate the bioprocess as soon as possible and thereafter as soon as possible reinitiate another batch (and hope for the best this time) to save time. However, it would not allow for small direct-feedback adjustments facilitated by the invention, which in turn would allow for trying to save the batch in question.

Furthermore, the system of the invention may comprise a reversed-phase desalting column, which allows for direct infusion of the target sample to the mass spectrometer (the current standard for comprehensive protein analysis and characterization).

Consequently, when utilizing the system, it becomes possible to sequentially perform all required sample preparation operations, with the resulting very short sampling-to- analysis time.

To enable the operator to directly couple the system to a mass spectrometry detector, the sample material may be treated in the online desalting column. Since most MS-instruments must operate either with salt-free samples, or with samples containing volatile salts, the desalting column traps the sample components and salt being present is removed. Elution with organic solvents makes the sample ready for MS analysis.

In one embodiment, the system further comprises a desalting micro column 24 prior to the processed sample outlet 17, the desalting micro column comprising an inlet, a column volume packed with a hydrophobic material, and an outlet, to desalt the fragments of mAh and mAh variants in a desalting step. In one embodiment, the outlet of the desalting column is connected to a mass spectrometric analyzer/instrument or fraction collector. The desalting micro column 24 may be arranged between the serial outlet 16 and sample outlet 17.

It must be pointed out that the inline sample preparation and directly coupled fragment analysis truly opens up a novel possibility to not only provide a quality control for the monitored batch, but (due to the rapid results) also allow for adjustment and control of the manufacturing process, directly acting upon the on-line sample analysis results.

Depending on the fragment of choice for analysis, it may be preferable to reduce the F(ab')2-fragments into Fd-fragments and Lc-fragments (as can be seen in Figure 1). As such, the system may further comprise a reduction unit for addition of the reduction agent to the fragment of choice, such as a confluence point or other fluid mixer.

In one embodiment, the system further comprises a reduction unit 26 for addition of the reduction agent to the fragments of mAh and mAh variants, either bound on the first capturing column 13 or the second capturing column 14 or in solution, such as a confluence pump or fluid mixer. The reduction unit 26 may be arranged between the serial outlet 16 and desalting micro column 24.

The system may further comprise a pre-column for enriching the mAh or mAb- variants before the fragmentation of the antibodies in said sample. Such a pre-column may contain a matrix with different antibody-binding entities, e.g. immobilized protein A, or protein G, or protein L.

In one embodiment, the system further comprises an enrichment column 27 arranged before the fragmenting column 12, for enrichment of the antibody. The enrichment column 27 may be arranged between the serial inlet 11 and the fragmenting column 12.

The system may comprise further columns, such as, but not limited to, a fourth packed with either Lc-Kappa selective material or CH1 selective material, for capturing of individual Lc-fragments or Fd-fragments respectively.

In one embodiment, the system further comprises a third capturing column, wherein the third capturing column is packed with either Lc-Kappa selective material or CHI selective material, for Capturing of Lc-fragments or Fd-fragments respectively. In one further embodiment, the affinity matrices are CaptureSelect matrices.

To be able to facilitate the reaction in the volume and time-scale of the method, a suitable column size has been found to be 1 to 100 pL, such as 5 to 50 pL, such as 10 to 25 pL, such as 15 pL. By having a small column size, a very well-defined peak can be obtained during elution of the target fragment of choice. This ensures a high peak concentration of the fragment, which helps the detection of the target fragment.

In one embodiment, the column volume of the fragmenting column 12, the first capturing column 13, the second capturing column 14, and/or optionally the third capturing column is between 1 to 100 pL per column, such as 5 to 50 pL per column, such as 10 to 25 pL per column, such as 15 pL per column. To ensure optimum performance of the catalytic enzyme, for instance used for mAb or mAb-variant fragmentation, the system further comprises a heating/cooling element and a temperature controller for maintaining an optimal column temperature. The heating/cooling element may optionally heat/cool the solutions used during sample treatment. Preferably, the heating element is a Peltier element, to facilitate both heating and cooling capacity, however, any suitable heating/cooling element will do. If the enzyme(s) used in the assay requires any cycling of the temperature, the effect of the heating/cooling element and the mass to be heated/cooled will dictate the speed of the temperature cycling. In such a case, it will be preferable to use a heating/cooling element of low mass (for instance a Peltier element) instead of a water cooling system of substantial mass (such as a cooling system using a large volume of water as heating/cooling media). If the enzyme used is IdeS, a preferred reaction temperature is 35 to 39 degrees, preferably 37 °C .

In one embodiment, the system further comprises a heating/cooling element and a temperature controller to keep columns at a desired temperature. In one further embodiment, the heating/cooling element is a Peltier element.

To maximize the lifetime of the enzymes used in the fragmenting column, the temperature of the heating/cooling element may be lowered when the system is not in use. Preferably, the temperature of the heating/cooling element is cycled between a preferred reaction temperature (such as 25 to 45 degrees, such as 35 to 39 degrees, such as 37 °C (preferred for the enzyme IdeS) and an idle temperature where the enzyme activity is lowered (such as 2 - 10 °C, preferably 4 °C), to prolong the enzyme long term stability.

In one embodiment, the preferred reaction temperature of the heating/cooling element is 25 to 45 degrees, such as 35 to 39 degrees, preferably 37 °C. In one further embodiment, the preferred idle temperature (when the system is not running a reaction) of the heating/cooling element is 2 - 10 °C, preferably 4 °C. In one embodiment, the temperature of the heating/cooling element is cycled between a reaction temperature and an idle temperature.

In the mAbLAB-system, the columns may be integrated into a disposable reagent cassette. This assists in keeping the dead volume of the system to a minimum.

Furthermore, it makes it easier to attach the columns adjacent to the heater/cooler element. The cassette is a block made from a non-flexible plastic material compatible with the reagents and column matrices. Each column comprises a column inlet, a column volume and a column outlet. The inlet and/or outlet may further comprise a 10 micrometer filter unit.

In one embodiment, at least two columns, such as al least the fragmenting column, the first capturing column, and the second capturing column, are integrated into a disposable reagent cassette. In one embodiment, the columns of the disposable reagent cassette are equipped with filter units at both ends of each column, wherein the filter units are 2 to 20 pm filters, such as 10 pm filters.

As can be seen in Figure 8, the system utilizes multiport valves, pumps and tubing to enable and direct the movement of solutions from port to port via tubing and tubing connectors. The valves may be standard HPLC quality multiport valves, the tubing is preferably also standard HPLC quality tubing. By having tubing and valves which do not expand/contract to a big extent and thus maintaining an even pressure, optimum column performance is achieved. The pumps are preferably syringe pumps which may be used with positive and/or negative displacement, to enable control of the liquid flow. However, other suitable pumps may also be used, such as HPLC-standard piston pumps.

In one embodiment, the system further comprises at least one, preferably two, multiport valve(s) 28, preferably low dead volume multiport valves, and at least one, preferably two, pump(s) 29 to enable controlled movement of solutions in the system.

The multiport valves 28 may direct the flow from and to different columns or components of the system, through utilizing different flow switching patterns.

The system may also comprise one or several sample loops. A sample loop is a loop of tubing, where both ends of the tubing are connected to one (or two) valve(s), enabling the tubing volume to be used to hold a sample which will be directed to a column, component or outlet in the system. The loop may for instance be made of HPLC steel tubing, fused silica, or PEEK tubing. The sample loop volume is easily varied by changing the inner diameter and length of the sample loop tubing. It is common that a sample loop is similar to the sample volume, or several times larger than the sample volume, such as 2.5 times the sample volume.

In figure 7b, the system 200 is shown with one valve. Here, individual outlet l9a. l9b, l9c of each column 12, 13, 14 could connect to the valve to direct the flow. Also, the system could utilize two valves, where the second valve could manage the output flow from the each column 12, 13, 14 individual outlet l9a. 19b, l9c. In this way, a sample can be eluted from any column 12, 13, 14 and transferred to any component (such as the desalting column 24 or MS instrument 25). By having all required solutions (i.e. water, running buffer (RB), elution buffer (EB), cleaning solution (CS), optionally desalting solution (DS), reducing solution (RS) and volatile solvent (VS)) in hermetically sealed bags, it is possible to transfer fluid from the bags in a closed system (no need for air intake from the surroundings). In addition, any low-pressure build-up resulting from fluid extraction in the solution vessel is avoided.

In one embodiment, all solutions (except for the sample solution) are in sealed compression bags 30, to make it possible to run the system as a closed system, where the only active inlets/outlets are the sample inlet 11 and the processed sample outlet 17.

In one embodiment, the mAh and mAb-variant fragment analysis takes place within a closed system.

As such, the mAbLAB-system enables dedicated monitoring and control of biopharmaceutical production, mainly of therapeutic proteins. The described system is suitable to completely autonomously handle all involved steps during the bioprocess, from reactor sampling through sample preparation including inline sample analysis to the delivery of a sample preparation ready for mass spectrometric analysis.

Figure 7 shows a schematic overview of a system 200 with more components added. Here, the instrument is directly coupled to an antibody production reactor 23 and a MS-instrument 25. In this configuration, the system 200 further comprises a pre- column 27, a valve 28 and a pump 29 to enable controlled movement of solutions in the system, sealed bags 30 containing solutions used during operations, a reduction unit 26, and a desalting column 24 to facilitate MS-analysis of high quality samples of antibody fragments from the antibodies of the antibody production reactor 23 for direct analysis in the MS-instrument 25.

Production of biomolecules is a dynamic process. It should be kept in mind that today, large reactors are used for production of mAbs and that the demands on reproducibility between batches and within a long-lasting batch are very high. If a deviation in e.g. the glycosylation pattern or a truncated peptide sequence is starting to appear, then one needs to act rapidly to stop the process and harvest the product.

Alternatively, changes might have happened to a large proportion of the mAbs, and then the whole batch is worthless and needs to be destroyed. So, fast and sensitive assays are essential for keeping quality control of the production. With shorter lead times, higher demands are raised. The concept with the integrated system makes on-line process control possible. As far as we know, there is currently no system or method available for analysis of quality of produced monoclonal antibodies that covers the complete workflow, from reactor sampling to final advanced sample analysis in a timeframe suitable for online monitoring.

Although the present invention has been described above with reference to (a) specific embodiment(s), it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and, other embodiments than the specific above are equally possible within the scope of these appended claims.

In the claims, the term "comprises/comprising" does not exclude the presence of other elements or steps. Furthermore, although individually listed, a plurality of means, elements or method steps may be implemented by e.g. a single unit or processor.

Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. The terms "a", "an",“first”, “second” etc do not preclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way.

Materials and methods

The intrinsic variability of biopharmaceutical production induces the need for continuous monitoring of the quality attributes, such as protein glycosylation profiles. Such monitoring would certainly be important, since it would allow in-process corrections to occur when an undesired protein characteristic is detected.

Even though there is a need for real-time monitoring, the technological developments and implementations in this direction are still in the early stage [1], with considerable achievements in real-time glycosylation monitoring [2] Nevertheless, there is significant progress in high-throughput analysis aimed at reducing cost and manual labour whilst increasing the speed of analysis. However, the majority of these methods are focused on studying the enzymatically released N-glycans [3] The main directions in the development of high-throughput analysis is mostly focused on offline methods using either 96-well plate format approach or microfluidic technologies for sample preparation. With the increasing demand for continuous monitoring of biopharmaceutical production processes, an automated flow-based analytical platform was invented and developed for the in-production monitoring of mAh quality. The platform enables parallel with production, proteolytic fragmentation of the antibody and analysis of both the F_c- and F(ab')2 (i.e. Fd)-glycosylation profiles (Fig.l). Measuring the intact glycoproteins, instead of released glycans, maintains the information regarding onsite specific glycosylation [4]. The fully-automated mAbLAB system, based on the

CapSenze FlowSystem concept, coupled with subsequent high-resolution mass spectrometry (HR-MS) detection allows rapid profiling of the glycosylation

heterogeneity of a series of ten different therapeutic mAbs and F_c-fusion proteins.

The automated flow-based mAbLAB platform (Figs. 2 and 3) utilizes two syringe pumps and two 12+ multiport valves (Fig. 6). These pumps execute the liquid handling which enable the movement of solutions from port to port through the valves. The disposable reagent cassette, which holds five micro-columns (each with a volume 15 pL), are equipped with 10 pm cut off filter units at both ends, are packed with immobilized IdeS enzyme (lst column), CH1 -specific matrix (2nd column) and F_c- specific matrix (3nd column), all matrices which are based on regular beaded Sepharose material. The tubing (250 pm ID) which is used to connect the pumps, valves and to the disposable reagent-cassette is made of polypropylene. During operation, the reagent cassette is kept at 37 °C using two integrated Peltier elements and one temperature- controller. The automated mAbLAB assay sequence, used e.g. to perform a scheduled sampling scheme from a bioreactor, is controlled by a tailor-made software program developed by CapSenze Biosystems AB (Lund, Sweden).

When initiating a typical mAbLAB workflow, a first step is always to rinse and wash columns, valve ports and all connected tubing with a certain amount of running buffer (RB, 10 mM PBS, pH 7.4) at a flow rate of 360 pL/min to ensure the removal of possible sample cross contamination. In this step, both the elution and sample ports are primed (200 pL at 600 pL/min) with elution buffer (EB, 0.1 M glycine, pH 3.0) and sample respectively followed by a thorough rinsing of the injection loop with RB (1 mL at 6 mL/min).

The assay sample volume (typical volume 10-50 pL) is pulled to the injection loop from the bioreactor or autosampler at a flow rate of 60 pL/min and is introduced to the disposable reagent-cassette at a flow rate of 18 pL/min. The sample solution initially passes through the first micro column, containing immobilized IdeS, and is fragmented at 37 °C with a residence time in the column of 1-3 min. The true F_c-fragments generated in the first micro column are passing unhindered through the second micro column (containing CH1 -specific affinity matrix) and are captured on the F_c-specific affinity matrix in the third micro column, while F(ab')2-fragments, semi-cleaved and intact mAbs are captured in the second micro column (Fig. 2). To wash away loosely bound molecules including non-specifically adsorbed material, a washing step with 1 mL RB at a flow rate of 600 pL/min is thereafter invoked. The captured F(ab')2- fragments, semi-cleaved and intact mAbs on the second micro column are then subsequently eluted with 100 pL EB at a flow rate of 60 pL/min and are passed to waste. The micro columns were thereafter rinsed with 1 mL RB (600 pL/min) in order to remove any remaining traces of F(ab')2-containing fragments.

The captured F_c-fragments on the third micro column are thereafter eluted with 100 pL EB at a flow rate of 60pL/min and passed to the equilibrated desalting column (100 pL Cl 8 material packed in a stainless-steel micro column equilibrated with 500 pL RB at 180 pL/min) which is placed inline with the mass spectrometer. The F_c-fragments now bound inside the desalting column are then washed with lmL RB at a flowrate of 300 pL/min. Finally, the analytically purified F_c-fragments are eluted to the high- resolution mass spectrometer using 200 pL of a MeOH/H20 solution containing 0.1 % Triflouroacetic acid at a flowrate of 50 pL/min.

The above example shows a workflow where the target is set to the F_c-fraction and the glycol-profiling of this fragment. However, should the target instead be e.g. the Fd-subunit of the mAh (i.e. the heavy-chain part of the F(ab')2 molecule after IdeS digestion, see Fig. 3), which in some mAh variants also are glycosylated, a slight variation of the previously described workflow can be exerted. Here, the material in the second and third micro columns are exchanged (second micro column is then filled with Fc-specific affinity matrix and the third column with CHl-specifie matrix). In addition, the mAbLAB workflow is modified to also involve an inline reduction step, where the eluted F(ab')2-target from the third column is joined via a confluence point with 75 pL of a 100 mM TCEP solution at a flowrate of 60 pL/min prior to the desalting step and mass spectrometric analysis. In all other steps, the two described workflows are the same.

In this latter workflow however, all versions of the mAh (F_c-fragments, semi- cleaved and intact mAh) that contains an F_c-part, will be captured on the second micro column. Hence, only true F(ab')2-fragments will pass unhindered to the third micro column containing CHl-specific affinity matrix. Results

Glycoprofiling of Fp-subunits

When applying the mAh LAB workflow for the targeting of F_c-fragments and the glycoprofiling of these subunits, the therapeutic monoclonal antibody Trastuzumab (for treatment of Her2-sensitive breast tumour cancer) was used as a model substance. A comprehensive study was performed with focus on the repeatability of the Trastuzumab F_c-glycan profile. The main objective was to prove that when processing a multitude of samples over time from the same source (bioreactor) using the mAbLAB system, a clear beneficial effect on the quality of the analytical data could be achieved.

During this study, 18 consecutively processed samples were compiled over three days and successively analysed using high-resolution mass spectrometric analysis. From Fig. 4, it becomes clear that the mAbLAB system can process numerous consecutive samples with maintained small variations and error margins. From the mass

spectrometric raw data, it also becomes evident that the exceedingly pure fraction of F_c- subunits generated by the inventive mAbLAB workflow, where only true F_c-fragments are allowed to pass the second“guard column”, clearly enhances the quality of the final glycoprofiling data generated.

Similar to the application where F_c-subunits are assessed on basis of their glycan content, the profiling of mAbs containing Fd-glycosylation was performed using the mAbLAB system. Cetuximab (trade name Erbitux) is such a mAh pharmaceutical, which target cancer tumours in the colon system of humans, and was used as model target when visualizing the mAbLAB workflow for Fd-glycoprofiling with an automated inline reduction step

In this case, the micro column architecture within the reagent cassette was the following: 1. Immobilized IdeS, 2. F_c-specific affinity matrix (guard column) and 3.

CHl-specifie affinity matrix. Hence, when sampling (50 pL) from an active mammalian cell culture containing 500 pg per mL Cetuximab, only true F(ab')2-fragments will pass unhindered to the third micro column, while all versions of the mAh (F_c-fragments, semi-cleaved and intact mAh) that contains an F_c-part, will be captured on the second micro column. Reduction of the eluted F(ab')2-fragments with TCEP introduces both Lc- and Fd-subunits to the desalting column prior to HR-MH analysis. In Fig. 5, is shown the results where the fully automated bioreactor-mAbLAB-MS workflow was executed over a 48h period with a variable sampling interval of 1 to 3 hours. High reproducibility throughout the course of the study can be seen from the deconvoluted mass spectrometric data presented. References

1. P. O’Mara, A. Farrell, J. Bones, K. Twomey. Staying alive! Sensors used for monitoring cell health in bioreactors. Talanta, 2018, 176, 130-139.

2. Sheun Oshinbolu, Louisa J. Wilson, Will Lewis, Rachana Shah, Daniel G. Bracewell. Measurement of impurities to support process development and manufacture of biopharmaceuticals. Trends in Analytical Chemistry, 2018, 101, 120-128.

3. Florian Cymer, Hermann Beck, Adelheid Rohde, Dietmar Reuse. Therapeutic monoclonal antibody N-glycosylation - Structure, function and therapeutic potential. Biologicals, 2018, 52, 1-11. 4. Eric Largy, Fabrice Cantais, Gery Van Vyncht, Alain Beck, Amaud Delobel.

Orthogonal liquid chromatography-mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level. Journal of Chromatography A. 2017, 1498, 128-146.

Claims

1. A method for providing fragments of mAbs and mAb variants for analysis, the method comprising the steps of, in a serial order;

(1) obtaining a sample comprising an antibody,

(2) fragmenting the antibody in said sample to obtain F_c-fragments and F(ab')2- fragments, and

(3) capturing the antibody fragments in two steps:

(3.1) capturing the F(ab')2-fragments,

(3.2) capturing the F_c-fragments,

the method further comprising a washing and elution step (4) subsequent to step (3), wherein the captured antibody fragment of choice for analysis is washed and eluted in the washing and elution step (4),

thereby providing an analytical quality sample of said antibody fragment.

2. Method according to claim 1, wherein step (3.2) precedes step (3.1).

3. Method according to claim 1, wherein step (3.1) precedes step (3.2).

4. Method according to any of claims 1 to 3, wherein the method further comprises a desalting step (5) subsequent to the step of washing and eluting the captured fragment, the desalting step (5) comprising

(5.1) capturing a F_c-fragment and/or a F(ab')2-fragment,

(5.2) washing the captured fragment, and

(5.3) eluting the fragment,

thereby providing an MS analysis quality sample of said antibody fragment.

5. Method according to any of claims 1 or 4, wherein the method further comprises a reduction step, wherein F(ab')2-fragments are reduced to Fd- fragments and Lc-fragments.

6. Method according to any one of claims 1 to 5, wherein the fragments of mAb and mAb variants for analysis are provided in 1 hour or less, such as 50 minutes or less, such as 45 minutes or less, such as 30 minutes or less.

7. Method according to any one of claims 4 to 6, wherein the method further comprises an analysis step, wherein the provided fragments of mAh and mAh variants is analyzed using mass spectrometry.

8. Method according to any one of claims 1 to 7, wherein the method steps take place within a closed system.

9. The method according to any one of claims 1 to 8, wherein the

fragmentation in step (2) is performed by a fragmenting enzyme, the fragmenting enzyme being a proteolytic enzyme selected from the group consisting of, IdeS, IgdE, SpeB, KGP, papain, and pepsin or any combination thereof.

10. A method according to any one of claims 1 to 9, wherein the method further comprises a mAh or mAh variant enrichment step prior to step (1) or step (2).

11. A method according to any one of claims 1 to 10, wherein the fragments of mAh and mAh variants for provided for analysis is used for glycoprofiling, post- translational modification (PTM) analysis, protein primary structure analysis.

12. The method according to any of claims 1 to 11, wherein the method uses a continuous low flow during steps 1 to 3, such as 5 to 400 pL per minute, such as 10 to 100 pL per minute, such as 6 to 30 pL per minute.

13. A system (100, 200) for providing fragments of mAbs and mAh variants for analysis according to any one of claims 1 to 12, comprising at least one sample inlet (11), at least a fragmenting column (12), a first capturing column (13) and a second capturing column (14) arranged in a serial arrangement with a serial inlet (15) in the beginning of the fragmenting column (12) and a serial outlet (16) from the end of the second capturing column (14), and at least one processed sample outlet (17),

wherein each of the columns (12, 13, 14) furthermore comprises an individual inlet (l8a, 18b, l8c) and outlet (l9a. l9b, l9c), which can be either opened or closed, to enable individual washing of, and elution from, a specific column (12, 13, 14),

whereby analytical quality fragments bound to the columns (12, 13, 14) may be washed and eluted from any of said columns (12, 13, 14) by means of a washing circuit (20a, 20b, 20c) and an elution circuit (2 la, 2 lb, 2lc), respectively, and eluted analytical quality fragments are further conveyed, optionally after further processing, to the processed sample outlet (17).

14. A system according to claim 13, wherein the fragmenting column (12) comprises immobilized mAb-active catalytic enzyme, the first capturing column (13) or second capturing column (14) comprises an affinity matrix for capturing of the F_c- fragments, the remaining capturing column comprises an affinity matrix for capturing of the F(ab’)2 fragments.

15. A system according to claim 14, wherein the mAb-active catalytic enzyme is IdeS for antibody fragmentation, the affinity matrix for capturing of the F_c-fragments is F_c -specific matrix, the affinity matrix for capturing of the F(ab')2-fragments is CH1- specific matrix.

16. A system according to any one of claims 13 to 15, wherein the system is provided with means for direct connection (22) to at least one antibody production reactor (23), whereby a mammalian cell culture in an antibody production reactor may be automatically sampled and produced antibody analyzed; the system preferably comprising at least one antibody production reactor.

17. The system according to any one of claims 13 to 16, wherein the system further comprises a desalting micro column (24) prior to the processed sample outlet, the desalting micro column (24) comprising an inlet, a column volume packed with a hydrophobic material, and an outlet, to desalt the fragments of mAh and mAh variants in a desalting step.

18. A system according to claim 17, wherein the outlet of the desalting column (24) is connected to a mass spectrometric analyzer/instrument (25) or fraction collector.

19. A system according to any one of claims 13 to 18, wherein the system further comprises a reduction unit (26) for addition of the reduction agent to the fragments of mAbs and mAh variants, either bound on the first capturing column (13) or the second capturing column (14) or in solution, such as a confluence pump or fluid mixer.

20. A system according to any one of claims 13 to 19, further comprising an enrichment column (27) arranged before the fragmenting column (12), for enrichment of the antibody.

21. A system according to any one of claims 13 to 20, further optionally comprising a third capturing column, wherein the column volume of the fragmenting column (12), the first capturing column (13), the second capturing column (14), and/or the third capturing column is between 1 to 100 pL per column, such as 5 to 50 pL per column, such as 10 to 25 pL per column, such as 15 pL per column.

22. A system according to any one of claims 13 to 21, wherein the system further comprises at least one, preferably two, multiport valve(s) (28), and at least one, preferably two, pump(s) (29) to enable controlled movement of solutions in the system.