Improved method for isolating and characterizing proteins

Abstract

The present disclosure provides novel methods for assessing product related impurities, such as protein aggregation, in a biological sample comprising a size exclusion chromatography (SEC) and an automated capillary electrophoresis (CE) western. Specifically, the present disclosure provides method for assessing protein aggregation in an unpurified biological sample comprising an in vivo or in vitro produced protein and / or aggregates thereof.

Description

IMPROVED METHOD FOR ISOLATING AND CHARACTERIZING PROTEINSCROSS-REFERENCE TO RELATED APPLICATION(0001 ] The present application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 621,829, filed January 17, 2024, the contents of which are hereby incorporated by reference in their entirety.FIELD OF THE DISCLOSURE10002 J The present disclosure relates to the field of therapeutic protein manufacturing and qualitative and quantitative assessment of therapeutic protein compositions. Specifically, the disclosure relates to the quantitative assessment of therapeutic protein impurities, e.g., aggregates, in compositions comprising in vivo or in vitro produced therapeutic proteins.BACKGROUND

[0003] The use therapeutic proteins, such as antibodies and their derivatives, is an established approach for treating many diseases and conditions. However, the development and production of these therapeutic proteins remain challenging because of manufacturing issues. One such manufacturing issue is the presence of product related impurities in therapeutic protein compositions. A common product related impurity is aggregates of the therapeutic protein. Such aggregates typically need to be removed from the biopharmaceutical preparations before the preparations can be used. This is because therapeutic protein aggregates can trigger immunogenic responses in patients, reduce the biologically activity of the therapeutic protein, decrease the therapeutic product yields, and / or increase commercial manufacturing costs. For example, therapeutic protein aggregates can trigger the production of antibodies against the therapeutic protein.

[0004] It is well established that therapeutic proteins, such as antibodies, may form protein aggregates and particulates. Therapeutic protein aggregates can be formed at different stages of the manufacturing process, such as for example, during protein synthesis in the cell, after protein secretion in the cell supernatant, or animal blood, during downstream protein purification, during protein formulation, and / or during the protein formulation storage.

[0005] However, therapeutic protein aggregation also occurs intracellularly. Protein aggregation during protein synthesis can be caused by mis-pairing of engineered disulfidebonds, protein misfolding, and / or other protein biophysical properties. Therapeutic proteins are also sensitive to conditions, such as pH, ionic strength, thermal stress, reactive oxygen species (ROS), temperature, shear and interfacial stresses, all of which can lead to aggregation.|0006| While several techniques for the removal of protein aggregates in a biological or biopharmaceutical sample exist, such removal can be challenging because of the physical, chemical, and / or biophysical similarities between the therapeutic protein, which is often a monomer, and the therapeutic protein aggregates, which are often multimer of the therapeutic protein. The therapeutic protein is often a monomer, while the therapeutic protein aggregates, can often be a multimer of the therapeutic protein. Furthermore, techniques known in the art can further induce protein aggregation during purification. In addition, many of these techniques are not sensitive enough to identify therapeutic protein and their aggregates that are found at very low concentration in an unpurified (e.g., crude) biological sample or an unpurified cell culture supernatant.100071 Accordingly, there is a need for an improved, specific, and sensitive method for assessing product related impurities, particularly a, a method for assessing the presence and / or quantity of protein aggregates in an unpurified biological sample comprising an in vivo produced therapeutic protein or an unpurified cell culture supernatant comprising an in vitro produced therapeutic protein. The present disclosure addresses this need.SUMMARY OF THE INVENTION

[0008] One aspect of the present disclosure provides a method for assessing product related impurities, e.g., protein aggregation, in a biological sample comprising an in vivo produced protein and / or related impurities, the method comprising: (a) fractionating the biological sample from a subject expressing the in vivo produced protein and / or impurities thereof using a size exclusion chromatography (SEC); (b) collecting eluate fractions of the in vivo produced protein; (c) electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western; and (d) detecting the in vivo produced protein and / or related impurities in the SEC eluate fractions using fluorescent detection or chemiluminescent detection. In some embodiments, the product relatedimpurities comprise protein aggregates. In some embodiments, the eluate fractions comprise a predicted size range.

[0009] Another aspect of the present disclosure provides a method for assessing protein aggregation, in a biological sample comprising an in vivo produced protein and / or aggregates thereof, the method comprising: (a) fractionating the biological sample from a subject expressing the in vivo produced protein and / or aggregates thereof using a size exclusion chromatography (SEC); (b) collecting eluate fractions of the in vivo produced protein; (c) electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western; and (d) detecting the in vivo produced protein and / or aggregates thereof in the SEC eluate fractions using fluorescent detection or chemiluminescent detection. In some embodiments, the eluate fractions comprise a predicted size range.

[0010] In some embodiments, detecting the in vivo produced protein and / or aggregates thereof comprises: (a) contacting the SEC eluate fractions with an antibody that binds to the in vivo produced protein; (b) incubating the SEC eluate fractions with the antibody for about 10 minutes to about 120 minutes; and (c) detecting the binding of the antibody to the SEC eluate fractions using fluorescent detection or chemiluminescent detection.

[0011] In some embodiments, prior to step (a)-(c), the SEC eluate fractions are electrophoretically separated by weight and immobilized on a solid support. In some embodiments, the solid support is a capillary wall of a microfluidic device. In some embodiments, the automated capillary electrophoresis (CE) western is a closed-loop automated capillary-based immunoassay system.

[0012] In some embodiments, the SEC eluate fractions and the antibody are incubated for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.

[0013] In some embodiments of the method described herein, the detecting the binding of the antibody to the in vivo produced protein comprises a dual antibody CE western or a singleantibody CE western. In that embodiment, the dual antibody CE western comprises: (a) a primary antibody that targets the in vivo produced protein or aggregates thereof; (b) a secondary antibody that targets the primary antibody, wherein the secondary antibody is operably linked to a chemiluminescent or a fluorescent molecule; and (c) a chemiluminescent or a fluorescent molecule substrate.

[0014] In some embodiments of the method described herein, the single antibody western comprises: (a) a secondary antibody that targets the in vivo produced protein or aggregate thereof, wherein the secondary antibody is operably linked to a chemiluminescent or a fluorescent molecule; and (b) a chemiluminescent or a fluorescent molecule substrate. In that embodiment, the in vivo produced protein or aggregates thereof comprises a constant domain or a Fc domain of an antibody. In one embodiment, the secondary antibody selectively binds to the constant domain or the Fc domain of the in vivo produced protein.

[0015] In some embodiments of the method described herein, the detecting the in vivo produced protein and / or aggregates thereof in the SEC fractions comprises chemiluminescent detection. In some embodiments, the method detects a nanogram or a picogram (pg) of the in vivo produced protein and aggregates thereof in the biological sample.

[0016] In some embodiments, the method detects at least about 0.0001 ng- 1. Ong, at least about O.OOOlng-O.lng, at least about O.lpg-l.Opg, at least about 0.5pg-2.5, at least about O. lpg-lOpg, at least about 5pg-50pg, at least about lOpg-lOOpg, or more than lOOpg of the in vivo produced protein or aggregates thereof in the biological sample. In some embodiments, the method detects at least about O.lpg, at least about 0.2pg, at least about 0.3pg, at least about 0.4pg, at least about 0.5 pg, at least about 0.6pg, at least about 0.7pg, at least about 0.8pg, at least about 0.9pg, at least about l.Opg, at least about l.lpg, at least about 1.5pg, at least about 5.0pg, at least about lOpg, at least about 20pg, at least about 30pg, at least about 40pg, at least about 50pg, at least about 60pg, at least about 70pg, at least about 80pg, at least about 90pg, or at least about lOOpg of the in vivo produced protein or aggregates thereof in the biological sample.

[0017] In some embodiments of the method described herein, the SEC comprises a resin selected from the group consisting of Superdex®, Bio-Sep™, TSKgel™, Sephacryl™, Superose™, and Sephadex™.

[0018] In some embodiments, the SEC has a separation range of about 0.1 kilo Dalton (KDa) to lOOKDa, about 1 KDa to about 150 KDa, about 5 KDa to about 100 KDa, about 5 KDa to about 700 KDa, about 5 KDa to about 1000 KDa, about 5 KDa to about 5,000 KDa, or about 10 KDa to about 10,000 KDa. In some embodiments, the SEC separation range is under a native or a denatured condition.

[0019] In some embodiments of the method described herein, the biological sample is fractionated at a flow rate of about 0.01 ml / min, about 0.05 ml / min, about 0.075 ml / min, about 0.1 ml / min, about 0.15ml / min, about 0.2ml / min, about 0.3ml / min, about 0.4 ml / min, about 0.5 ml / min, about 0.6 ml / min, about 0.7 ml / min, about 0.8 ml / min, about 0.9 ml / min, about 1.0 ml / min, about 1.5 ml / min, or about 2.0ml / min or more. In some embodiments, the biological sample is fractionated at a flow rate of about 0.5 ml / min.

[0020] In some embodiments, an aggregate of the in vivo expressed protein: (a) comprises a dimer, a trimer, a tetramer, a multimer, or a higher order oligomer of the in vivo expressed protein; and / or (b) is an homo-aggregate or a hetero-aggr egate.

[0021] In some embodiments, prior to the SEC fractionation, the biological sample is: (a) not purified; (b) not pre-treated with a process selected from the group consisting of affinity chromatography, low pH viral inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration, ultrafiltration, and tangential flow filtration; or (c) not purified using a Protein A- or a Protein G-based affinity chromatography.

[0022] In some embodiments of the method described herein, the in vivo produced protein is generated by administering a polynucleotide encoding the in vivo produced protein to the subject.

[0023] In that embodiment, (a) the polynucleotide is a DNA, or an RNA; (b) the polynucleotide is an mRNA; or (c) the polynucleotide further comprises a modified nucleotide, a cap structure, a poly A tail, a 5' untranslated region, and / or a 3' untranslated region.

[0024] In some embodiments, the subject is administered about Ipg to about 50 pg, about 10 pg to about 50 pg, about 5 pg to about 25 pg, about 1 pg to about 15 pg, about 1 pg to about 10 pg, about 5 pg to about 15 pg, about 6 pg to about 15 pg, or about 5 pg to about 20 pg of a mRNA encoding the inv vivo expressed protein.

[0025] In some embodiments of the method described herein, the biological sample comprises: (a) at least about 0.1 pg / ml to about 50 pg / ml, at least about 0.1 pg / ml to about 25 pg / ml, at least about 0.5 pg / ml to about 25 pg / ml, at least about 0.1 pg / ml to about 15 pg / ml, at least about 0.1 pg / ml to about 10 pg / ml, or at least about 0.1 pg / ml to about 10 pg / ml of the in vivo produced protein; or (b) at least about 0.1 pg / ml, at least about 0.2 pg / ml, at least about 0.3 pg / ml, at least about 0.4 pg / ml, at least about 0.5 pg / ml, at least about 0.6 pg / ml, at least about 0.7 pg / ml, at least about 0.8 pg / ml, at least about 0.9 pg / ml, at least about 1.0 pg / ml, at least about 1.25 pg / ml, at least about 1.5 pg / ml, at least about 2.0 pg / ml, at least about 2.25 pg / ml, about 2.3 pg / ml, at least about 2.5 pg / ml, at least about 2.75 pg / ml, or at least about 3.0 pg / ml of the in vivo produced protein.

[0026] In one embodiment, the polynucleotide comprises a first and a second nucleic acids, and optionally wherein the first and the second nucleic acids encode different subunits or domains of the in vivo produced protein. In that embodiment, the subject is administered a predetermined molar ratio of the first and the second nucleic acids; and when administered to the subject, the first and the second nucleic acids generate a fully assembled protein.

[0027] In some embodiments of the method described herein, the subject is: (a) a cell, and administering comprises transfecting the cell with the polynucleotide; or (b) a whole organism selected from a mammal, a non-human primate, or a human.10028] Another aspect of the present disclosure provides a method of determining an in vivo produced antibody quality and / or quantity, the method comprising obtaining a biological sample comprising the in vivo produced antibody from a subject and determining the antibody aggregation using the methods described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows a schematic illustrating the problem solved by the present disclosure. Namely, how to assess the quality and quantity of in vivo expressed or produced biologies or protein therapeutics. Specifically, whether in vivo expressed or produced biologies or protein therapeutics (e.g., antibodies or multi-specific antibodies) aggregate in vivo, or whether multi-specific antibody chains pair correctly when produced in vivo.

[0030] FIG. 2 shows a schematic illustrating the novel method of assessing protein aggregation disclosed herein. The method comprises characterizing, qualifying, andquantifying protein aggregates in an unpurified biological sample comprising: (1) a size exclusion chromatography (SEC) step to initially fractionate the biological sample by protein size combined with (2) an automated capillary electrophoresis (CE) western (e.g., Simple Western™, or ProteinSimple Jess™) to identify, characterize, and quantify the target protein and / or aggregates thereof in the biological sample. The CE western comprises a further protein size separation step (e.g., a size distribution profiling), a protein immobilization step, and a protein detection step using fluorescent detection and / or chemiluminescent detection. This combination of SEC and CE western techniques provides a sensitive aggregate characterization, qualification, and quantification method with enhanced resolution and size separation range. The improved resolution is mediated in part by the double size separation steps (size distribution profiling): a first size separation step with the SEC and the second size separation step with the CE-westem. This is the first time these two techniques are used together, in series, to assess aggregates in a protein composition, rather than being used as orthogonal analytical techniques ( ie., an alternative to each other). An automated capillary electrophoresis (CE) western, such as Simple Western™, was used because CE can be more sensitive than an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of targets proteins in a serum sample from immunized animals. While ELISA only provides protein binding information, CE western techniques provide additional information about the target protein, such as molecular weight estimation (e.g., as size, size distributions) and protein intactness (e.g., isomers, fragments, and aggregates).

[0031] FIGs. 3A-B shows electropherograms of in vivo expressed protein from a serum sample and purified proteins analyzed using an automated capillary electrophoresis (CE) western (e.g., Simple Western™, Wes™, or Jess™ (ProteinSimple)). The target protein was either expressed in Expi293™ cells or was produced in vivo following the administration of a mRNA encoding the protein to mice. FIG. 3A shows a Jess™ electropherogram of a purified fusion protein X (bottom) and a Jess™ electropherogram of the same protein detected in a crude in vivo serum sample from a mice (top). FIG. 3B shows a Jess™ electropherogram of a purified fusion protein Y (bottom) and a Jess™ electropherogram of the same protein detected in a crude in vivo serum sample from a mice expressing the protein(top).

[0032] FIGs. 4A-D show the quantitative analysis of fusion protein X using Jess™. The purification standard protein concentrations was 0.625 to 5 ug / mL and the protein wasanalyzed using a goat anti -human Fc antibody (FIG. 4A) or a rabbit anti-fusion protein (FIG. 4C) FIGs. 4B and 4D show linear regression curves demonstrating the linear relationship between the peak area and the concentration of the protein shown in FIGs. 4A and 4C.

[0033] FIGs. 5A-B show quantitative PK analysis of fusion protein X from mouse serum illustrating that concentrations obtained using Jess™ were substantially similar to concentrations obtained using an ELISA assay. FIG. 5A shows a linear relationship between the area under the curve of a standard protein and the standard protein concentration. FIG. 5B shows a protein expression in a serum sample days after the administration of a nucleic acid encoding the protein to mice, as assessed by Jess™.

[0034] FIGs. 6A-C show Jess™ electropherograms of fusion protein X analyzed in serum samples using an anti-Fc antibody and an anti-fusion protein antibody illustrating that the fusion protein X remained intact throughout the in vivo study time course. FIG. 6A shows that fusion protein X was monomeric in the serum sample at 6 hrs. FIG. 6B shows the time course of the fusion protein X in the serum sample using the anti-Fc antibody. FIG. 6C shows the time course of the fusion protein X in the serum sample using the anti-fusion protein antibody.](HI35[ FIGs. 7A-C show the quantitative analysis of fusion protein Y from a mouse serum sample illustrating a mRNA dose-dependent expression of the fusion protein Y in BALB / c mice following administration. FIG. 7A shows Jess™ electropherogram demonstrating that the fusion protein Y had two peaks, peak 1 comprised 92% of the protein and peak 2 comprised 7% of the protein. FIG. 7B shows bar graphs showing that the administration of a high mRNA dose (15 ug MFG) produced about 22 ug / ml of protein and a low mRNA dose (6ug MFG) produced less than 5 ug / ml of protein. FIG. 7C shows a linear regression demonstrating the linear relationship between the area under the curve and the concentration of the standard protein.

[0036] FIGs. 8A-B show size exclusion chromatography (SEC) chromatograph elution pattern of a BALB / c mouse serum sample comprising an in vivo produced protein (FIG. 8B) when compared to a SEC chromatograph elution pattern of molecular size marker (BioRad SEC standards; FIG. 8A). In the BALB / c mouse serum sample, a single dominant peak was observed in fractions 19-24, which corresponded to proteins with sizes of about 44KDa toabout 158 kDa. Minor peaks were observed in SEC fractions 11-15, which corresponded to protein sizes of about 670KDa or more (z.e., multimers or aggregates). The SEC column used to fractionate the mouse serum sample based on size comprised 6 HR 10 / 30 (Superose™ 6 Increase 10 / 300 GL) (10mm by 300mm) equilibrated with PBS buffer. Superose ™6 10 / 300 GL column has a separation range from 5 to 5,000 kilo Dalton (kDa) of globular proteins. The SEC was run at a flow rate of about 0.5ml / min, and about 0.5mL per fractions was collected for elution.

[0037] FIGs. 9A-C show a SEC-Jess™ analysis of a murine serum sample comprising an in vivo expressed fusion protein X illustrating that the in vivo (SCID) expressed fusion protein X was monomeric based on Jess™-positive SEC fraction number. FIG. 9A shows a gel view of a CE western demonstrating the presence of the protein X in SEC fractions 20-24, which corresponded to protein sizes of about 44KDa to about 158KDa. FIG. 9B shows a CE western electropherogram of the serum sample with a single protein X dominant peak. FIG. 9C shows CE western electropherograms of a naive serum (negative control; no peak), SEC fractions 11, 12, 13, 20 and 21 from a serum sample comprising the protein X; and demonstrates that SEC fractions 20 and 21 contained the same single dominant peak at different intensity, but factions 11-13 showed background noise.

[0038] FIGs. 10A-B show a SEC chromatograph demonstrating the aggregation of a protein Z expressed in a cell culture, purified with Protein A (ProA Elution), and analyzed by SEC. FIG. 10A shows a schematic of the protein A purification step. FIG. 10B shows the SEC chromatograph of the ProA elution illustrating that the purified ProA elution sample comprised at least two species of target proteins: a monomeric protein peak (MP) and a high molecular weight (BMW) protein peak. These figures demonstrate that protein purification using protein A (Protein A pull down), which was eluted using a mild acid at pH 3-4, may induce protein aggregation. These data further indicate that purification induced protein aggregation can confound the protein aggregation assessment. Thus, it may be impossible to decouple the aggregation already formed during expression and the aggregation that is formed during acid elution.

[0039] FIGs. 11A-C show SEC- Jess™ analysis of a fusion protein Z from ProA Eluate as described in FIG. 10, illustrating that Protein A (ProA) purification prior to SEC fractionation induced protein aggregation of a target protein. FIG. HA shows a gel view of aCE western demonstrating the presence of the purified and ProA eluted protein Z in SEC fractions 10-18, with fractions 10-13 comprising aggregated proteins; and fractions 15-17 comprising the monomeric protein Z. FIG. 11B shows a CE western electropherogram of the ProA elution showing a single protein Z peak. FIG. 11C shows CE western electropherograms of SEC fractions 12 (HMW or aggregate), 16, and 17 from the ProA elution comprising the purified protein Z and demonstrating that SEC fractions 12, 16, and 17 contained the same single protein peak, but with different intensity. Surprisingly, fraction 12 contained a weak monomeric protein peak with an intensity of about 105,446.10040] FIGs. 12A-C show SEC- Jess™ analysis of a fusion protein Z from a cell culture supernatant demonstrating that the fusion protein Z shown in FIG. 11 was expressed as a monomer when analyzed without Protein A purification. FIG. 12A shows a gel view of a CE western demonstrating the presence of the protein Z in SEC fractions 14-19 comprising the monomeric protein Z. FIG. 12B shows a CE western electropherogram of the cultured supernatant showing a single protein Z peak. FIG. 12C shows CE western electropherograms of SEC fractions 12, 16, and 17 from the cultured supernatant comprising the protein Z and demonstrating that SEC fractions 16, and 17 contained the same single protein peak, but with different intensity. In contrast to FIG. 11C, fraction 12 contained a blip with an intensity of about 628 (no protein Z; background).10041 ] FIGs. 13A-E show SEC- Jess™ analysis of an in vivo expressed fusion protein Z from BALB / c mouse serum sample demonstrating that BALB / c in vivo expressed fusion protein Z eluted in the same fractions as the expected in vitro expressed protein. FIG. 13A shows a gel view of a CE western demonstrating the presence of the protein Z in SEC fractions 14-19 (protein size of about 150KDa to about 670KDa) comprising the monomeric protein Z. FIG. 13B shows a CE western electropherogram of the BALB / c mouse serum sample showing multiple dominant peaks identified as the fusion protein Z and non-specific serum proteins. BALB / c mouse was administered with a mRNA encoding a fusion protein Z. FIG. 13C shows CE western electropherograms of SEC fractions 12 (protein size > 670 KDa), 16 (protein size between 158KDa and 670 KDa), and 17 (protein size between 158KDa and 670 KDa) from the serum sample comprising the protein Z and demonstrating that SEC fractions 16, and 17 contained the same single dominant peak, but with different intensity. In contrast to FIG. 11C, fraction 12 contained a blip with an intensity of about 298(no protein Z). FIG. 13D shows a CE western electropherogram of SEC fraction 19 (protein size between 158KDa and 670 KDa) from the serum sample, with a dominant peak that did correspond to protein Z size, and thus demonstrating the presence of non-specific binding of serum proteins to the antibody. FIG. 13E shows a CE western electropherogram of a serum sample from a naive mouse, which was not administered with the fusion protein Z mRNA, showing multiple non-specific peaks.(0042] FIGs. 14A-C show SEC- Jess™ analysis of a naive BALB / c serum sample demonstrating that identified backgrounds peaks in FIG. 13, were triggered by non-specific binding of the BALB / s mouse immunoglobulins (Ig) and non-specific serum proteins to the antibody used to identify the fusion protein Z. Specifically, the Horseradish Peroxidase (HRP) conjugated antibody used in the CE western crossed-reacted with the mouse Ig in the absence of specific human Fc. FIG. 14A shows a gel view of a CE western demonstrating the presence of background proteins in SEC fractions 17-22 (protein size between 44KDa and 670KDa). FIG. 14B shows a CE western electropherogram of the naive BALB / c mouse serum sample showing multiple dominant peaks identified as the mouse immunoglobulin and non-specific serum proteins. FIG. 14C shows CE western electropherograms of SEC fractions 18 (protein size between 158KDa and 670KDa), 20 (protein size of about 158KDa), and 21 (protein size between 44KDa and 158KDa) from the serum sample.100431 FIG. 15 shows a CE western electropherogram of non-specific binding of the HRP conjugated antibody to a murine Ig was enhanced in the absence of a target (human) antibody; and demonstrating that the concentration of a target protein should be higher than the limit of the background murine Ig detection (e.g., higher than 2ug / mL) to significantly reduce non-specific background serum proteins.100441 FIGs. 16A-C show schematics illustrating the mechanism of the single antibody western method using CE western described herein, where a secondary antibody or a primary is used to bind to serum antibodies compared to a canonical (dual antibody) western (FIG. 16B vs FIG. 16C). In particular, a secondary antibody is usually used in a canonical western blot to target a constant region or a Fc region of primary antibody (FIG. 16B). However, one aspect of the present disclosure describes a single antibody CE western where the secondary antibody directly binds a fusion or chimeric protein (FIG. 16A top) comprising the constant region or a Fc region of an antibody (FIG. 16C), thereby bypassing the requirement of aprimary antibody in the CE western. FIG. 16A shows schematics of exemplary in vivo produced proteins that can be assessed using the method disclosed herein (e.g., a whole monomeric or multi-specific antibody or fragment thereof or fusion or chimeric protein comprising a fragment of an antibody).|0045| FIGs. 17A-D show results from a single-antibody CE western blot method illustrating that the binding of an anti-human secondary antibody produced a single dominant signal (z.e., strong signals) by CE western, and showed virtually no non-specific background (FIGs. 17A- B and D). FIG. 17A shows a schematic of the single-antibody CE western blot method. FIG. 17B shows that binding of an anti-human HRP conjugated secondary antibody to a biological sample comprising a chimeric Fc fusion protein (FIG. 16A top) produced a strong signal with no non-specific background. FIG. 17D shows that binding of an anti-human HRP conjugated secondary antibody to a purified chimeric Fc fusion protein produced a strong signal with no non-specific background. FIG. 17C shows that the anti-human HRP- secondary antibody produced no signal when added to a biological sample from a mouse that did not express the chimeric Fc fusion protein (z.e., naive mouse serum).

[0046] FIGs. 18A-D show results from a dual-antibody (canonical) CE western blot method using a biological sample comprising a BALB / c serum sample and illustrating that the primary antibody or the secondary antibody cross-reacted with non-specific serum proteins. FIG. 18A shows a schematic of the dual-antibody CE western blot method. FIG. 18C shows CE western results of a serum sample from naive mouse illustrating that the antibodies bound to the mouse immunoglobulin, and non-specific proteins (e.g., albumin or other serum protein). FIG. 18B shows CE western results of a serum sample from a mouse expressing a target chimeric Fc fusion protein (treated mouse) illustrating that the antibodies strongly bound to the target chimeric fusion protein, but they also bound to the mouse immunoglobulin, and non-specific proteins. Nonetheless, the dominant peak in the CE electropherogram identified the target chimeric Fc fusion protein. FIG. 18D shows CE western results of a purified target chimeric Fc fusion protein illustrating that the antibodies selectively bound to the target chimeric Fc fusion protein.

[0047] FIGs. 19A-C show results from a dual-antibody (canonical) CE western blot method similar to FIG. 18 using a different primary antibody and / or secondary antibody and illustrating that a dual-antibody CE western blot method required the availability of a goodantibody pair to obtain results with virtually no non-specific background. FIG. 19A shows CE western results of a serum sample from a mouse expressing a target chimeric Fc fusion protein (treated mouse). FIG. 19B shows CE western results of a serum sample from naive mouse. FIG. 19C shows CE western results of a purified target chimeric Fc fusion protein.DETAILED DESCRIPTIONI. OVERVIEW

[0048] Biologies or protein therapeutics are currently an established approach of treating many diseases and conditions. The production of these protein therapeutics is quickly moving toward in vivo protein production, where a nucleic acid encoding the target protein is administered to an animal as a single vector or multiple vectors. The target protein is then produced by the body in vivo. However, novel fusion proteins and antibodies may be prone to aggregation during production in the cells, either in vitro or in vivo, which might be attributed to stability or design issues.

[0049] Several questions regarding the quality of these in vivo produced protein therapeutics remain as shown in FIG. 1. For example, the quality and quantity the expressed target protein in vivo is not known. It is not known whether the in vivo produced protein is 100% monomeric or whether they comprise aggregates and to what extent. If the in vivo produced protein comprises multiple subunits (e.g., an antibody or a multi-specific antibody) that are co-administered to an animal, it is not known whether the subunits of the target protein paired correctly.[00501 Specifically, for an antibody or a multi-specific antibody, the question is whether the in vivo expressed or produced antibodies or multi-specific antibodies aggregate in vivo, or whether multi-specific antibody chains are paired correctly when produced in vivo. As used herein “Aggregate” means any non-native higher molecular weight molecule derived from a protein of interest (i.e., a target protein). Aggregates are of particular interest in the development of biologies or protein therapeutics because they can reduce drug efficacy. In some cases, aggregates can elicit an immune response. Thus, it is important to assess therapeutic protein aggregation during manufacturing.A. Canonical methods for analyzing protein aggregation of therapeutic proteins require protein purification.

[0051] Several tools are known in the art for in vitro assessment of protein quality during manufacturing as shown for example in Table 1. The standard methods for determining the amount of protein (product related) aggregation are size-exclusion chromatography (SEC) and light scattering methods. SEC can be used alone or in combination with High- performance liquid chromatography (HPLC) (SEC-HPLC) to separate compounds in a biological sample and to identify protein aggregation (e.g., multi -mer, monomer, and / or a population of protein fragments). SEC can be used to separate proteins and species of proteins based on their sizes using differential exclusion from pores of a stationary phase matrix. (FIG. 9). The availability of standard molecules facilitates the analysis of aggregates in a biological sample. (FIG. 9A). However, SEC alone can be limiting because the separation range of a typical SEC column may be insufficient to resolve larger aggregates. As such, several orthogonal techniques are used in lieu of SEC.

[0052] Furthermore, methods known in the art characterized purified proteins. As such, these methods can only be accurate if the protein sample is pure (z.e., free of other non-related proteins). To remove separate non-related proteins (z.e., impurities) from the target protein, the best method is to specifically pull down the target protein using a Protein A column (ProA). During the Protein A pull down, unrelated protein impurities are washed out, so a pure target protein (e.g., an antibody) can be eluted by applying mild acid at pH 3-4. (FIGs. 10A-C). As such, fusion proteins that may be naturally unstable at low pH tend to aggregate during the elution step. This purification-induced aggregation undoubtedly confounds the aggregate analysis because it is difficult to decouple the aggregation formed during protein synthesis (z.e., expression) and the aggregation that is purification- induced (z.e., formed during acid elution).B. SEC / Capillary electrophoresis (CE) Western aggregation assessment workflow

[0053] To circumvent these problems, the present disclosure provides a novel method of assessing protein aggregation. The new method comprises separating the therapeutic protein mixture (e.g., a cell culture supernatant, a blood sample, plasma, or serum from a subject expressing the therapeutic protein) by size (molecular weight) using a size exclusionchromatography (SEC) and then analyzing the SEC fractions using an automated and high- resolution western blot method (such as e.g., ProteinSimple Jess™), which further separate SEC eluates by size.

[0054] FIG. 2 shows a schematic illustrating the novel method of assessing protein aggregation disclosed herein. The method comprises characterizing and quantifying protein aggregates in a biological sample comprising a size exclusion chromatography (SEC) step to initially fractionate the biological sample by protein size combined with an automated capillary electrophoresis (CE) western (e.g., Simple Western™) to identify, characterize, and quantify the target protein and / or aggregates of the target protein (aggregates thereof) in the biological sample. The CE western comprises a size separation step in conjunction with a protein detection step using fluorescent detection and / or chemiluminescent detection. This combination of SEC and CE western techniques provides a sensitive aggregate characterization and quantification method with enhanced resolution and size separation range.

[0055] CE-westem techniques are orthogonal methods of analyzing protein in a biological sample. They can complement traditional fluorescent detection and / or chemiluminescent detection methods such as an ELISA method. In contrast to ELISA which only provides binding information, CE Western methods can generally provide additional information such as molecular weight (MW) estimation and protein “intactness” , and / or protein distribution profiling. As shown in FIGs. 3-7, a CE western technique (e.g., Jess™) can readily separate and identify an in vivo expressed protein from a crude (non-purified) same (FIG. 3A-B). CE western can provide other analytical information such as the relationship between the concentration of a nucleic acid administered to a mouse and the amount of in vivo protein produced (FIGs. 4-5 and 7). Furthermore, the serum sample remained stable during the entire analysis period. (FIG. 6).

[0056] The improved resolution was mediated in part by the double size separation steps: a first size separation step with the SEC and the second size separation step with the CE- western. The SEC-CE western method disclosed herein can analyze a therapeutic protein in an unpurified cell culture supernatant (FIG. 12) or an unpurified serum sample from a mouse expressing the therapeutic molecule (FIGs. 9, and 13-14). Because CE western is inherently an fluorescent detection and / or chemiluminescent detection method, the sensitivity of themethod described herein may depend on the availability of a good antibody to identify the target protein in the biological sample. However, the method disclosed herein is sensitive enough to distinguish background signal from a real signal, as shown in FIGs. 13-14. Once more, the double size separation steps ensures that background proteins and / or aggregates are distributed in different SEC fractions and CE band, thereby facilitating the characterization and / or quantification of aggregates, non-specific binding protein, and the target protein (See e.g, FIG. 13A, C, and D) FIG. 13B shows a sample comprising multiples peaks. Further characterization shows that SEC fraction 12 did not contain any significant aggregates (mfi = 298) and SEC fractions 16 (mfi= 2,941) and 17 (mfi= 2323) contained the target protein. While SEC fraction 19 captured low molecular weight protein fragments (background / non- specific protein) that are further expanded in FIG. 13D.

[0057] The novel method described herein comprises characterizing and quantifying protein aggregates in a biological sample comprising a size exclusion chromatography (SEC) step to initially fractionate the biological sample by protein size combined with an automated capillary electrophoresis (CE) western (e.g., Simple Western™) to identify, characterize, and quantify the target protein and / or aggregate of the target protein (aggregate thereof) in the biological sample. This is the first time these two techniques are used together, in series, to assess aggregate in a protein composition, rather than being used as an alternative to each other (i.e., as orthogonal analytical techniques).

[0058] The present disclosure provides a novel method of assessing protein aggregates in a biological sample. The biological sample can comprise a purified protein, a cell culture supernatant from cells expressing a recombinant protein, or an in vivo produced protein from a serum sample of an animal administered a nucleic acid encoding the in vivo produced protein.

[0059] One aspect of the present disclosure provides a method for assessing product related impurities, e.g., protein aggregation, in a biological sample comprising an in vivo produced protein and / or related impurities, the method comprising: (a) fractionating the biological sample from a subject expressing the in vivo produced protein and / or impurities thereof using a size exclusion chromatography (SEC); (b) collecting eluate fractions of the in vivo produced protein; (c) electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western; and (d) detecting the invivo produced protein and / or related impurities in the SEC eluate fractions using fluorescent detection or chemiluminescent detection. In some embodiments, the product related impurities comprise protein aggregates. In some embodiments, the eluate fractions comprise a predicted size range.100601 Another aspect of the present disclosure provides a method for assessing protein aggregation in a biological sample comprising an in vivo produced protein and / or aggregates thereof, the method consisting of, consisting, or comprising: fractionating the biological sample from a subject expressing the in vivo produced protein and / or aggregates thereof using a size exclusion chromatography (SEC); collecting eluate fractions of the in vivo produced protein; electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western; and detecting the in vivo produced protein and / or aggregates thereof in the SEC eluate fractions using fluorescent detection or chemiluminescent detection. In one embodiment, the eluate fractions comprise a predicted size range.II. SIZE EXCLUSION CHROMATOGRAPHY

[0061] In some embodiments of the method disclosed herein, the biological sample from a subject comprising an in vivo produced protein and / or aggregates thereof is fractionated using a size exclusion chromatography (SEC). The method can begin with the collection of whole blood from a subject. In some embodiments, about 1-2 mL are collected. The subject can be a human, a non-human primate, a rat, a mouse, a dog, a goat, a rabbit, or any other mammal, as defined herein. A serum sample can be separated from whole blood by centrifugation using any methods known to those of skill in the art. The serum sample from the subject can be aliquoted into vessels for analysis (e.g., Eppendorf tubes). In some embodiments, the serum sample can be analyzed by size exclusion chromatography (SEC). In some embodiments, a supernatant from a cell culture can be analyzed by SEC.

[0062] In some embodiments, the size exclusion chromatography (SEC) separate the protein aggregates from the monomeric protein. Because of the differential molecular weight of the monomer and the aggregate, the monomeric protein and the protein aggregates can be eluted in different SEC fractions. SEC can be used to separate proteins and species of proteins based on their sizes using differential exclusion from pores of a stationary phase matrix. (FIG. 9).Thus, protein species of various molecular weights can be distributed in various fractions and these species can be further separated, quantified and identified using the CE Western described herein. The CE Western can also determine the molecular weight of the various aggregate species. However, when used alone, SEC can be a slow process. SEC can have a poor capacity and can require disproportionately large amount columns. For example, SEC columns can require superior packing skills, and large buffer volumes.(0063] However, SEC remains useful in the present disclosure because its selectivity is directly related to protein size and a crude or an unpurified protein sample is analyzed using the methods of the present disclosure. Thus, in the first step of the method disclosed herein, the SEC distributes the amount of proteins to be analyzed in different eluate fractions based on size. The target protein and / or aggregates of various sizes can be distributed across various eluate fractions. For example, aggregates that are dimers, or trimers of the therapeutic protein can be found in SEC fractions that are eluted immediately prior to fractions containing the main monomeric therapeutic protein. High molecular weight aggregates (HMW), such as e.g., pentamers, can be eluted in fractions that are eluted prior to fractions containing dimers and trimers.(0064] In some embodiments of the method disclosed herein, the SEC comprises a resin selected from the group consisting of Superdex®, Bio-Sep™, TSKgel™, Sephacryl™, Superose™, and Sephadex™. Superdex® is a dextran-agarose composite resin for SEC. The SEC column can be Superdex™ 200 column or Superdex™ 200 Increase column. Sephacryl is an allyl dextran-N,N'methylene bisacrylamide composite gel filtration for SEC. The SEC column can be Sephacryl™ S-200, Sephacryl™ 100-HR, or Sephacryl™ 200-HR. Superose is a cross-linked agarose resin for SEC. The SEC column can be Superose® 6 Increase. Sephadex® is a cross-linked dextran resin chromatography. The SE column can be Sephadex® G-25, Sephadex® G-100, or Sephadex® LH-20. BioSep-SEC-S is Silica-based SEC columns. The SEC column can be PREP SEC 300, BioSep-SEC-s2000, BioSep-SEC-s3000, or BioSep-SEC-s4000.

[0065] In some embodiments, the resin of the size exclusion chromatography is Superose. Superose ™6 10 / 300 GL column has a separation range from 5 to 5,000 kilo Dalton (kDa) of globular proteins. In some embodiments, the SEC has a separation range of about 0.1 kilo Dalton (KDa) to lOOKDa, about 1 KDa to about 150 KDa, about 5 KDa to about 100 KDa,about 5 KDa to about 700 KDa, about 5 KDa to about 1000 KDa, about 5 KDa to about 5,000 KDa, or about 10 KDa to about 10,000 KDa. In some embodiments, the SEC has a separation range of about 5 KDa to about 5,000 KDa. In that embodiment, the SEC separation range is under a native condition or a denatured condition.10066] In some embodiments, the biological sample is fractionated at a flow rate of about 0.01 ml / min, about 0.05 ml / min, about 0.075 ml / min, about 0.1 ml / min, about 0.15ml / min, about 0.2ml / min, about 0.3ml / min, about 0.4 ml / min, about 0.5 ml / min, about 0.6 ml / min, about 0.7 ml / min, about 0.8 ml / min, about 0.9 ml / min, about 1.0 ml / min, about 1.5 ml / min, or about 2.0ml / min or more. In some embodiments, the biological sample is fractionated at a flow rate of about 0.5 ml / min.

[0067] Thus, in some embodiments of the method disclosed herein, a biological sample or cell culture supernatant of up to lOOul can be loaded onto a AKTA system mounted with a SEC column (e.g., Superdex® 200 or Superose® 6 HR 10 / 30 (10mm by 300mm) equilibrated with PBS buffer. In that embodiment, the flow rate of about 0.5ml / min can be used for the separation of the proteins by molecular weight. In some embodiments, 0.5mL per fractions can be collected for elution volume from 7mL to 19mL.III. CE WESTERNA. Size distribution profiling(0068] The present disclosure provides a novel method of assessing protein aggregation. The novel method comprises a first step of separating the therapeutic protein mixture (e.g., cell culture supernatant, a blood sample, a plasma sample, or a serum sample from a subject expressing the therapeutic protein) by size (molecular weight) using a size exclusion chromatography (SEC), as described herein. The second step comprises collecting the SEC eluate fractions for qualitative analysis. The third and fourth steps comprise analyzing the SEC fractions using an automated and high-resolution western blot method (e.g., the ProteinSimple Jess™). In step 3 of the method described herein (FIG. 2), the SEC eluates fractions are further separated by size. Thus, the improved resolution of the method disclosed herein depend in part on the double size separation steps: a first size separation step with the SEC and the second size separation step with the CE-westem. This is the first time these twotechniques are used together, in series, to assess aggregate in a protein composition, rather than being used as an alternative to each other (z.e., as orthogonal analytical techniques).

[0069] Accordingly, in step 3, the sample can be prepared for Jess™ (or equivalent thereof) and can be loaded into a Jess™ plate. Specifically, one or more SEC eluate fractions comprising the therapeutic protein (e.g., in vitro produced or in vivo produced) can be placed in a single well along with a serum diluent or a buffer, a primary antibody that targets the in vivo produced protein (e.g., a therapeutic protein) and / or a secondary antibody conjugated to a chemiluminescent or a fluorescent molecule, and Jess™ reagents (Standard Equipment for Western Assays),. The chemiluminescent or the fluorescent molecule can be obtained from any known supplier. Jess™ reagents can be obtained from the manufacturer.

[0070] The Jess™ plate can be loaded in a ProteinSimple Jess™ instrument. Thus, in some embodiments of the method disclosed herein, the method comprises electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western. In some embodiments, the automated capillary electrophoresis (CE) western is a closed-loop automated capillary-based immunoassay system. In some embodiments, the SEC eluate fractions are electrophoretically separated by weight. In some embodiments, separated proteins are immobilized on a solid support. In some embodiments, the solid support is a capillary wall of a microfluidic device. In some embodiments, electrophoretically separating the proteins from the SEC eluate fractions provides a size distribution profiling of the proteins found in the SEC eluate fraction.B. Immunodetection

[0071] In some embodiments of the method disclosed herein, the method comprises detecting the in vivo produced protein and / or aggregates thereof in the SEC eluate fractions using fluorescent detection or chemiluminescent detection. In some embodiments, detecting the in vivo produced protein and / or aggregates thereof comprises the steps of: (a) contacting the SEC eluate fractions with an antibody that binds to the in vivo produced protein; (b) incubating the SEC eluate fractions with the antibody; and (c) detecting the binding of the antibody to the SEC eluate fractions using fluorescent detection or chemiluminescent detection.

[0072] In some embodiments, the SEC eluate fractions and the antibody are incubated for about 10 minutes to about 120 minutes. In some embodiments, the SEC eluate fractions and the antibody are incubated for about 10 minutes, about 15 minutes, about 20 minutes, about25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.

[0073] In some embodiments, the detecting the binding of the antibody to the in vivo produced protein comprises a dual antibody CE western or a single antibody CE western. In some embodiments, the dual antibody CE western comprises: (a) a primary antibody that targets the in vivo produced protein or aggregates thereof; (b) a secondary antibody that targets the primary antibody; and (c) a chemiluminescent or a fluorescent molecule substrate. In some embodiments, the secondary antibody that targets the primary antibody is operably linked to a chemiluminescent or a fluorescent molecule.

[0074] In some embodiments, the single antibody western comprises: (a) a secondary antibody that targets the in vivo produced protein or aggregates thereof and (b)a chemiluminescent or a fluorescent molecule substrate. In that embodiment, the secondary antibody is operably linked to a chemiluminescent or a fluorescent molecule. In those embodiments, the in vivo produced protein or aggregates thereof can comprise a constant domain or a Fc domain of an antibody. In some embodiments, the secondary antibody selectively binds to the constant domain or the Fc domain of the in vivo produced protein or aggregates thereof.

[0075] In some embodiments, the single antibody western comprises: (a) a primary antibody that targets the in vivo produced protein or aggregates thereof and (b) a chemiluminescent or a fluorescent molecule substrate. In that embodiments, the primary antibody is operably linked to a chemiluminescent or a fluorescent molecule.

[0076] In some embodiments of the method disclosed herein, the methods can comprise detecting the binding of the therapeutic protein present in the SEC eluate fractions by fluorescence and / or chemiluminescent detection. In those embodiments, the presence of thefluorescence signal and / or the chemiluminescent signal indicates the presence of the in vivo or in vitro produced protein (e.g., target therapeutic protein) in the SEC eluate fractions.

[0077] In some embodiments, the target protein is detected using chemiluminescent detection. In some embodiments, the detecting the in vivo produced protein and / or aggregates thereof in the SEC fractions comprises chemiluminescent detection. Chemiluminescent detection can be a more sensitive detection method for detecting target proteins that may be in low abundance in a biological sample. For example, chemiluminescent detection can maximize immunodetection output by detecting protein that are present in the sample at picogram concentration.100781 In some embodiments, the method described herein detects a nanogram or a picogram (pg) of the in vivo produced protein and / or aggregates thereof in the biological sample. In some embodiments, the method detects at least about 0.0001 ng- 1. Ong, at least about O.OOOlng-O. lng.

[0079] In some embodiments, the method detects at least about 0.1 pg- 1.Opg, at least about 0.5pg-2.5, at least about O. lpg-lOpg, at least about 5pg-50pg, at least about lOpg-lOOpg, or more than lOOpg of the in vivo produced protein or aggregates thereof in the biological sample. In some embodiments, the method detects at least about 0. Ipg, at least about 0.2pg, at least about 0.3pg, at least about 0.4pg, at least about 0.5pg, at least about 0.6pg, at least about 0.7pg, at least about 0.8pg, at least about 0.9pg, at least about l.Opg, at least about 1. Ipg, at least about 1.5pg, at least about 5. Opg, at least about lOpg, at least about 20pg, at least about 30pg, at least about 40pg, at least about 50pg, at least about 60pg, at least about 70pg, at least about 80pg, at least about 90pg, or at least about lOOpg of the in vivo produced protein or aggregates thereof in the biological sample.

[0080] One aspect of the present disclosure provides a method for assessing protein aggregation in a biological sample comprising an in vivo produced protein and / or aggregates thereof, the method comprising: (a) fractionating the biological sample from a subject expressing the in vivo produced protein and / or aggregates thereof using a size exclusion chromatography (SEC); (b) collecting eluate fractions of the in vivo produced protein; (c) electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western; and (d) detecting the in vivo produced protein and / oraggregates thereof in the SEC eluate fractions using a detection agent. In one embodiment, the eluate fractions comprise a predicted size range.

[0081] In some embodiments, the detection agents are capable of binding to or interacting with the in vivo produced protein and aggregates thereof in the SEC eluate fraction. Nonlimiting examples of detection agents include proteins, peptides, antibodies, enzyme substrates, transition state analogs, cofactors, nucleotides, polynucleotides, aptamers, lectins, small molecules, ligands, inhibitors, drugs, and other biomolecules as well as nonbiomolecules capable of binding the analyte to be detected. In some embodiments, the detection agent is an antibody. The antibody can be an IgM and / or an IgG antibody.100821 In some embodiments, the detection agents can comprise one or more label moieties. In some embodiments, the detection agents can comprise two or more label moieties. In that embodiment, each label moiety can be the same or different.

[0083] In some embodiments, the label moiety may comprise a fluorescent label or a chemiluminescent label (e.g., fluorescent detection and / or chemiluminescent detection). The chemiluminescent label can comprise any entity that provides a light signal and that can be used in accordance with the methods and devices described herein. A wide variety of such chemiluminescent labels are known in the art. See, e.g., U.S. Pat. Nos. 6,689,576, 6,395,503, 6,087,188, 6,287,767, 6,165,800, and 6,126,870.

[0084] Suitable labels include enzymes that are capable of reacting with a chemiluminescent substrate to induce a photon emission by chemiluminescence. The labels may include peroxidase, beta-galactosidase, phosphatase, or others for which a chemiluminescent substrate is available. In some embodiments, the chemiluminescent label can be selected from any of a variety of classes of luminol, or isoluminol. In some embodiments, the detection agents comprise one or more chemiluminescent substrates.

[0085] The label moiety can also comprise a bioluminescent compound. As used herein, bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent compound is determined by detecting the presence of luminescence. Suitable bioluminescent compounds can include luciferin, luciferase and aequorin.

[0086] In some embodiments, the label moiety comprises a fluorescent dye. The fluorescent dye comprises a resonance-delocalized system or aromatic ring system that absorbs light at a first wavelength and emits fluorescent light at a second wavelength in response to the absorption event. A wide variety of such fluorescent dye molecules are known in the art. For example, fluorescent dyes can be selected from any of a variety of classes of fluorescent compounds, non-limiting examples include xanthenes, rhodamines, fluoresceins, cyanines, phthalocyanines, squaraines, bodipy dyes, coumarins, oxazines, and carbopyronines. In some embodiments, for example, where detection agents contain fluorophores, such as fluorescent dyes, their fluorescence is detected by exciting them with an appropriate light source and monitoring their fluorescence by a detector sensitive to their characteristic fluorescence emission wavelength. In some embodiments, the detection agents comprise fluorescent dye labeled antibodies.

[0087] In some embodiments, the fluorescent detection and / or chemiluminescent detection comprises a Horseradish Peroxidase (HRP), streptavidin-HRP, luminol-S, peroxide, LacZ, biotin, or luciferase. In some embodiments, the detection agents comprise chemiluminescent labeled antibodies. In some embodiments, the fluorescent detection and / or chemiluminescent detection comprises a horseradish peroxidase (HRP) conjugated anti-IgG antibody or anti- IgM antibody.

[0088] The detection of the in vivo or in vitro produced protein can include detecting the presence or absence, measurement, and / or characterization of an analyte (e.g., the in vivo or in vitro produced protein and / or aggregates thereof). Typically, an analyte (e.g., the in vivo or in vitro produced protein and / or aggregates thereof) is detected by detecting a signal from a label. Such detection can include, but is not limited, to detecting isotopic labels, immune labels, optical dyes, enzymes, particles and combinations thereof such as chemiluminescent labeled antibodies and fluorescent labeled antibodies.

[0089] Moreover, the methods disclosed herein allow a person of skill in the art to monitor a signal in real time. A real time monitoring to allow the user to rapidly determine whether an in vivo or in vitro produced protein and / or aggregates thereof is present in the biological sample and / or aggregate thereof, and / or the amount or activity of the in vivo or in vitro produced protein and / or aggregates thereof.[00901 In some embodiments, the signal can be measured from at least two different time points. In some embodiments, the signal can be monitored continuously or at several selected time points. Alternatively, the signal can be measured at a predetermined end-point. For example, the signal can be measured after a certain amount of time. Alternatively, the signal can be compared against a control signal (e.g., sample without the in vivo or in vitro produced protein and / or aggregates thereof; or a sample without biological material), threshold signal, or standard curve. The amount of the signal generated may not be critical and can vary over a broad range. The only requirement may for the signal to be measurable by the detection system being used. In some embodiments, a signal can be at least 2-fold greater than the background signal (e.g., control signal; sample without analyte; or a sample without biological material). In some embodiments, a signal can be 2 to 10-fold greater than the background. In some embodiments, a signal can be 10-fold greater than the background.

[0091] The results generated by the ProteinSimple Jess™ instrument can be analyzed using a Compass software. Thus, the protein band images and quantitative results can be generated by Compass software after running one or more samples on the Jess™ capillary electrophoresis system (ProteinSimple) and / or using chromatogram output data. See e.g., chrome-extension: / / efaidnbmnnnibpcajpcglclefindmkaj / https: / / www.bio-techne.com / pdf- download-arena-document / brochure / pl6-0002 / 25.

[0092] One aspect of the present disclosure provides a method of determining an in vivo produced antibody quality and / or quantity, the method comprising obtaining a biological sample comprising the in vivo produced antibody from a subject and determining the antibody aggregation using any of the methods for assessing protein aggregation in a biological sample comprising an in vivo produced protein and / or aggregates thereof described herein.IV. SAMPLES

[0093] In some embodiments, the biological sample can be selected from the group consisting of a cell lysate, a cell culture supernatant, a whole blood, a serum sample, a plasma sample, urine, seminal fluid, cerebrospinal fluid, and saliva. In some embodiments, the biological sample is a serum sample.

[0094] In some embodiments, the biological sample is diluted to about 1 :20; about 1 :40; about 1 :50; about 1 :75, about 1 : 100; about 1 : 150; about 1 : 120; about 1 :250; about 1 :300; about 1 : 350; about 1 : 375; about 1 : 400; about 1 : 500; about 1 : 750; about 1 :800; about 1 : 1000; about 1 : 1100; or about 1 : 1200. In some embodiments, the biological sample is diluted at about 1 :75 or about 1 :300.[00951 In some embodiments, the in vivo or in vitro produced protein can be used at a concentration of about O. lug; about 0.125ug; about 0.150ug; about 0.2ug; about 0.225ug; about 0.25ug; about 0.3ug; about 0.315ug; about 0.325ug; about 0.350ug; about 0.4ug; about 0.415ug; about 0.425ug; about 0.450ug; about 0.5ug; about 0.515ug; about 0.52ug; about 0.55ug; or about 0.6ug.

[0096] In some embodiments, the biological sample may be unpurified, at an intermediate level of purity, or highly purified. In some embodiments, an intermediate level of purity of the sample may be in a range of from about 40% to about 90% purity. In some embodiments, the high level of purity of the sample may be in a range of from about 90% or greater.

[0097] In some embodiment of the method disclosed herein, the biological sample is not purified prior to the SEC fractionation described herein. In some embodiments, prior to the SEC fractionation, the biological sample is not pre-treated with a process selected from the group consisting of affinity chromatography, low pH viral inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration, ultrafiltration, and tangential flow filtration. In some embodiments, prior to the SEC fractionation, the biological sample is not purified using a Protein A- or a Protein G-based affinity chromatography.

[9098] In some embodiments, the biological sample is a cell culture supernatant. The cell culture supernatant can be generated using a cell culture production medium. In some embodiments, the cell culture production medium is a cell line. In some embodiments, the cell culture production medium comprises a cell line selected from the group consisting of Chinese Hamster Ovary (CHO) cells, GS-CHO, CHOK1SV GS-KO, CHO DUX-B11, CHO- Kl, BSC-1, NSO myeloma cells, CV-1 in Origin carrying SV40 (COS) cells, COS-1, COS-7, P3X3Ag8.653, SP2 cells, human embryonic kidney (HEK 293) cells, baby hamster kidney (BHK 21) cells, African green monkey kidney VERO-76 cells, HELA cells, human lung cells (W138), Retinal cells, and human hepatoma line (Hep G2). VERO, BHK, MDCK, W138cells, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, HsS78Bst cells, PER.C6, SP2 / 0-Agl4, a myeloma cell line, a hybridoma cell line, human lung cells (W138), Retinal cells, human hepatoma line (Hep G2), CH0-K1 (ATCC CCL-61), DGI 44, SH87 cellCHO-DXBl l, CHO designated ECACC 85050302, CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1 / SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), CHOKlsv, CHO-S, dihydrofolate reductase negative CHO cells (CHO / -DHFR), and dpl2.CHO cells; monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells; baby hamster kidney cells (BHK, ATCC CCL-10); CAP cell, AGE1.HN cell, monkey kidney cells (CV 1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3 A, ATCC CRL-1442); TRI cells; MCR 5 cells; and FS4 cells ;and hybridoma cells.

[0099] In some embodiments, the biological sample is a protein preparation such as, a cell culture harvest, a cell culture supernatant, an antibody-containing solution derived from a cell culture, or an antibody-containing solution from a previous stage of protein purification. In some embodiments, the protein preparation is an antibody-containing solution from a previous stage of protein purification. In some embodiments, the protein preparation is an eluate from a chromatography column.A. The in vivo produced protein

[0100] One aspect of the present disclosure provides a method for assessing protein aggregation in a biological sample comprising an in vivo produced protein and / or aggregates thereof. In some embodiments, the in vivo produced protein is an antibody. In some embodiments, the in vivo produced protein is a mono-specific antibody, a bi-specific antibody, or a tri-specific antibody.

[0101] In some embodiments, the in vivo produced protein comprises a multi-specific antibody that binds two or more antigens comprising: (a) a first polypeptide comprising an immunoglobulin heavy chain (VH) of an antibody that targets a first antigen; and (b) a second polypeptide comprising an immunoglobulin light chain (VL) of an antibody that targets the first antigen. In some embodiments, the VH is operably linked to an immunoglobulin Fc fragment; at least one antigen binding domain that binds to a second antigen; and an immune modulator. In some embodiments, the VL is operably linked to: a linker; an immune modulator molecule; and at least one antigen binding domain that binds the second antigen.[01021 In some embodiments, the first or the second antigen is selected from the group consisting of CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CDl lb, CD18, CD169, CDlc, CD33, CD38, CD138, and CD13. In some embodiments, the first or second antigen is CD 19 or CD20. In some embodiments, the first antigen is CD 19 and the second antigen is CD20. Alternatively, the first antigen is CD20 and the second antigen is CD 19.

[0103] In some embodiments, the first or second antigen is expressed on cancer cells. In some embodiments, the first and / or second antigen are expressed on cancer cell, such as for example, B cell lymphoma cell. In some embodiments, the first and / or second antigen are B cell lymphoma tumor antigens.

[0104] In some embodiments, the in vivo produced protein comprises antigen binding domain is selected from the group consisting of a Fab, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the in vivo produced protein comprises an antigen binding domain that targets an antigen on a tumor cell. In some embodiments, the in vivo produced protein comprises antigen binding domain that targets an antigen selected from the group consisting of CD 19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CDl lb, CD18, CD169, CDlc, CD33, CD38, CD 138, and CD13. In some embodiments, the antigen is CD 19 or CD20. In some embodiments, the antigen is CD 19. In some embodiments, the antigen is CD20. In some embodiments, the in vivo produced protein comprises two antigen binding domains. In some embodiments, the first antigen binding domain targets CD20 and the second antigen binding domain targets CD 19.[0105| In some embodiments, the in vivo produced protein comprises an immune modulator capable of binding to a molecule selected from the group consisting of BTLA, HVEM, CSF1R, CCR4, CD39, CD40, CD73, CD96, CXCR2, CXCR4, CD200, GARP, CD47, CXCL9, CXCL10, CXCL11, and CD155. In some embodiments, the immune modulator capable of binding to CD47 is signal regulatory protein alpha (SIRPa).[0106| In some embodiments, the in vivo produced protein comprises an immunoglobulin Fc fragment. In some embodiments, the immunoglobulin Fc fragment is a human immunoglobulin G1 (IgGl) Fc fragment. In some embodiments, the immunoglobulin Fc fragment has a modified effector function, for example, an enhanced or increased effector function. In some embodiments, the immunoglobulin Fc fragment has an enhanced effector function.

[0107] In some embodiments, the in vivo produced protein comprises an immunoglobulin Fc fragment and an immune modulator capable of binding to a molecule selected from the group consisting of BTLA, HVEM, CSF1R, CCR4, CD39, CD40, CD73, CD96, CXCR2, CXCR4, CD200, GARP, CD47, CXCL9, CXCL10, CXCL11, and CD155. In some embodiments, the immunoglobulin Fc fragment is a human immunoglobulin G1 (IgGl) Fc fragment and the immune modulator capable of binding to CD47 is signal regulatory protein alpha (SIRPa). In some embodiments, the in vivo produced protein comprises an immunoglobulin Fc fragment operably linked to one or more SIRPa molecules.[01081 As used herein, the term “Antibody,” refers to an immunoglobulin molecules and / or molecules comprising one or more immunologically active portions of immunoglobulin (Ig) molecules, which specifically bind with an antigen. The term “antibody” is used in the broadest sense and specifically covers single (mono-specific) monoclonal antibodies (including agonist and antagonist antibodies), antibodies with polyepitopic specificity (bispecific, tri-specific, or multi-specific), antibody fragments, and chimeric or fusion molecules comprising one or more antibody fragments or a portion of an immunoglobulin molecule, so long as they exhibit the desired biological activity of an immunoglobulin molecule.

[0109] A multi-specific antibody means an engineered molecule comprising two or more binding domains that specifically binds to an antigen. The multi-specific antibodies are engineered to target distinct antigen epitopes simultaneously on the same or different celltypes. For example, a multi-specific antibody can comprise 2, 3, 4, 5, 6, 7, 8, or more domains that bind to one or more antigens on the same cell or different cells. The binding domain can be a fragment or a portion of an immunoglobulin molecule, or the binding domain of any molecule, including but not limited to immune cell receptors, or tumor antigen receptors. In some embodiments, the multi-specific antibody is a bi-specific, trispecific antibody, a tetra-specific antibody, a penta-specific antibody, or a hexa-specific antibody.

[0110] In some embodiments, the multi-specific antibody is a tri-specific (or trispecific) antibody. In some embodiments, the tri-specific antibody comprises three different binding domains. In some embodiments, one binding domain is specific for a receptor on an immune cell (e.g., T cells or a B cell). A second binding domain is specific for a tumor associated antigen including but not limited to CD 19, CD20, ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD33, CD123, CD22, CD30. A third binding domain can be specific for an immune checkpoint modulator, such as PDL1, PD1, 0X40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, Light, HVEM, CD73, or CD39.

[0111] In some embodiments, the trispecific antigen comprises three different binding domains; and one binding domain is specific for a receptor on an immune cell (e.g., CD47; CD20; or CD 19); a second binding domain is specific for a first tumor associated antigen (e.g., CD 19 or CD20); and the third binding domain is specific for a second tumor associated antigen (e.g., CD20; or CD 19). In some embodiments, at least one of the three binding domains targets an immune receptor. In some embodiments, at least one of the three binding domains targets a tumor antigens.

[0112] Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, chimeric molecules comprising a portion of an antibody, antibody fragments (e.g., Fv, Fab, F(ab')2, as well as single chain antibodies (scFv)) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In:Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0113] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGl, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0114] An antibody may comprise a VH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VH domain of any of the antibodies shown in the appended sequence listing, and / or comprising a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of any of those antibodies. Algorithms that can be used to calculate % identity of two amino acid sequences include e.g., BLAST, FASTA, or the Smith-Waterman algorithm, e.g., employing default parameters. Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and / or insertion of an amino acid residue). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab')2 fragments, scFv, and an Fab expression library.B. Producing the in vivo produced protein

[0115] In some embodiments, the in vivo produced protein is generated by administering a polynucleotide encoding the in vivo produced protein to the subject. In some embodiments, the polynucleotide is a DNA, or an RNA. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide further comprises a modified nucleotide, a cap structure, a poly A tail, a 5' untranslated region, and / or a 3' untranslated region.

[0116] In some embodiments, the subject is administered at least about Ipg to about 50 pg, at least about 10 pg to about 50 pg, at least about 5 pg to about 25 pg, at least about 1 pg toabout 15 pg, at least about 1 pg to about 10 pg, at least about 5 pg to about 15 pg, at least about 6 pg to about 15 pg, or at least about 5 pg to about 20 pg of a mRNA encoding the inv vivo expressed protein.

[0117] In some embodiments, the biological sample comprises at least about 0.1 pg / ml to about 50 pg / ml, at least about 0.1 pg / ml to about 25 pg / ml, at least about 0.5 pg / ml to about 25 pg / ml, at least about 0.1 pg / ml to about 15 pg / ml, at least about 0.1 pg / ml to about 10 pg / ml, or at least about 0.1 pg / ml to about 10 pg / ml of the in vivo produced protein.

[0118] In some embodiments, the biological sample comprises at least about 0.1 pg / ml, at least about 0.2 pg / ml, at least about 0.3 pg / ml, at least about 0.4 pg / ml, at least about 0.5 pg / ml, at least about 0.6 pg / ml, at least about 0.7 pg / ml, at least about 0.8 pg / ml, at least about 0.9 pg / ml, at least about 1.0 pg / ml, at least about 1.25 pg / ml, at least about 1.5 pg / ml, at least about 2.0 pg / ml, at least about 2.25 pg / ml, about 2.3 pg / ml, at least about 2.5 pg / ml, at least about 2.75 pg / ml, or at least about 3.0 pg / ml of the in vivo produced protein.

[0119] In some embodiments, the polynucleotide comprises a first and a second nucleic acids. In that embodiment, the first and the second nucleic acids encode different subunits or domains of the in vivo produced protein. In those embodiments, the subject is administered a predetermined molar ratio of the first and the second nucleic acids. In those embodiments, when administered to the subject, the first and the second nucleic acids generate a fully assembled protein.

[0120] In some embodiments, the subject is a cell, and administering comprises transfecting the cell with the polynucleotide. In some embodiments, the subject is a whole organism selected from a mammal, a non-human primate, or a human. In that embodiment, the subject is systemically administered the first and the second nucleic acids as described herein. In some embodiments, systemic administration comprises an intravenous or an intraperitoneal administration.

[0121] In some embodiments, the subject has a disease or condition. The disease or condition can be selected multiple myeloma malignant plasma cell neoplasm, Hodgkin’s lymphoma, nodular lymphocyte predominant Hodgkin’s lymphoma, Kahler’s disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia,B-cell non-Hodgkin’s lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt’s lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom’s macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma. In some embodiments, the subject has B-cell lymphoma or refractory B-cell lymphoma.V. AGGREGATES

[0122] In some embodiments, an aggregate of the in vivo expressed protein comprises a dimer, a trimer, a tetramer, a multimer, or a higher order oligomer of the in vivo expressed protein. In some embodiments, an aggregate of the in vivo expressed protein is an homoaggregate or a hetero-aggregate. In some embodiments, the biological sample can be substantially free of aggregates. The aggregate content of the biological sample can be less than about 5%. In some embodiments, the aggregate content of the biological sample can be expected to be less than about 1%. In some embodiments, the aggregate content of the biological sample can be expected to be less than about 0.5%. In some embodiments, the aggregate content of the biological sample can be expected to be less than about 0.1%. In some embodiments, the aggregate content of the biological sample can be expected to be below the detection limit of the method disclosed herein.(0123] In one or more of the preceding embodiments, the aggregates comprise homoaggregates of the desired protein. In one or more of the preceding embodiments, the aggregates comprise hetero-aggregates of the desired protein and a contaminant.

[0124] In some embodiments, the contaminant is a nucleic acid, nucleotide, endotoxin, metal ion, protein, lipid, or cell culture media component. In some embodiments, the aggregates in the sample include both homo-aggregates and hetero-aggregates. In some embodiments, the desired protein is an antibody or antibody fragment.VI. DEFINITIONS

[0125] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0126] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.(0127] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0128] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, forexample, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0129] Headings, e.g., (a), (b), (i), are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0130] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0131] As used herein the term “Aggregate” means any non-native higher molecular weight molecule derived from a protein of interest (z.e., a target protein). The protein of interest can be a monomeric molecule. In some embodiments, Aggregate can be soluble, thermodynamically and / or kinetically stable species with a molecular weight at least twice that of the therapeutically active species. For example, if the protein of interest is an antibody, the aggregate is a multimer of a monomeric antibody (e.g., monomeric IgG monoclonal antibody). In some embodiments, the aggregate can also be an insoluble species of a protein of interest (ie., a target protein) that can be larger than ~60 nm. In that embodiment, the aggregate can be at the lower limit of detection for most submicron particle characterization techniques. In such an embodiment, the aggregate can be a ‘particulate matter.’

[0132] In some embodiments, Aggregate means an association of two or more molecules that is stable at physiological conditions and may remain stable over a wide range of pH and conductivity conditions. Aggregates can comprise at least one biomolecule such as a protein, nucleic acid, or lipid and another molecule or metal ion. The association may occur through any type or any combination of chemical interactions. Aggregates of antibodies may be classified into two categories:10.1.33] As used herein, the term “Animal” refers to a mammal. The animals of the presentdisclosure can be “genetically modified” or “transgenic.” A transgenic animal can be a genetically modified animal comprising one or more exogenous transgenes, or other foreign DNA, added or incorporated, or one or more endogenous genes modified, including, targeted, recombined, interrupted, deleted, disrupted, replaced, suppressed, enhanced, or otherwise altered, to mediate a genotypic or phenotypic effect in at least one cell of the animal and typically into at least one germ line cell of the animal. The animal may have the transgene integrated on one allele of its genome (heterozygous transgenic). Alternatively, the animal may have the transgene on two alleles (homozygous transgenic).101341 As used herein, the term “Antibody,” refers to an immunoglobulin molecules and / or molecules comprising one or more immunologically active portions of immunoglobulin (Ig) molecules, which specifically bind with an antigen. The term “antibody” is used in the broadest sense and specifically covers single (mono-specific) monoclonal antibodies (including agonist and antagonist antibodies), antibodies with polyepitopic specificity (bi- specific, tri-specific, or multi-specific), antibody fragments, and chimeric or fusion molecules comprising one or more antibody fragments or a portion of an immunoglobulin molecule, so long as they exhibit the desired biological activity of an immunoglobulin molecule.

[0135] In some embodiments, “Antibody” refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, antibody fragment (Fv), fragment antigenbinding regions (Fab) and F(ab)2, single chain variable fragment (scFv) and humanized antibodies. In some embodiments, antibody refers to such assemblies (e.g., intact antibody molecules, immunoadhesins, or variants thereof), which have significant known specific immunoreactive activity to an antigen of interest (e.g., a tumor associated antigen).Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0136] As used herein, the term “Antigen” or “Ag” is defined as a molecule that provokes an immune response.

[0137] As used herein, the term “binding” a molecule to an chromatography resin is meant exposing the molecule to chromatography resin under appropriate conditions (pH / conductivity) such that the molecule is reversibly immobilized in or on the chromatography resin by virtue of ligand-protein interactions. Non-limiting examples include ionic interactions between the molecule and a charged group or charged groups of the ion exchange material and a bispecific interaction between Protein A and an immunoglobulin.

[0138] As used herein, the term “cell culture supernatant” refers to a medium in which cells are cultured and into which proteins are secreted provided they contain appropriate cellular signals. The cellular signal can be a signal peptide. In some embodiments, recombinant protein expressing cells are cultured with serum. In some embodiments, recombinant protein expressing cells are cultured under serum-free culture conditions. Thus, the cell culture supernatant can be devoid of animal-serum derived components. Most preferably, the cell culture medium is a chemically defined medium.10139] As used herein, “clarified” refers to a sample (i.e., a cell suspension) having undergone a solid-liquid separation step involving one or more of centrifugation, microfiltration and depth filtration to remove host cells and / or cellular debris. A clarified fermentation broth may be a cell culture supernatant. Clarification is sometimes referred to as a primary or initial recovery step and typically occurs prior to any chromatography or a similar step.101.401 As used herein, the term “Contacting” means mixing, admixing, or incubating one or more components for the purpose of stimulating an interaction and / or binding of two or more of the components. In particular, contacting can also refer to bringing one or more components in closed proximity to trigger an interaction (e.g., binding) between the two or more components.[01411 As used herein, the term “Chromatography” refers to the separation of chemically different molecules in a mixture from one another by percolation of the mixture through an absorbent, which absorbs or retains different molecules more or less strongly. Molecules that are least strongly absorbed to or retained by the absorbent are released from the absorbent under conditions where those more strongly absorbed or retained are not. In someembodiments, chromatography means a technique used to separate a target protein of interest (e.g., a monomer of a monoclonal mAh) from other molecules present in a mixture.

[0142] As used herein, the term “chromatography resin” or “chromatography media” are used interchangeably herein and refer to any kind of solid phase which separates an analyte of interest (e.g., an Fc region containing protein such as an immunoglobulin) from other molecules present in a mixture. Usually, the analyte of interest is separated from other molecules as a result of differences in rates at which the individual molecules of the mixture migrate through a stationary solid phase under the influence of a moving phase, or in bind and elute processes. Non-limiting examples include cation exchange resins, affinity resins, anion exchange resins, anion exchange membranes, hydrophobic interaction resins and ion exchange monoliths. The volume of the resin, the length and diameter of the column to be used, as well as the dynamic capacity and flow-rate depend on several parameters such as the volume of fluid to be treated, concentration of protein in the fluid to be subjected to the process of the invention. Determination of these parameters for each step is well within the average skills of the person skilled in the art.

[0143] As used herein, the terms “Detection,” “Detect,” and “Detecting” mean to discover the presence or existence of (e.g., an analytes such as an antibody).

[0144] As used herein, the term “elute” refers to a process which removes a protein of interest from a chromatography resin by altering the solution conditions such that buffer competes with the molecule of interest for the ligand sites on the chromatography resin. A non-limiting example is to elute a molecule from an ion exchange resin by altering the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material.

[0145] As used herein, the term “Electrophoresis” refers to the movement of suspended or dissolved molecules through a fluid or gel under the action of an electromotive force applied to electrodes in contact with a fluid.

[0146] “As used herein, the term “Endotoxin” refers to a toxic heat-stable lipopolysaccharide substance present in the outer membrane of gram-negative bacteria that is released from the cell upon lysis. Endotoxins can be generally acidic due to their high content of phosphate andcarboxyl residues and can be highly hydrophobic due to the fatty acid content of the lipid-A region. Endotoxins can offer extensive opportunity for hydrogen bonding.

[0147] As used herein, the term “Homo-aggregate” refers to a stable association of two or more proteins of identical composition.10148] As used herein, the term “Hetero-aggregate” refers to a stable association of one or more proteins of identical or different composition, optionally associated with one or more non-protein molecules. The non-protein component may consist of one more entities from the group consisting of a nucleotide, an endotoxin, a metal ion, a lipid, or a cell culture media component.10149] As used herein, the terms “Identification,” “Identify,” and “Identifying” mean to recognize exposure to a specific pathogen or agent in sample from a subject.

[0150] As used herein, the term “Incubating” encompasses maintaining or providing conditions favorable for or conducive to a desired reaction such as but not limited to, providing a reaction component or reagent. It is recognized that conditions favorable for or conducive to one reaction may differ from conditions favorable for another reaction. Suitable conditions for different reactions and reaction types are known in the art.

[0151] As used herein, the term “z z vivo produced protein” refers to a protein that is produced in a whole animal following the administration of a nucleic acid (e.g., DNA or RNA) encoding the protein to the animal. Such in vivo produced protein is synthesized in a cell and released in the blood of the animal.

[0152] As used herein, the term “ / / / vitro produced protein” refers to a protein that is produced in an ex vivo cell culture. In some embodiments, the in vitro produced protein is produced in a cell line following the transfection of a nucleic acid (e.g., DNA or RNA) encoding the protein into the cell line. In some embodiments, the in vitro produced protein is produced from a culture of hybrid cells, such as an hybridoma cell line.

[0153] As used herein, the term “Immobilizing” refers to substantially reducing or eliminating the motion of molecules in a fluid path. The immobilization can be via covalent bonds or non-covalent means, such as by hydrophobic or ionic interaction. In some embodiments, the resolved analytes (e.g., antibodies) present in the sample are immobilizedin the fluid path by isoelectric focusing (e.g., according to the Jess™ system specification).

[0154] As used herein the term “high molecular weight aggregates,” or “HMW,” as used interchangeably herein, refers to a higher order fraction of protein aggregates, i.e., pentamers and above.10155 [ As used herein, the term “non-specific binding” describes interactions between a molecule of interest (e.g., a target protein has described herein) and a ligand, or other compound bound to a solid support (e.g., Protein A bound to a solid phase matrix or resin), through electrostatic forces, hydrogen bonding, hydrophobic forces, and / or van der Waals forces at an interaction site, but lacking structural complementarity that enhances the effects of the nonstructural forces. Examples of non-specific interactions include, but are not limited to, electrostatic, hydrophobic, and van der Waals forces as well as hydrogen bonding.

[0156] As used herein, “Orthogonal analytical technique” means a technique that exploits a different chemical or physical principle to measure the same parameter using a different method. In some embodiments, the technique may provide a different way of assessing the same analytical problem. Orthogonal analytical technique can mean, an alternative technique to solve the same problem.

[0157] As used herein, the term “Protein” refers to a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed principally of one or more chains of amino acids linked by peptide bounds. The protein may be of natural or recombinant origin. Proteins may be modified with non-amino acid moieties such as through glycosylation, pegylation, or conjugation with other chemical moieties. Examples of proteins include but are not limited to antibodies, antibody fragments, clotting factors, enzymes, and peptide hormones. In some embodiments, “protein” has its ordinary meaning.10158] As used herein, “Protein A” refers to a protein originally discovered in the cell wall of Stapphylococcus and binds to an Fc portion or a variable domain of an antibody. In some embodiments, Protein A binds to a domain from VH3 family (e.g., a VH3 domain of IgG antibody). For purposes of the invention, "Protein A" is any protein identical or substantially similar to StapphylococcalVmX.Q,m A, including commercially available and / or recombinant forms of Protein A. For purposes of the invention, the biological activity of Protein A for thepurpose of determining substantial similarity is the capacity to bind to an Fc portion or a variable domain (e.g., VH3) of IgG antibody. “Protein A,” and “ProA” are used interchangeably herein and encompass Protein A recovered from a native source thereof, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have a CH2 / CH3 region, such as an Fc region. Protein A is generally immobilized on a solid phase support material. The term “ProA” also refers to an affinity chromatography resin or column containing chromatographic solid support matrix to which is covalently attached Protein A.101591 As used herein, the term “purified” is used to indicate that the relative concentration (weight of component or fraction divided by the weight of all components or fractions in the mixture) of a protein of interest or target protein e.g., an antibody) is increased by at least about 20%. In one series of embodiments, the relative concentration is increased by at least about 40%, about 50%, about 60%, about 75%, about 100%, about 150%, or about 200%. A component or fraction can also be said to be purified when the relative concentration of components from which it is purified (weight of component or fraction from which it is purified divided by the weight of all components or fractions in the mixture) is decreased by at least about 20%, about 40%, about 50%, about 60%, about 75%, about 85%, about 95%, about 98% or 100%. In still another series of embodiments, the component or fraction is purified to a relative concentration of at least about 50%, about 65%, about 75%, about 85%, about 90%, about 97%, about 98%, or about 99%.

[0160] As used herein, the term “Sample” means a biological sample of a subject. The biological sample may comprise any number of macromolecules, for example, cellular macromolecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample can include one or more cells. The sample can include one or more microbes. The biological sample may be a nucleic acid sample or protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may be a cell-free or cell free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodilysample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.

[0161] A sample can be any substance containing or presumed to contain one or more analytes of interest (e.g., antibodies). The sample can be of natural or synthetic origin and can be obtained by any means known to those of skill in the art. Samples can also be synthetic and include but are not limited to in vitro cell culture constituents including but not limited to conditioned medium, recombinant cells, and cell components. The sample can be a tissue, a cell, an organ, or a fluid isolated from a subject. The fluid may include but not be limited to, plasma, serum, whole blood, cerebrospinal fluid (CSF), semen, amniotic fluid, lymph fluid, synovial fluid, urine, or tears.

[0162] In some embodiments of the methods disclosed herein, the sample is a body fluid, which may include, for example, blood, plasma, serum, urine, saliva, tears, synovial fluid, and / or lymphatic fluid. In some embodiments, the sample is blood or whole blood. In some embodiments, the sample comprises plasma or serum. The sample can be serum.101631 Accordingly, in some embodiments of the methods disclosed herein, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, seminal fluid, cerebrospinal fluid and saliva. Alternatively, the biological sample is whole blood, or a serum.

[0014] The term “specific binding” as used herein, such as to describe interactions between a target protein (e.g., an Fc region containing protein) and a ligand bound to a solid support (e.g., Protein A bound to a solid phase matrix or resin), refers to the generally reversible binding of a protein of interest to a ligand through the combined effects of spatial complementarity of protein and ligand structures at a binding site coupled with electrostatic forces, hydrogen bonding, hydrophobic forces, and / or van der Waals forces at the binding site. Generally, the greater the spatial complementarity and the stronger the other forces at the binding site, the greater will be the binding specificity of a protein for its respective ligand. Non-limiting examples of specific binding includes antibody-antigen binding, enzymesubstrate binding, enzyme-cofactor binding, metal ion chelation, DNA binding protein-DNA binding, regulatory protein-protein interactions, and the like

[0165] As used herein, the term “Subject” means any organism including, without limitation,a mammal. For example, the mammal can be a farm animal, a livestock animal, a pet, a primate, a non-human primate, or a monkey. In some embodiments, the mammal can be a human, a rhesus monkey, a cynomolgus monkey, a dog, a cat, a mouse, a rat, a fox, a deer, a ferret, a guinea pig, a rabbit, a pig, a goat, a cow, a baboon, or a horse. In a preferred embodiment, the subject is a human being. In some embodiments, the mammal is a non- human primate (e.g., a monkey or a baboon).

[0166] As used herein, the terms “target protein” and “protein of interest” as used interchangeably herein, and refer to a protein or polypeptide, including but not limited to, a recombinant monoclonal antibody that is to be assessed by a method described herein, from a biological sample comprising a mixture of proteins, and optionally, other materials such as cell debris, DNA, host cell proteins, media components, and the like.VII ENUMERATED EMBODIMENTS

[0167] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

[0168] Embodiment 1 provides a method for assessing product related impurities, e.g., protein aggregation, in a biological sample comprising an in vivo produced protein and / or aggregates thereof, the method comprising: (a) fractionating the biological sample from a subject expressing the in vivo produced protein and / or aggregates thereof using a size exclusion chromatography (SEC); (b) collecting eluate fractions of the in vivo produced protein; (c) electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western; and (d) detecting the in vivo produced protein and / or aggregates thereof in the SEC eluate fractions using fluorescent detection or chemiluminescent detection.

[0169] Embodiment 2 provides the method of embodiment 1, wherein detecting the in vivo produced protein and / or aggregates thereof comprises: (a) contacting the SEC eluate fractions with an antibody that binds to the in vivo produced protein; (b) incubating the SEC eluate fractions with the antibody for about 10 minutes to about 120 minutes; and (c) detecting the binding of the antibody to the SEC eluate fractions using fluorescent detection or chemiluminescent detection.

[0170] Embodiment 3 provides the method of embodiment 2, wherein prior to step (a)-(c), the SEC eluate fractions are electrophoretically separated by weight and immobilized on a solid support.

[0171] Embodiment 4 provides the method of embodiment 3, wherein the solid support is a capillary wall of a microfluidic device.

[0172] Embodiment 5 provides the method of any one of embodiments 1-4, wherein the automated capillary electrophoresis (CE) western is a closed-loop automated capillary-based immunoassay system.

[0173] Embodiment 6 provides the method of any one of embodiments 2-5, wherein the SEC eluate fractions and the antibody are incubated for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.

[0174] Embodiment 7 provides the method of any one of embodiments 2-6, wherein the detecting the binding of the antibody to the in vivo produced protein comprises a dual antibody CE western or a single antibody CE western.10175 [ Embodiment 8 provides the method of embodiment 7, wherein the dual antibody CE western comprises: (a) a primary antibody that targets the in vivo produced protein or aggregates thereof; (b) a secondary antibody that targets the primary antibody, wherein the secondary antibody is operably linked to a chemiluminescent or a fluorescent molecule; and (c) a chemiluminescent or a fluorescent molecule substrate.

[0176] Embodiment 9 provides the method of embodiment 7, wherein the single antibody western comprises: (a) a secondary antibody that targets the in vivo produced protein or aggregate thereof, wherein the secondary antibody is operably linked to a chemiluminescent or a fluorescent molecule; and (b) a chemiluminescent or a fluorescent molecule substrate.

[0177] Embodiment 10 provides the method of embodiment 9, wherein the in vivo produced protein or aggregates thereof comprises a constant domain or a Fc domain of an antibody.

[0178] Embodiment 11 provides the method of embodiment 10, wherein the secondary antibody selectively binds to the constant domain or the Fc domain of the in vivo produced protein.[0.179] Embodiment 12 provides the method of any one of embodiments 1-11, wherein the detecting the in vivo produced protein and / or aggregates thereof in the SEC fractions comprises chemiluminescent detection.

[0180] Embodiment 13 provides the method of any one of embodiments 1-12, wherein the method detects a nanogram or a picogram (pg) of the in vivo produced protein and aggregates thereof in the biological sample.

[0181] Embodiment 14 provides the method of any one of embodiments 1-13, wherein the method detects at least about 0.0001 ng- 1. Ong, at least about O.OOOlng-O.lng, at least about O. lpg-l.Opg, at least about 0.5pg-2.5, at least about O. lpg-lOpg, at least about 5pg-50pg, at least about lOpg-lOOpg, or more than lOOpg of the in vivo produced protein or aggregates thereof in the biological sample.

[0182] Embodiment 15 provides the method of any one of embodiments 1-14, wherein the method detects at least about O.lpg, at least about 0.2pg, at least about 0.3pg, at least about 0.4pg, at least about 0.5pg, at least about 0.6pg, at least about 0.7pg, at least about 0.8pg, at least about 0.9pg, at least about l.Opg, at least about l.lpg, at least about 1.5pg, at least about 5.0pg, at least about lOpg, at least about 20pg, at least about 30pg, at least about 40pg, at least about 50pg, at least about 60pg, at least about 70pg, at least about 80pg, at least about 90pg, or at least about lOOpg of the in vivo produced protein or aggregates thereof in the biological sample.

[0183] Embodiment 16 provides the method of any one of embodiments 1-15, wherein the SEC comprises a resin selected from the group consisting of Superdex®, Bio-Sep™, TSKgel™, Sephacryl™, Superose™, and Sephadex™.

[0184] Embodiment 17 provides the method of any one of embodiments 1-16, wherein the SEC has a separation range of about 0.1 kilo Dalton (KDa) to lOOKDa, about 1 KDa to about 150 KDa, about 5 KDa to about 100 KDa, about 5 KDa to about 700 KDa, about 5 KDa to about 1000 KDa, about 5 KDa to about 5,000 KDa, or about 10 KDa to about 10,000 KDa.

[0185] Embodiment 18 provides the method of embodiment 17, wherein the SEC separation range is under a native or a denatured condition.

[0186] Embodiment 19 provides the method of any one of embodiments 1-18, wherein the biological sample is fractionated at a flow rate of about 0.01 ml / min, about 0.05 ml / min, about 0.075 ml / min, about 0.1 ml / min, about 0.15ml / min, about 0.2ml / min, about 0.3ml / min, about 0.4 ml / min, about 0.5 ml / min, about 0.6 ml / min, about 0.7 ml / min, about 0.8 ml / min, about 0.9 ml / min, about 1.0 ml / min, about 1.5 ml / min, or about 2.0ml / min or more.10187] Embodiment 20 provides the method of any one of embodiments 1-19, wherein the biological sample is fractionated at a flow rate of about 0.5 ml / min.10188] Embodiment 21 provides the method of any one of embodiments 1-20, wherein an aggregate of the in vivo expressed protein: (a) comprises a dimer, a trimer, a tetramer, a multimer, or a higher order oligomer of the in vivo expressed protein; and / or (b) is an homoaggregate or a hetero-aggregate.

[0189] Embodiment 22 provides the method of any one of embodiments 1-21, wherein prior to the SEC fractionation, the biological sample is: (a) not purified; (b)not pre-treated with a process selected from the group consisting of affinity chromatography, low pH viral inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration, ultrafiltration, and tangential flow filtration; or (c) not purified using a Protein A- or a Protein G-based affinity chromatography.

[0190] Embodiment 23 provides the method of any one of embodiments 1-22, wherein the in vivo produced protein is generated by administering a polynucleotide encoding the in vivo produced protein to the subject.

[0191] Embodiment 24 provides the method of embodiment 23, wherein: (a) the polynucleotide is a DNA, or an RNA; (b) the polynucleotide is an mRNA; or (c) the polynucleotide further comprises a modified nucleotide, a cap structure, a poly A tail, a 5' untranslated region, and / or a 3 ' untranslated region.

[0192] Embodiment 25 provides the method of embodiment 23 or 24, wherein the subject is administered about Ipg to about 50 pg, about 10 pg to about 50 pg, about 5 pg to about 25 pg, about 1 pg to about 15 pg, about 1 pg to about 10 pg, about 5 pg to about 15 pg, about 6 pg to about 15 pg, or about 5 pg to about 20 pg of a mRNA encoding the inv vivo expressed protein.

[0193] Embodiment 26 provides the method of any one of embodiments 1-25, wherein the biological sample comprises: (a) at least about 0.1 pg / ml to about 50 pg / ml, at least about 0.1 pg / ml to about 25 pg / ml, at least about 0.5 pg / ml to about 25 pg / ml, at least about 0.1 pg / ml to about 15 pg / ml, at least about 0.1 pg / ml to about 10 pg / ml, or at least about 0.1 pg / ml to about 10 pg / ml of the in vivo produced protein; or (b) at least about 0.1 pg / ml, at least about 0.2 pg / ml, at least about 0.3 pg / ml, at least about 0.4 pg / ml, at least about 0.5 pg / ml, at least about 0.6 pg / ml, at least about 0.7 pg / ml, at least about 0.8 pg / ml, at least about 0.9 pg / ml, at least about 1.0 pg / ml, at least about 1.25 pg / ml, at least about 1.5 pg / ml, at least about 2.0 pg / ml, at least about 2.25 pg / ml, about 2.3 pg / ml, at least about 2.5 pg / ml, at least about 2.75 pg / ml, or at least about 3.0 pg / ml of the in vivo produced protein.

[0194] Embodiment 27 provides the method of any one of embodiments 23-26, wherein the polynucleotide comprises a first and a second nucleic acids, and optionally wherein the first and the second nucleic acids encode different subunits or domains of the in vivo produced protein.

[0195] Embodiment 28 provides the method of embodiment 27, wherein the subject is administered a predetermined molar ratio of the first and the second nucleic acids; and wherein when administered to the subject, the first and the second nucleic acids generate a fully assembled protein.

[0196] Embodiment 29 provides the method of any one of embodiments 23-28, wherein the subject is: (a) a cell and administering comprises transfecting the cell with thepolynucleotide; or (b) a whole organism selected from a mammal, a non-human primate, or a human.

[0017] Embodiment 30 provides a method of determining an in vivo produced antibody quality and / or quantity, the method comprising obtaining a biological sample comprising the in vivo produced antibody from a subject and determining the antibody aggregation using the method of any one of embodiments 1-28.EXAMPLES

[0198] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0199] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure and are not to be construed as limiting in any way the remainder of the disclosure.Example 1: SEC / WB aggregation assessment workflow

[0200] Novel fusion proteins and antibodies may be prone to aggregation during production in the cells, either in vitro or in vivo, which might be attributed to stability or design issues. The standard methods for determining the amount of protein (product related) aggregation are size-exclusion chromatography or light scattering methods. Additional non-limiting methods as shown in Table 1.

[0201] But these methods can only be accurate if the protein sample is pure, (z.e., free of other non-related protein). To remove impurities, the best method is to specifically pull down the target protein using a Protein A column. During the Protein A pull down, unrelated protein impurities are washed out, so a pure target protein (e.g., an antibody) can be eluted by applying mild acid at pH 3-4. However, some fusion proteins may be naturally unstable at low pH and tend to aggregate during the elution step. See e.g., FIGs. 10-11.

[0202] FIGs. 10A-B show a SEC chromatograph demonstrating the aggregation of a protein Z expressed in a cell culture, purified with Protein A (ProA Elution), and analyzed by SEC- CE. FIG. 10A shows a schematic of the protein A purification step. FIG. 10B shows the SEC chromatograph of the ProA Elution illustrating that the purified protein sample comprised at least two species of target proteins: a monomeric protein peak (MP) and a high molecular weight (BMW) protein peak. These figures demonstrate that protein purification using protein A (Protein A pull down), which was eluted using a mild acid at pH 3-4, may induce protein aggregation.

[0203] FIGs. 11A-C show SEC- Jess™ analysis of a fusion protein Z from ProA Eluate as described in FIG. 10 and illustrate that Protein A (ProA) purification prior to SEC fractionation induced protein aggregation of a target protein. FIG. HA shows a gel view of aCE western demonstrating the presence of the purified and ProA eluted protein Z in SEC fractions 10-18, with fractions 10-13 comprising aggregated protein; and fractions 15-17 comprising the monomeric protein Z. FIG. 11B shows a CE western electropherogram of the ProA elution showing a single protein Z peak. FIG. 11C shows CE western electropherograms of SEC fractions 12 (HMW or aggregate), 16, and 17 from the ProA elution comprising the purified protein Z and demonstrating that SEC fractions 12, 16, and 17 contained the same single protein peak, but with different intensity. Surprisingly, fraction 12 contained a weak monomeric protein peak with an intensity of about 105,446.10204] Thus, this protein purification-induced aggregate formation can undoubtedly confound the analysis because it is difficult to decouple the aggregation already formed during protein expression and the aggregation that is formed during acid elution (e.g., ProA elution).

[0205] To circumvent this problem, the inventors of the present disclosure proposed separating the therapeutic protein mixture by size (molecular weight) using a size exclusion chromatography (SEC) and then analyzing the fractions using an automated and high- resolution western blot method, such as the ProteinSimple Jess™.Example 2: Methods

[0206] Size exclusion chromatography .

[0207] Up to lOOul of the sample, either serum or plasma or cell culture supernatant, was loaded onto a AKTA system mounted with a SEC column, such as superdex200 or Superose™ 6 HR 10 / 300 (10mm by 300mm) equilibrated with PBS buffer. A flow rate at 0.5ml / min was used for the separation, and 0.5mL per fractions was collected for elution volume from 7mL to 19mL. Superose ™6 10 / 300 GL column has a separation range from 5 to 5,000 kilo Dalton (kDa) of globular proteins (GE Healthcare, Indianapolis, Ind.).

[0208] FIGs. 8A-B show size exclusion chromatography (SEC) chromatograph of a BALB / c mouse serum sample comprising an in vivo produced protein (FIG. 8A) when compared to a SEC chromatograph of BioRad SEC standards. The SEC column used to fractionate the mouse serum sample based on size comprised 6 HR 10 / 30 (Superose™ 6 Increase 10 / 300 GL) (10mm by 300mm) equilibrated with PBS buffer. The SEC was run at a flow rate of about 0.5ml / min, and about 0.5mL per fractions was collected for elution.[0209| Jess™ can be used as a quantitative analysis tool to assess the presence of a target protein in serum sample.

[0210] An automated capillary electrophoresis (CE) western, such as Simple Western™, was used because CE can be more sensitive than an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of targets proteins in a serum sample from immunized animals. While ELISA only provides protein binding information, CE western techniques provide additional information about the target protein, such as molecular weight estimation (e.g., as size, size distributions) and protein intactness (e.g., isomers, fragments, and aggregates).[02111 The protocol for sample preparation and equipment setup was followed according to manufacturer’s recommendations. For detecting the human Fc, the secondary HRP conjugated antibody from ProteinSimple (Biotechne, cat number 043-491) was identified as the best antibody for analyzing culture supernatant and mouse serum because it produced the lowest background during detection (due to non-specific binding to the endogenous serum protein). As for analyzing serum samples from cynomolgus monkey, an antibody from BioRad (cat number MCA5748G) conjugated with HRP in-house using the AbCam HRP conjugation kit (cat number ab 102890), was selected. The anti-SIRPa antibody from ThermoFisher (cat number PA5-29544) was used as the primary antibody to probe for the SIRPa moiety of the fusion constructs. The Jess™ protocol is known to a person of skill in the art, for example as disclosed by the manufacturer. In addition, any ProteinSimple products or Simple Western™ products (e.g., Wes™, Abby™, Sally Sue™, PeggySue™, or NanoPro™ 1000) or CE western products can be used, bio-techne.com / resources / instrument- applications; bio-techne.eom / p / simple-western / jess_004-650.[02121 FIGs. 3A-B shows electropherograms of in vivo expressed protein from a serum sample and purified proteins analyzed using an automated capillary electrophoresis (CE) western (e.g., Simple Western™, Wes™, or Jess™ (ProteinSimple)). The target protein was either expressed in Expi293™ cells or was produced in vivo following the administration of a mRNA encoding the protein to mice. FIG. 3A shows a Jess™ electropherogram of a purified fusion protein X (bottom) and a Jess™ electropherogram of the same protein detected in a crude in vivo serum sample from a mice (top). FIG. 3B shows a Jess™ electropherogram of apurified fusion protein Y (bottom) and a Jess™ electropherogram of the same protein detected in a crude in vivo serum sample from a mice expressing the protein(top).

[0213] FIGs. 4A-D show the quantitative analysis of fusion protein X using Jess™. The purification standard protein concentrations was 0.625 to 5 ug / mL and the protein was analyzed using a goat anti -human Fc antibody (FIG. 4A) or a rabbit anti-fusion protein (FIG. 4C) FIGs. 4B and 4D show linear regression curves demonstrating the linear relationship between the peak area and the concentration of the protein shown in FIGs. 4A and 4C.

[0214] FIGs. 5A-B show quantitative PK analysis of fusion protein X from mouse serum illustrating that concentrations obtained using Jess™ were substantially similar to concentrations obtained using an ELISA assay. FIG. 5A shows a linear relationship between the area under the curve of a standard protein and the standard protein concentration. FIG. 5B shows a protein expression in a serum sample days after the administration of a nucleic acid encoding the protein to mice, as assessed by Jess™.

[0215] FIGs. 6A-C show Jess™ electropherograms of fusion protein X analyzed in serum samples using an anti-Fc antibody and an anti-fusion protein antibody illustrating that the fusion protein X remained intact throughout the in vivo study time course. FIG. 6A shows that fusion protein X was monomeric in the serum sample at 6 hrs. FIG. 6B shows the time course of the fusion protein X in the serum sample using the anti-Fc antibody. FIG. 6C shows the time course of the fusion protein X in the serum sample using the anti-fusion protein antibody.

[0216] FIG. 7A-C show the quantitative analysis of fusion protein Y from a mouse serum sample illustrating a mRNA dose-dependent expression of the fusion protein Y in BALB / c mice following administration. FIG. 7A shows Jess™ electropherogram demonstrating that the fusion protein Y had two peaks, peak 1 comprised 92% of the protein and peak 2 has 7% of the protein. FIG. 7B shows bar graphs showing that the administration of a high mRNA dose (15 ug MFG) produced about 22 ug / ml of protein and a low mRNA dose (6ug MFG) produced less than 5 ug / ml of protein. FIG. 7C shows a linear regression demonstrating the linear relationship between the area under the curve and the concentration of the standard protein.Example 3: Qualitative analysis of a target protein in a culture supernatant or a serum sample using a combination of SEC and CE Western

[0217] FIGs. 9A-C show a SEC-Jess™ analysis of a murine serum sample comprising an in vivo expressed fusion protein X illustrating that the in vivo (SCID) expressed fusion protein X was monomeric based on JESS-positive SEC fraction number. FIG. 9A shows a gel view of a CE western demonstrating the presence of the protein X in SEC fractions 20-24. FIG. 9B shows a CE western electropherogram of the serum sample with a single protein X peak. FIG. 9C shows CE western electropherograms of a naive serum (negative control), SEC fractions 11, 12, 13, 20 and 21 from a serum sample comprising the protein X; and demonstrates that SEC fractions 20 and 21 contained the same single protein peak at different intensity.

[0218] Purification of a protein from the biological sample or cell culture supernatant induced protein aggregation

[0219] FIGs. 10A-B show a SEC chromatograph demonstrating the aggregation of a protein Z expressed in a cell culture and purified with Protein A (ProA Elution) and analyzed by SEC. FIG. 10A shows a schematic of the protein A purification step. FIG. 10B shows the SEC chromatograph of the ProA Elution illustrating that the purified ProA elution sample comprised at least two species of target proteins: a monomeric protein peak (MP) and a high molecular weight (HMW) protein peak. These figures demonstrate that protein purification using protein A (Protein A pull down), which was eluted using a mild acid at pH 3-4 may induce protein aggregation. These data further indicate that purification induced protein aggregation can confound the protein aggregation assessment. Thus, it may be impossible to decouple the aggregation already formed during expression and the aggregation that is formed during acid elution.

[0220] FIGs. 11A-C show SEC- Jess™ analysis of a fusion protein Z from ProA Eluate as described in FIG. 10, illustrating that Protein A (ProA) purification prior to SEC fractionation induced protein aggregation of a target protein. FIG. HA shows a gel view of a CE western demonstrating the presence of the purified and ProA eluted protein Z in SEC fractions 10-18, with fractions 10-13 comprising aggregated protein; and fractions 15-17 comprising the monomeric protein Z. FIG. 11B shows a CE western electropherogram of the ProA elutionshowing a single protein Z peak. FIG. 11C shows CE western electropherograms of SEC fractions 12 (HMW or aggregate), 16, and 17 from the ProA elution comprising the purified protein Z and demonstrating that SEC fractions 12, 16, and 17 contained the same single protein peak, but with different intensity. Surprisingly, fraction 12 contained a weak monomeric protein peak with an intensity of about 105,446.

[0221] SEC-Jess™ combination bypassed the need for protein purification prior to protein aggregates assessment.

[0222] FIGs. 12A-C show SEC- Jess™ analysis of a fusion protein Z from a cell culture supernatant demonstrating that the fusion protein Z shown in FIG. 11 was expressed as a monomer when analyzed without Protein A purification. FIG. 12A shows a gel view of a CE western demonstrating the presence of the protein Z in SEC fractions 14-19 comprising the monomeric protein Z. FIG. 12B shows a CE western electropherogram of the cultured supernatant showing a single protein Z peak. FIG. 12C shows CE western electropherograms of SEC fractions 12, 16, and 17 from the cultured supernatant comprising the protein Z and demonstrating that SEC fractions 16, and 17 contained the same single protein peak, but with different intensity. In contrast to FIG. 11C, fraction 12 contained a blip with an intensity of about 628 (no protein Z).(0223] SEC-Jess™ remained sensitive even when the antibody bound non-specific serum proteins.

[0224] FIGs. 13A-E show SEC-Jess™ analysis of an in vivo expressed fusion protein Z from BALB / c mouse serum sample demonstrating that BALB / c in vivo expressed fusion protein Z eluted in the same fractions as the expected in vitro expressed protein. FIG. 13A shows a gel view of a CE western demonstrating the presence of the protein Z in SEC fractions 14-19, comprising the monomeric protein Z. FIG. 13B shows a CE western electropherogram of the BALB / c mouse serum sample showing multiple peaks identified as the fusion protein Z and non-specific serum proteins. BALB / c mouse was administered with a mRNA encoding a fusion protein Z. FIG. 13C shows CE western electropherograms of SEC fractions 12, 16, and 17 from the serum sample comprising the protein Z and demonstrating that SEC fractions 16, and 17 contained the same single protein peak, but with different intensity. In contrast to FIG. 11C, fraction 12 contained a blip with an intensity of about 298 (no protein Z). FIG.13D shows a CE western electropherogram of SEC fraction 19 from the serum sample demonstrating the presence of non-specific binding of serum proteins to the antibody. FIG. 13E shows a CE western electropherogram of a serum sample from a naive mouse that was not administered with the fusion protein Z mRNA.102251 FIGs. 14A-C show SEC- Jess™ analysis of a naive BALB / c serum sample demonstrating that identified backgrounds peaks in FIG. 13, were triggered by non-specific binding of the BALB / s mouse immunoglobulins (Ig) and non-specific serum proteins to the antibody used to identify the fusion protein Z. Specifically, the HRP conjugated antibody used in the CE western crossed-reacted with the mouse Ig in the absence of specific human Fc. FIG. 14A shows a gel view of a CE western demonstrating the presence of background proteins in SEC fractions 17-22. FIG. 14B shows a CE western electropherogram of the naive BALB / c mouse serum sample showing multiple peaks identified as the mouse immunoglobulin and non-specific serum proteins. FIG. 14C shows CE western electropherograms of SEC fractions 18, 20, and 21 from the serum sample.

[0226] FIG. 15 shows a CE western electropherogram of non-specific binding of the HRP conjugated antibody to a murine Ig was enhanced in the absence of a target (human) antibody; and demonstrating that the concentration of a target protein should be higher than the limit of the background murine Ig detection (e.g., higher than 2ug / mL) to significantly reduce non-specific background serum proteins.

[0227] Single-antibody CE western blot method using a Horseradish peroxidase (HRP) conjugated anti-human secondary antibody produced a strong single signal and virtually no non-specific background

[0228] FIG. 16A-C show schematics illustrating the mechanism of the single antibody western method using CE western described herein, where a secondary antibody is used to bind to serum antibodies compared to a canonical western (FIG. 16B vs FIG. 16C). In particular, a secondary antibody is usually used in a canonical western blot to target a constant region or a Fc region of primary antibody (FIG. 16B). However, one aspect of the present disclosure describes a single antibody CE western where the secondary antibody directly binds a fusion or chimeric protein (FIG. 16A top) comprising the constant region or a Fc region of an antibody (FIG. 16C), thereby bypassing the requirement of a primaryantibody in the CE western. FIG. 16A shows schematics of exemplary in vivo produced proteins that can be assessed using the method disclosed herein (e.g., a whole monomeric or multi-specific antibody or fragment thereof or fusion or chimeric protein comprising a fragment of an antibody).|0229| FIGs. 17A-D show results from a single-antibody CE western blot method illustrating that the binding of an anti-human secondary antibody produced a single dominant signal (z.e., strong signals) by CE western, and showed virtually no non-specific background (FIGs. 17A- B and D). FIG. 17A shows a schematic of the single-antibody CE western blot method. FIG. 17B shows that binding of an anti-human HRP conjugated secondary antibody to a biological sample comprising a chimeric Fc fusion protein (FIG. 16A top) produced a strong signal with no non-specific background. FIG. 17D shows that binding of an anti-human HRP conjugated secondary antibody to a purified chimeric Fc fusion protein produced a strong signal with no non-specific background. FIG. 17C shows that the anti-human HRP- secondary antibody produced no signal when added to a biological sample from a mouse that did not express the chimeric Fc fusion protein (z.e., naive mouse serum).

[0230] FIGs. 18A-D show results from a dual-antibody (canonical) CE western blot method using a biological sample comprising a BALB / c serum and illustrating that the primary antibody or the secondary antibody cross-reacted with non-specific serum proteins. FIG. 18A shows a schematic of the dual-antibody CE western blot method. FIG. 18C shows CE western results of a serum sample from naive mouse illustrating that the antibodies bound to the mouse immunoglobulin, and non-specific proteins (e.g., albumin or other serum protein). FIG. 18B shows CE western results of a serum sample from a mouse expressing a target chimeric Fc fusion protein (treated mouse) illustrating that the antibodies strongly bound to the target chimeric fusion protein, but they also bound to the mouse immunoglobulin, and non-specific proteins. Nonetheless, the dominant peak in the CE electropherogram identified the target chimeric Fc fusion protein. FIG. 18D shows CE western results of a purified target chimeric Fc fusion protein illustrating that the antibodies selectively bound to the target chimeric Fc fusion protein.[02311 FIGs. 19A-C show results from a dual-antibody (canonical) CE western blot method similar to FIG. 18 using a different primary antibody and / or secondary antibody and illustrating that a dual-antibody CE western blot method required the availability of a goodantibody pair to obtain results with virtually no non-specific background. FIG. 19A shows CE western results of a serum sample from a mouse expressing a target chimeric Fc fusion protein (treated mouse). FIG. 19B shows CE western results of a serum sample from naive mouse. FIG. 19C shows CE western results of a purified target chimeric Fc fusion protein.EQUIVALENTS

[0232] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0233] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.INCORPORATION BY REFERENCE

[0234] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A method for assessing product related impurities, in a biological sample comprising an in vivo produced protein and / or aggregates thereof, the method comprising:(a) fractionating the biological sample from a subject expressing the in vivo produced protein and / or aggregates thereof using a size exclusion chromatography (SEC);(b) collecting eluate fractions of the in vivo produced protein;(c) electrophoretically separating the proteins from the SEC eluate fractions using an automated capillary electrophoresis (CE) western; and(d) detecting the in vivo produced protein and / or aggregates thereof in the SEC eluate fractions using fluorescent detection or chemiluminescent detection.

2. The method of claim 1, wherein detecting the in vivo produced protein and / or aggregates thereof comprises:(a) contacting the SEC eluate fractions with an antibody that binds to the in vivo produced protein;(b) incubating the SEC eluate fractions with the antibody for about 10 minutes to about 120 minutes; and(c) detecting the binding of the antibody to the SEC eluate fractions using fluorescent detection or chemiluminescent detection.

3. The method of claim 2, wherein prior to step (a)-(c), the SEC eluate fractions are electrophoretically separated by weight and immobilized on a solid support.

4. The method of claim 3, wherein the solid support is a capillary wall of a microfluidic device.

5. The method of claim 1, wherein the automated capillary electrophoresis (CE) western is a closed-loop automated capillary-based immunoassay system.

6. The method of claim 1, wherein the SEC eluate fractions and the antibody are incubated for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes,about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes.

7. The method of claim 1, wherein the detecting the binding of the antibody to the in vivo produced protein comprises a dual antibody CE western or a single antibody CE western.

8. The method of claim 7, wherein the dual antibody CE western comprises:(a) a primary antibody that targets the in vivo produced protein or aggregates thereof;(b) a secondary antibody that targets the primary antibody, wherein the secondary antibody is operably linked to a chemiluminescent or a fluorescent molecule; and(c) a chemiluminescent or a fluorescent molecule substrate.

9. The method of claim 7, wherein the single antibody western comprises:(a) a secondary antibody that targets the in vivo produced protein or aggregate thereof, wherein the secondary antibody is operably linked to a chemiluminescent or a fluorescent molecule; and(b) a chemiluminescent or a fluorescent molecule substrate.

10. The method of claim 9, wherein the in vivo produced protein or aggregates thereof comprises a constant domain or a Fc domain of an antibody.

11. The method of claim 10, wherein the secondary antibody selectively binds to the constant domain or the Fc domain of the in vivo produced protein.

12. The method of claim 1, wherein the detecting the in vivo produced protein and / or aggregates thereof in the SEC fractions comprises chemiluminescent detection.

13. The method of claim 1, wherein the method detects a nanogram or a picogram (pg) of the in vivo produced protein and aggregates thereof in the biological sample.

14. The method of claim 1, wherein the method detects at least about 0.0001 ng- 1. Ong, at least about O.OOOlng-O. lng, at least about O. lpg-l.Opg, at least about 0.5pg-2.5, at least about O.lpg-lOpg, at least about 5pg-50pg, at least about lOpg-lOOpg, or more than lOOpg of the in vivo produced protein or aggregates thereof in the biological sample.

15. The method of claim 1, wherein the method detects at least about O.lpg, at least about 0.2pg, at least about 0.3 pg, at least about 0.4pg, at least about 0.5 pg, at least about 0.6pg, at least about 0.7pg, at least about 0.8pg, at least about 0.9pg, at least about l.Opg, at least about l.lpg, at least about 1.5pg, at least about 5.0pg, at least about lOpg, at least about 20pg, at least about 30pg, at least about 40pg, at least about 50pg, at least about 60pg, at least about 70pg, at least about 80pg, at least about 90pg, or at least about lOOpg of the in vivo produced protein or aggregates thereof in the biological sample.

16. The method of claim 1, wherein the SEC comprises a resin selected from the group consisting of Superdex®, Bio-Sep™, TSKgel™, Sephacryl™, Superose™, and Sephadex™.17 The method of claim 1, wherein the SEC has a separation range of about 0.1 kilo Dalton (KDa) to lOOKDa, about 1 KDa to about 150 KDa, about 5 KDa to about 100 KDa, about 5 KDa to about 700 KDa, about 5 KDa to about 1000 KDa, about 5 KDa to about 5,000 KDa, or about 10 KDa to about 10,000 KDa.

18. The method of claim 17, wherein the SEC separation range is under a native or a denatured condition.

19. The method of claim 1, wherein the biological sample is fractionated at a flow rate of about 0.01 ml / min, about 0.05 ml / min, about 0.075 ml / min, about 0.1 ml / min, about 0.15ml / min, about 0.2ml / min, about 0.3ml / min, about 0.4 ml / min, about 0.5 ml / min, about 0.6 ml / min, about 0.7 ml / min, about 0.8 ml / min, about 0.9 ml / min, about 1.0 ml / min, about 1.5 ml / min, or about 2.0ml / min or more.

20. The method of claim 1, wherein the biological sample is fractionated at a flow rate of about 0.5 ml / min.

21. The method of claim 1, wherein an aggregate of the in vivo expressed protein:(a) comprises a dimer, a trimer, a tetramer, a multimer, or a higher order oligomer of the in vivo expressed protein; and / or(b) is an homo-aggregate or a hetero-aggregate.

22. The method of claim 1, wherein prior to the SEC fractionation, the biological sample is:(a) not purified;(b) not pre-treated with a process selected from the group consisting of affinity chromatography, low pH viral inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration, ultrafiltration, and tangential flow filtration; or(c) not purified using a Protein A- or a Protein G-based affinity chromatography.

23. The method of claim 1, wherein the in vivo produced protein is generated by administering a polynucleotide encoding the in vivo produced protein to the subject.

24. The method of claim 23, wherein:(a) the polynucleotide is a DNA, or an RNA;(b) the polynucleotide is an mRNA; or(c) the polynucleotide further comprises a modified nucleotide, a cap structure, a poly A tail, a 5' untranslated region, and / or a 3' untranslated region.

25. The method of claim 23, wherein the subject is administered about Ipg to about 50 pg, about 10 pg to about 50 pg, about 5 pg to about 25 pg, about 1 pg to about 15 pg, about 1 pg to about 10 pg, about 5 pg to about 15 pg, about 6 pg to about 15 pg, or about 5 pg to about 20 pg of a mRNA encoding the inv vivo expressed protein.

26. The method of claim 1, wherein the biological sample comprises:(a) at least about 0.1 pg / ml to about 50 pg / ml, at least about 0.1 pg / ml to about25 pg / ml, at least about 0.5 pg / ml to about 25 pg / ml, at least about 0.1 pg / ml to about 15 pg / ml, at least about 0.1 pg / ml to about 10 pg / ml, or at least about 0.1 pg / ml to about 10 pg / ml of the in vivo produced protein; or(b) at least about 0.1 pg / ml, at least about 0.2 pg / ml, at least about 0.3 pg / ml, at least about 0.4 pg / ml, at least about 0.5 pg / ml, at least about 0.6 pg / ml, at least about 0.7 pg / ml, at least about 0.8 pg / ml, at least about 0.9 pg / ml, at least about 1.0 pg / ml, at least about 1.25 pg / ml, at least about 1.5 pg / ml, at least about 2.0 pg / ml, at least about 2.25 pg / ml, about 2.3 pg / ml, at least about 2.5 pg / ml, at least about 2.75 pg / ml, or at least about 3.0 pg / ml of the in vivo produced protein.

27. The method of claim 23, wherein the polynucleotide comprises a first and a second nucleic acids, and optionally wherein the first and the second nucleic acids encode different subunits or domains of the in vivo produced protein.

28. The method of claim 27, wherein the subject is administered a predetermined molar ratio of the first and the second nucleic acids; and wherein when administered to the subject, the first and the second nucleic acids generate a fully assembled protein.

29. The method of claim 23, wherein the subject is:(a) a cell, and administering comprises transfecting the cell with the polynucleotide; or(b) a whole organism selected from a mammal, a non-human primate, or a human.

30. A method of determining an in vivo produced antibody quality and / or quantity, the method comprising obtaining a biological sample comprising the in vivo produced antibody from a subject and determining the antibody aggregation using the method of claim 1.

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