Reagents and Antibodies Used

The drug surrogate IgG1 for this study was produced in-house. The polyclonal ADA surrogate pAb<CDR> was generated by immunization of rabbits in-house (13). Sodium chloride (1.06404) was obtained from Merck KGaA (Germany). Histidine was ordered from Sigma (H6034 and 56190). Antibodies and buffers used to generate the in vivo dosing solutions were tested and approved for endotoxin levels.

3H-Labeling of Drug

The drug surrogate was transferred into a Slide-A-Lyzer Cassette (G2 Dialysis Cassettes, 10 K MWCO, 3 mL, Thermo Fisher Scientific, 87730) and buffer exchanged into DPBS (14 mg, 97.9 nmol, in 2.8 mL formulation buffer) (Thermo Fisher Scientific, 14190-094). The buffer was changed 4 times after 30 min and stored overnight at 6°C. The protein solution was warmed to 22°C; a protein concentration determination (Eppendorf BioSpectrometer®) resulted in 4.9 mg/mL. Twenty-five millicuries (925 MBq, 44.5 µg, 245 nmol) [3H]NSP (Pharmaron, Cardiff, Wales, UK) as a solution in toluene was transferred in portions into a 5-mL Eppendorf LoBind tube. Toluene was removed by an argon stream, and the solid residue was dissolved in 35 µL DMSO. A total of 2.8 mL of the protein solution was added, and the tube was shaken horizontally at 150 rpm for 30 min. The solution was transferred into a Slide-A-Lyzer Cassette and buffer exchanged into formulation buffer (20 mM L-histidine and L-histidine monohydrochloride monohydrate (Sigma, H-8000/H-5659), 140 mM sodium chloride (Sigma, 71376), pH 6.0). The buffer was changed 4 times after 30 min and stored overnight at 6°C. The solution was transferred to a 5-mL Eppendorf LoBind tube at 22°C and resulted in a protein concentration of 4.4 mg/mL in a volume of 2.9 mL. A total activity of 10.7 mCi (396 MBq) was obtained, resulting in a specific activity of 839 µCi/mg (31.0 MBq/mg). Radiolabeled antibody was analyzed using an Agilent 1200 series HPLC system (Santa Clara, CA, USA), equipped with β-radioactivity HPLC detector RAMONA* with internal liquid scintillator admixture (Elysia-raytest, Straubenhardt, Germany). Absorbance at 280 nm and 320 nm was used for detection and quantification. For size-exclusion chromatography (SEC), a TSKgel G3000 SWXL column (Tosoh Bioscience, Tokyo, Japan), 7.8 × 300 mm, 5 μm with 0.2 M potassium phosphate, 0.25 M potassium chloride, and pH 7.0 as the mobile phase at a flow rate of 0.5 mL/min, was used. The injection volume was 10 μL and the protein concentration 1 mg/mL. The target concentration of 1 mg/mL was set by adding the eluent to the protein stock solution. The radiochemical purity using SEC was 98.1%; the low molecular weight impurity was 1%.

Analytical FcRn affinity chromatography was carried out with an FcRn affinity column (Roche Custom Biotech, Mannheim, Germany), column volume of 0.5 mL containing 1.5 mg FcRn protein (14). The 45-min analytical method was applied with a flow rate of 0.5 mL/min under the following conditions: Eluent [A] was 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) (with 140 mM sodium chloride, pH 5.5) and eluent [B] was 20 mM tris(hydroxymethyl)aminomethane (Tris) (with 140 mM sodium chloride, pH 8.8). Starting with isocratic conditions of 20% [B] for 5 min, a linear gradient to 100% [B] followed over 35 min to keep 100% [B] for 5 min. The injection volume was 30 μL and the protein concentration 1 mg/mL. The target concentration of 1 mg/mL was set by adding eluent [A] to the protein stock solution. Liquid scintillation counting was accomplished using a HIDEX 300 SL (Mainz, Germany) and an ULTIMATE GOLD cocktail (PerkinElmer Inc., Waltham, MA, USA). A comparison of unlabeled with the tritium-labeled protein using FcRn affinity chromatography revealed no shift in retention times, which indicates that the label has no influence on FcRn affinity. The protein solution was sterile-filtered using a Millex-GV Durapore (PVDF) filter (Merck Millipore, Darmstadt, Germany), divided into two portions, and stored at − 80°C.

Generation and Characterization of Dosing Solutions

An endotoxin-free 20 mM histidine/HCl buffer (with 140 mM NaCl, pH 6) was used to generate the dosing solutions for the in vivo studies. For the control groups, 2 mg/mL drug was prepared. For the IC groups, a solution of 2 mg/mL drug and 3 mg/mL ADA was prepared and incubated for 1 h at 22°C on a shaker at 450 rpm before administration. Aliquots of the solutions were stored at − 80°C for further analysis by SEC using a Dionex Ultimate 3000 system like described previously (7, 10).

Study Design QWBA (Quantitative Whole-Body Autoradiography)

This study was performed at a contractor research organization. Tissue distribution and kinetics as well as blood distribution were investigated after i.v. administration of the radiolabeled drug to female rats. For this purpose, CRL:WI rats were dosed with 3H-drug (4 mg/kg) or 3H-drug complexed with pADA<CDR> (4 mg/kg and 6 mg/kg, respectively) (n = 1) and sacrificed 0.25 h after administration. The body of the animals was embedded as a whole in a frozen state. The concentration of radioactivity in tissues and organs was determined in whole-body sections of the animals by means of the QWBA technique. Just before the sacrifice, blood was taken from the sublingual vein under anesthesia (K3-EDTA). The aliquots were separated and used for the determination of total radioactivity in the blood by liquid scintillation counting (LSC) after solubilization using tissue solubilizer Solvable (PerkinElmer). Immediately, after blood sampling, the animals were sacrificed by an overdose of carbon dioxide.

QWBA: Preparation of Animal Sections

The preparation of the whole-body animal sections was performed using a cryostat microtome Cryo Macrocut Leica CM 3600 XP (Leica Instruments GmbH, Nussloch, Germany). After sacrifice, the animals were immediately frozen as a whole at approximately − 80°C in a mixture of hexane in solid carbon dioxide until the animals were completely frozen (approximately 0.5 h). Thereafter, the frozen animals were stored for at least 1 day in the deep freezer at approximately − 20°C for equilibration. The animals were then processed as soon as possible. The tail and limbs of the carcass were trimmed in a frozen state. The carcass was placed on a specimen frame. An aqueous solution of Methylan (Henkel, Düsseldorf; 125 g/7 L tap water) was poured over the carcass. The specimen frame containing the animal was put into a freezer (− 20°C for at least 24 h). Prior to sectioning, the embedded animal was allowed to equilibrate for approximately 1–2 h to the temperature of the cryostat (− 20 ± 2°C). The frozen block was cut in a cryomicrotome at a temperature of − 20 ± 2°C. Sections of 40 μm thickness were automatically sliced to the section of interest. Prior to the removal of the section for radioluminography, a piece of adhesive transparent tape (Scotch Tape 810; 3M, Neuss, Germany) was firmly placed onto the frozen surface of the carcass in order to adhere the cut section to the tape. Sections were taken at different levels through the embedded animal. About 6–7 levels were selected to cover all tissues. The sections were dehydrated in the microtome at (− 20 ± 2°C) for at least 48 h. The sections were then treated with talcum to cover the unoccupied sections of the adhesive tape. A series of 8 blood calibration standards with different concentrations of [3H]glucose plus one blank prepared with control blood was used. The concentrations of radioactivity covered a range from 10,000 to 30,000,000 dpm/g. The radioactivity of each blood calibration standard was determined after dissolving using a tissue solubilizer followed by LSC. The mean value of each standard was used to establish a calibration curve for the correlation of PSL (photo-stimulated luminescence) and dpm (radioactivity).

Biodistribution Study

All studies were conducted with the approval of the local veterinary authorities in strict adherence to the Swiss federal regulations on animal protection and to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Tissue, serum, urine, and feces distribution and kinetics were investigated after i.v. administration into the tail vein of the radiolabeled drug to female Wistar rats (CRL:WI; age approximately 8 weeks old and body weight of 230 g). For this purpose, rats were dosed with 3H-drug (4 mg/kg) or 3H-drug complexed with pADA<CDR> (4 mg/kg and 6 mg/kg, respectively) (n = 5). Blood samples were taken from the sublingual vein under anesthesia after 0.25, 1, 3, 7, 24, 48, 72, and 168 h (n = 2 per time point). For organ collection, animals were sacrificed 0.25, 3, 7, and 168 h after administration (n = 1 per time point). The liver, lung, muscle, kidney, spleen, skin, heart, bone marrow, and pancreas were collected. Urine and feces from all rats were collected in 2 blocks, from 0 to 24 h and 24 to 48 h.

Determination of Radioactivity in Serum and Organs by Liquid Scintillation Counting (LSC)

Ten microliters of serum sample were directly pipetted into a 6-mL scintillation vial (PerkinElmer, polypropylene), and 3.5 mL of Scintillation Cocktail (PerkinElmer, Ultima Gold™, 6013329) was added. The tubes were rigorously mixed using a Vortex mixer, and the radioactivity was measured using a liquid scintillation analyzer (Packard A3100). For tissue, approximately 200 mg material was transferred into Precellys24 tubes, and 3 mL of a 1:1 Soluene350/2-propanol solution was added. Samples were homogenized with the Precellys24 homogenizer three times for 10 s. An aliquot of 250 µL of the homogenates was transferred into 20-mL scintillation vials. One milliliter of a 1:1 Soluene350/2-propanol solution was added to the sample and incubated at 40°C overnight. Depending on the organ, a bleaching step was needed (e.g., kidney). In this case, 50 µL hydrogen peroxide 35% was added to the sample, and the vial was incubated at 40°C for 2 h. Fourteen milliliters of scintillation cocktail was added to the vial and rigorously mixed using a Vortex mixer, and the radioactivity was measured using a liquid scintillation analyzer (Packard A3100). Following equation is used to determine the total radioactivity per gram collected organ.

Size-Exclusion Chromatography and Reconstruction of Complex Profile by Solid Scintillation Counting

Protein separation was carried out by SEC on an Agilent 1290 Infinity II UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) using an Acquity BEH SEC column (450 Å, 2.1 × 150 mm, Waters Corporation, Milford, MA, USA) with 100 mM sodium phosphate buffer at pH 7.4 containing 5% ethanol as mobile phase. Column oven temperature was set to 30°C. The method run time was 10 min at 250 μL/min isocratic flow. UV absorbance was measured with a diode array detector at 250 and 280 nm. Fractions were collected between 2.5 and 9.2 min every 0.07 min (17.5 μL) in a Deepwell LumaPlate-96 (PerkinElmer, Waltham, MA, USA), and radioactivity was measured by solid scintillation counting on a TopCount NXT HTS Microplate Counter (PerkinElmer, Waltham, MA, USA) after drying the plate in a rotational vacuum concentrator (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).

Peptide separation was carried out by SEC on an Agilent 1290 Infinity II UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) using a Superdex 30 Increase (3.2 × 300 mm, Cytiva Marlborough, MA, USA) with 100 mM sodium phosphate buffer at pH 7.4 containing 5% ethanol as mobile phase. Column oven temperature was set to 25°C. The method run time was 60 min at 400 μL/min isocratic flow. UV absorbance was measured with a diode array detector at 215 and 280 nm. Fractions were collected between 37.5 and 57.5 min every 0.052 min (20.8 μL) in a Deepwell LumaPlate-384 (PerkinElmer, Waltham, MA, USA), and radioactivity was measured by solid scintillation counting on a TopCount NXT HTS Microplate Counter (PerkinElmer, Waltham, MA, USA) after drying the plate in a rotational vacuum concentrator (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).