LARGE MOLECULE UNSPECIFIC CLEARANCE ASSAY
20230194547 · 2023-06-22
Assignee
Inventors
- Anna-Lena BOLENDER (Penzberg, DE)
- Martina DUSCHMALE (Bettingen, CH)
- Lisa EBELER (Munich, DE)
- Johannes FRAIDLING (Wessobrunn, DE)
- Thomas KRAFT (Munich, DE)
Cpc classification
International classification
Abstract
Herein is reported a method for determining non-specific clearance of an antibody comprising the steps of incubating the antibody, which is conjugated to a pH-sensitive fluorescent dye, with primary human endothelial cells, and determining the fluorescence intensity of the primary human endothelial cells, whereby an increase of the fluorescence intensity of the primary human endothelial cells above background level is indicative for non-specific clearance of the antibody.
Claims
1. A method for determining non-specific clearance of an antibody comprising the following steps: a) incubating the antibody, which is conjugated to a pH-sensitive fluorescent dye, with primary human endothelial cells, and b) determining the fluorescence intensity of the primary human endothelial cells of step a), whereby non-specific clearance of the antibody is detected if the fluorescence intensity of the primary human endothelial cells determined in step b) is higher than the fluorescence intensity of the primary human endothelial cells determined in the absence of the antibody.
2. The method according to claim 1 further comprising the following step: c) determining the fluorescence intensity of the primary human endothelial cells not incubated with/in the absence of the antibody.
3. The method according to any one of claims 1 to 2, wherein the primary human endothelial cells are washed prior to the determination of the fluorescence intensity.
4. The method according to any one of claims 1 to 3, wherein the dye has a fluorescence intensity change between a physiological pH of about 7 and an acidic pH in the range of pH 4 to 5 of about 10-fold determined at the same concentration of the dye and with the same excitation wavelength.
5. The method according to any one of claims 1 to 4, wherein the dye is pHAb of Formula I.
6. The method according to any one of claims 1 to 5, wherein the dye is conjugated to the antibody at residue 297 (numbering according to Kabat).
7. The method according to any one of claims 1 to 6, wherein the dye is conjugated to the antibody via a sulfo DBCO-PEG4-amine linker of Formula II.
8. The method according to any one of claims 1 to 7, wherein the dye is conjugated to a linker and the linker is conjugated to the antibody and has a structure of Formula III.
9. The method according to any one of claims 1 to 8, wherein the fluorescence intensity is determined by FACS by determining the shift of the fluorescence maximum.
10. The method according to any one of claims 1 to 9, wherein the fluorescence intensity is the geometric mean fluorescence intensity determined by FACS.
11. The method according to any one of claims 1 to 10, wherein the primary human endothelial cells are primary human liver endothelial cells.
12. The method according to any one of claims 1 to 11, wherein the incubating is for up to 4 hours.
13. The method according to any one of claims 1 to 12, wherein the incubating is at least for 0.5 hours.
14. The method according to any one of claims 1 to 13, wherein the antibody has an Fc-region of the human IgG1 or IgG4 subclass.
15. The method according to any one of claims 1 to 14, wherein the antibody is a bispecific antibody.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
I. Materials and Methods
Antibodies
[0177] The reference antibodies used in the experiments were an anti-pTau antibody that has the heavy chain amino acid sequence of SEQ ID NO: 01 and the light chain amino acid sequence of SEQ ID NO: 02 and an anti-Her 3 antibody that has the heavy chain amino acid sequence of SEQ ID NO: 03 and the light chain amino acid sequence of SEQ ID NO: 04.
[0178] Synthetic genes were produced at Geneart (Life technologies GmbH, Carlsbad, Calif., USA).
[0179] The monoclonal antibodies used herein were transiently expressed in HEK293 cells (see below) and purification was performed by protein A chromatography using standard procedures (see below).
[0180] The biochemical characterization included size exclusion chromatography (Waters BioSuite™ 250 7.8×300 mm, eluent: 200 mM KH.sub.2PO.sub.4, 250 mM KCl, pH 7.0) and analysis of the molecular weight distribution using the BioAnalyzer 2100 (Agilent technologies, Santa Clara, Calif., USA).
Expression Plasmids
[0181] For the expression of the above described antibodies, variants of expression plasmids for transient expression (e.g. in HEK293-F) cells based either on a cDNA organization with or without a CMV-Intron A promoter or on a genomic organization with a CMV promoter were applied.
[0182] Beside the antibody expression cassette the plasmids contained: [0183] an origin of replication which allows replication of this plasmid in E. coli, [0184] a β-lactamase gene which confers ampicillin resistance in E. coli., and [0185] the dihydrofolate reductase gene from Mus musculus as a selectable marker in eukaryotic cells.
[0186] The transcription unit of the antibody gene was composed of the following elements: [0187] unique restriction site(s) at the 5′ end [0188] the immediate early enhancer and promoter from the human cytomegalovirus, [0189] followed by the Intron A sequence in the case of the cDNA organization, [0190] a 5′-untranslated region of a human antibody gene, [0191] an immunoglobulin heavy chain signal sequence, [0192] the human antibody chain either as cDNA or as genomic organization with the immunoglobulin exon-intron organization [0193] a 3′ non-translated region with a polyadenylation signal sequence, and [0194] unique restriction site(s) at the 3′ end.
[0195] The fusion genes comprising the antibody chains were generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective plasmids. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfections larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).
Cell Culture Techniques
[0196] Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc.
Transient Transfections in HEK293-F System
[0197] The antibodies were generated by transient transfection with the respective plasmids (e.g. encoding the heavy chain, as well as the corresponding light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) were transfected with a mix of the respective expression plasmids and 293Fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seeded at a density of 1*10.sup.6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO.sub.2. The day after the cells were transfected at a cell density of ca. 1.5*10.sup.6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA (1 μg/mL) encoding the heavy chain, respectively and the corresponding light chain in an equimolar ratio and B) 20 ml Opti-MEM+1.2 mL 293 fectin or fectin (2 μL/mL). According to the glucose consumption glucose solution was added during the course of the fermentation. The supernatant containing the secreted antibody was harvested after 5-10 days and antibodies were either directly purified from the supernatant or the supernatant was frozen and stored. Some of the antibodies that have been produced accordingly:
TABLE-US-00002 antibody format Adalimumab IgG1 anti-human alpha synuclein antibody IgG1 Avelumab IgG1 TCB-1 (reference TCB) TCB CD-Ag-1/CD-AG-2 bispecific antibody CrossMab TCB-2 TCB wild-type IgG with sortase tag IgG1 non-binding antibody with sortase tag IgG1 TCB-3 TCB TCB-4 TCB anti-Her3 antibody IgG1 Ixekizumab IgG4 TCB-5 TCB TCB-6 TCB Motavizumab wt IgG1 Motavizumab-YTE IgG1 Nivolumab IgG1 Ofatumumab IgG1 Palivizumab IgG1 anti-human p(422)Tau antibody IgG1 Reslizumab IgG4 Ustekinumab IgG1 Vedolizumab IgG1
Purification
[0198] Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl-Sepharose (GE Healthcare, Sweden) and
[0199] Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.
[0200] Briefly, sterile filtered cell culture supernatants were captured on a MabSelectSuRe resin equilibrated with PBS buffer (10 mMNa.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate at pH 3.0. The eluted antibody fractions were pooled and neutralized with 2 M Tris, pH 9.0. The antibody pools were prepared for hydrophobic interaction chromatography by adding 1.6 M ammonium sulfate solution to a final concentration of 0.8 M ammonium sulfate and the pH adjusted to pH 5.0 using acetic acid. After equilibration of the butyl-Sepharose resin with 35 mM sodium acetate, 0.8 M ammonium sulfate, pH 5.0, the antibodies were applied to the resin, washed with equilibration buffer and eluted with a linear gradient to 35 mM sodium acetate pH 5.0. The antibody containing fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. The antibody containing fractions were pooled, concentrated to the required concentration using Vivaspin ultrafiltration devices (Sartorius Stedim Biotech S.A., France) and stored at −80° C.
[0201] Purity and antibody integrity were analyzed after each purification step by CE-SDS using microfluidic Labchip technology (Caliper Life Science, USA). Five μl of protein solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer's instructions and analyzed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software.
Mice
[0202] B6.Cg-Fcgrt.sup.tm/Dcr Tg(FCGRT)276Dcr mice deficient in mouse FcRn α-chain gene, but hemizygous transgenic for a human FcRn α-chain gene (muFcRn−/− huFcRn tg +/−, line 276) were used for the pharmacokinetic studies. Mouse husbandry was carried out under specific pathogen free conditions. Mice were obtained from the Jackson Laboratory (Bar Harbor, Me., USA) (female, age 4-10 weeks, weight 17-22 g at time of dosing). All animal experiments were approved by the Government of Upper Bavaria, Germany (permit number 55.2-1-54-2532.2-28-10) and performed in an AAALAC accredited animal facility according to the European Union Normative for Care and Use of Experimental Animals. The animals were housed in standard cages and had free access to food and water during the whole study period.
Pharmacokinetic Studies
[0203] A single dose of antibody was injected i.v. via the lateral tail vein at a dose level of 5 mg/kg. The mice were divided into 3 groups of 6 mice each to cover 9 serum collection time points in total (at 0.08, 2, 8, 24, 48, 168, 336, 504 and 672 hours post dose). Each mouse was subjected twice to retro-orbital bleeding, performed under light anesthesia with Isoflurane™ (CP-Pharma GmbH, Burgdorf, Germany); a third blood sample was collected at the time of euthanasia. Blood was collected into serum tubes (Microvette 500Z-Gel, Sarstedt, Nümbrecht, Germany). After 2 h incubation, samples were centrifuged for 3 min at 9.300 g to obtain serum. After centrifugation, serum samples were stored frozen at −20° C. until analysis.
Determination of Human Antibody Serum Concentrations
[0204] Concentrations of antibodies in murine serum were determined by specific enzyme-linked immunoassays. Biotinylated capture reagent specific for each of the antibodies and digoxygenin-labeled anti-human-Fc mouse monoclonal antibody (Roche Diagnostics, Penzberg, Germany) were used for capturing and detection, respectively. Streptavidin-coated microtiter plates (Roche Diagnostics, Penzberg, Germany) were coated with biotinylated capture reagent diluted in assay buffer (Roche Diagnostics, Penzberg, Germany) for 1 h. After washing, serum samples were added at various dilutions followed by another incubation step for 1 h. After repeated washings, bound antibodies were detected by subsequent incubation with detection antibody, followed by an anti-digoxygenin antibody conjugated to horseradish peroxidase (HRP; Roche Diagnostics, Penzberg, Germany). ABTS (2,2′Azino-di[3-ethylbenzthiazoline sulfonate]; Roche Diagnostics, Germany) was used as HRP substrate to form a colored reaction product. Absorbance of the resulting reaction product was read at 405 nm with a reference wavelength at 490 nm using a Tecan sunrise plate reader (Mannedorf, Switzerland).
[0205] All serum samples, positive and negative control samples were analyzed in duplicates and calibrated against reference standard.
PK Analysis
[0206] The pharmacokinetic parameters were calculated by non-compartmental analysis using WinNonlin™ 1.1.1 (Pharsight, CA, USA).
[0207] Briefly, area under the curve (AUC.sub.0-inf) values were calculated by logarithmic trapezoidal method due to non-linear decrease of the antibodies and extrapolated to infinity using the apparent terminal rate constant λz, with extrapolation from the observed concentration at the last time point.
[0208] Plasma clearance was calculated as Dose rate (D) divided by AUC.sub.0-inf. The apparent terminal half-life (T1/2) was derived from the equation T1/2=ln2/λz.
Example 1
Cynomolgus SDPK Studies
[0209] The pharmacokinetics of the test compounds was determined in cynomolgus monkeys following single intravenous administration at dose levels ranging from 0.3 mg/kg to 150 mg/kg. Serial blood samples were collected from the monkeys over several weeks and serum/plasma was prepared from the collected blood samples. Serum/plasma levels of test compounds were determined by ELISA. In case of linear pharmacokinetics pharmacokinetic parameter were determined by standard non-compartmental methods. Clearance was calculated according to following formula:
Clearance=Dose/Area under concentration-time curve
[0210] In cases of non-linear pharmacokinetics the linear fraction of the clearance was determined via following alternative methods: Either clearance values were estimated following IV administration at high dose levels, at which additional non-linear clearance pathways are virtually saturated. Alternatively, PK models comprising a linear and a non-linear, saturable clearance term were established. In these cases, the model-determined linear clearance fraction was used for correlations.
Example 2
Preparation of FcRn Affinity Column
Expression of FcRn in HEK293 Cells
[0211] FcRn was transiently expressed by transfection of HEK293 cells with two plasmids containing the coding sequence of FcRn and of beta-2-microglobulin. The transfected cells were cultured in shaker flasks at 36.5° C., 120 rpm (shaker amplitude 5 cm), 80% humidity and 7% CO.sub.2. The cells were diluted every 2-3 days to a density of 3 to 4*10.sup.5 cells/ml.
[0212] For transient expression, a 14 l stainless steel bioreactor was started with a culture volume of 81 at 36.5° C., pH 7.0±0.2, pO.sub.2 35% (gassing with N.sub.2 and air, total gas flow 200 ml min.sup.−1) and a stirrer speed of 100-400 rpm. When the cell density reached 20*10.sup.5 cells/ml, 10 mg plasmid DNA (equimolar amounts of both plasmids) was diluted in 400 ml Opti-MEM (Invitrogen). 20 ml of 293fectin (Invitrogen) was added to this mixture, which was then incubated for 15 minutes at room temperature and subsequently transferred into the fermenter. From the next day on, the cells were supplied with nutrients in continuous mode: a feed solution was added at a rate of 500 ml per day and glucose as needed to keep the level above 2 g/l. The supernatant was harvested 7 days after transfection using a swing head centrifuge with 11 buckets at 4000 rpm for 90 minutes. The supernatant (13 L) was cleared by a Sartobran P filter (0.45 μm+0.2 μm, Sartorius) and the FcRn beta-2-microglobulin complex was purified therefrom.
Biotinylation of Neonatal Fc Receptor
[0213] 3 mg FcRn beta-2-microglobulin complex were solved/diluted in 5.3 mL 20 mM sodium dihydrogenphosphate buffer containing 150 mM sodium chloride and added to 250 μL PBS and 1 tablet complete protease inhibitor (complete ULTRA Tablets, Roche Diagnostics GmbH). FcRn was biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions (Bulk BIRA, Avidity LLC). The biotinylation reaction was done at room temperature overnight.
[0214] The biotinylated FcRn was dialyzed against 20 mM MES buffer comprising 140 mM NaCl, pH 5.5 (buffer A) at 4° C. overnight to remove excess of biotin.
Coupling to Streptavidin Sepharose
[0215] For coupling to streptavidin Sepharose, 1 mL streptavidin Sepharose (GE Healthcare, United Kingdom) was added to the biotinylated and dialyzed FcRn beta-2-microglobulin complex and incubated at 4° C. overnight. The FcRn beta-2-microglobulin complex derivatized Sepharose was filled a 4.6 mm×50 mm chromatographic column (Repligen). The column was stored in 80% buffer A and 20% buffer B (20 mM Tris(hydroxymethyl)aminomethane pH 8.8, 140 mM NaCl).
Example 3
Chromatography Using FcRn Affinity Column and pH Gradient
Conditions:
[0216] column dimensions: 50 mm×4.6 mm [0217] loading: 30 μg sample [0218] buffer A: 20 mM MES, with 140 mM NaCl, adjusted to pH to pH 5.5 [0219] buffer B: 20 mM Tris/HCl, with 140 mM NaCl, adjusted to pH 8.8
[0220] 30 μg of samples were applied onto the FcRn affinity column equilibrated with buffer A. After a washing step of 10 minutes in 20% buffer B at a flow rate of 0.5 mL/min, elution was performed with a linear gradient from 20% to 70% buffer B over 70 minutes. The UV light absorption at a wavelength of 280 nm was used for detection. The column was regenerated for 10 minutes using 20% buffer B after each run.
[0221] For the calculation of relative retention times, a standard sample (anti-Her3 antibody (SEQ ID NO: 03 and 04), oxidized for 18 hours with 0.02% hydrogen peroxide according to Bertoletti-Ciarlet, A., et al. (Mol. Immunol. 46 (2009) 1878-1882) was run at the beginning of a sequence and after each 10 sample injections.
[0222] Briefly, the antibody (at 9 mg/mL) in 10 mM sodium phosphate pH 7.0 was mixed with H.sub.2O.sub.2 to a final concentration of 0.02% and incubated at room temperature for 18 hours. To quench the reaction the samples were thoroughly dialyzed into pre-cooled 10 mM sodium acetate buffer pH 5.0.
Example 4
Chromatography Using Heparin Affinity Column and pH Gradient
Conditions:
[0223] column dimensions: 50 mm×5.0 mm
loading: 20-50 μg sample
buffer A: 50 mM TRIS pH 7.4
buffer B: 50 mM TRIS pH 7.4, 1000 mM NaCl
[0224] 20 to 50 μg of protein samples in low-salt buffer (<25 mM ionic strength) were applied to a TSKgel Heparin-5PW Glass column, 5.0×50 mm (Tosoh Bioscience, Tokyo/Japan), which was pre-equilibrated with buffer A at room temperature. Elution was performed with a linear gradient from 0-100% buffer B over 32 minutes at a flow rate of 0.8 mg/mL. The UV light absorption at a wavelength of 280 nm was used for detection.
Example 5
Examination of Antibody Internalization
[0225] The method is based on a previously reported method that detects the internalized antibody using homogeneous fluorescence imaging of a pH-activated probe, which enables maximum fluorescence signals of antibody under intracellular acidic conditions without any fluorescence signals detected in the extracellular environments (Li, Z., et al., Int. Immunopharm. 62 (2018) 299-308).
[0226] Briefly, the respective antibody was conjugated with pHAb Amine Reactive dye and then diluted with cell culture medium. Meanwhile, cells were seeded into a 6-well plate (1×10.sup.5 cells per well), and 100 μL of medium containing pHAb Amine Reactive dyes-conjugated antibody (final concentration of 10 μg/mL) was added into each well. After incubation at 37° C., the internalization of the antibody was measured by flow cytometry at different time points (0 h, 1.5 h, 2 h, 4 h, 5.5 h, and/or 24 h).
Example 6
Antibody Labeling
[0227] Antibodies were labeled using the SiteClick™ Antibody Azido Modification Kit (Thermo Fisher Scientific) according to the manufactures instructions. Briefly, N-linked galactose residues of the Fc-region were removed by β-galactosidase and replaced by an azide-containing galactose (GalNaz) via β-1,4-galactosyltransferase (GalT). This azide modification enables a copper-free conjugation of sDIBO-modified dyes. The pH-sensitive amine-reactive dye (523 nm) was purchased from Promega and coupled to a sulfo DBCO PEG4 amine. Antibodies were labeled with a molar dye excess of 2. Excess dye was removed using the Amicon® Ultra-2 Centrifugal Filter with a MWCO of 50 kDa (EMD Millipore, #UFC200324) and antibodies were re-buffered in 20 mM histidine buffer (pH 5.5). The concentration of the labeled antibodies [1] as well as the dye to antibody ratio (DAR) [2] was determined with a Nanodrop spectrometer at 280 nm (A.sub.280nm) and 532 nm (A532 nm).
CAB=[A.sub.280nm−[A.sub.280nm*CFDye]]/ε.sub.mAb [1]
DAR=[A.sub.532nm*MW.sub.mAb]/[c.sub.mAb*ε.sub.Dye] [2]
εDye=47225
CF.sub.Dye=0.36
Example 7
Cell Maintenance and Preparation
[0228] Cryopreserved human liver-derived endothelial Cells (HLEC-P2) were purchased from Lonza (Lonza, #HLECP2). Cell were maintained in EBMT™-2 Endothelial Cell Growth Basal Medium-2 (Lonza, #CC-3156) supplemented with EGM™-2 MV Microvascular Endothelial Cell Growth Medium SingleQuots™ (Lonza, #CC-4176). Five days prior antibody treatment, cells were plated onto collagen I coated 100 mm culture dishes (Corning® BioCoat™, #354450) and two days prior treatment sub-cultured into collagen I coated 96-well plates (Corning® BioCoat™ #354407) at a cell density of 4×10.sup.4 cells/well to allow adherence for 48 hours. Medium was changed after 24 hours and cells were kept at 37° C. and 5% CO.sub.2.
[0229] On the day of the experiment, cells were washed twice with 200 μl pre-warmed medium and subsequently incubated with 400 nM labeled antibody or 20 mM histidine buffer (pH 5.5) as negative control in medium. After 2 and 4 hours, the antibody solution was removed and cells were washed once with 200 μl ice-cold DPBS (without Mg and Ca) and detached by applying 100 μl Trypsin (with EDTA) for 2.5 minutes at 37° C. Trypsin was inactivated by the addition of 100 μl FACS Buffer (20% FCS, 1 mM EDTA in DPBS).
Example 8
Flow Cytometry and Pharmacokinetic Analysis
[0230] The mean fluorescent intensity (MFI, more specifically the geometric mean (geo-mean)) of the internalized antibodies was acquired using the MACSQuant® Analyzer 10 (Miltenyi Biotec) equipped with a laser to excite at 488 nm and a filter to collect emitted light at 585 nm/540 nm. The exact same conditions, gains and gates were used for both times points (2 hours and 4 hours). Data extraction was performed using the FloJo_V10 software. Values of the negative control was subtracted from all geo-mean values followed by normalization to the DAR. The normalized geo-mean values from each antibody were plotted as linear regression curve using GraphPad Prism to extract the slope (Geo Mean MFI/min for 120 and 240 min). Two standard antibodies were selected to normalize the slopes: Motavizumab-YTE was set to 0 and a TCB was set to 1. The final slopes were plotted against published in vivo human, cynomolgus and hFcRn Tg32+/+ mouse clearance values using the TIBCO Spotfire software.
Example 9
Quality Control
[0231] Biophysical binding properties are key determinants affecting clearance mechanisms. Therefore, it is important to assess, whether the binding affinities of the antibodies changed during the labeling process. Heparin chromatography and neonatal Fc-receptor binding has been previously shown to allow prediction of antibody clearance in vitro (Kraft, T. E., et. al., MABS 12 (2020) e1683432). Herein, this method was used to account for potential aberrant binding properties introduced by the click label. Details for the methods are provided in Examples 3 and 4.
[0232] To confirm the absence of unbound dye and to verify the concentration measured at the spectrometer, a size exclusion chromatography of the labeled antibodies was performed. Samples were separated using a BioSuite Diol (OH) column (Waters, 186002165) with a potassium dihydrogen phosphate buffer (pH 6.2) as the mobile phase at a flow rate of 0.5 ml/min. Detectors at 280 nm and 532 nm were used to quantify and analyze the labeled antibodies. The area under the curve (AUC) at 280 nm and 532 nm was extracted to calculate the concentration. The geo-mean of the AUC from all antibodies was computed and the deviation from each antibody to this geo-mean was identified. For an antibody to be reliable within the assay according to the invention, the difference from the geo-mean was expected to be below 15%.