All sample handling steps for isolation of charge variant fractions were aseptically performed under a laminar flow hood. All materials and reagents used for this study were ensured to be pyrogen-free, either by LAL testing or material certification. The mAb analyzed was expressed in Chinese hamster ovary (CHO) cells and was a humanized IgG1 that does not react with the rat counterpart of its human target antigen, and shows activity in vivo. This purified mAb was characterized using ion exchange chromatography and the profile showed the presence of three distinct components: the acidic (20%) and basic variants (12%) and the main peak (68%).
Displacement chromatography and characterization of charge variant fractions.
Separation of charge variants. mAb charge variant fractions were isolated using displacement chromatography. Briefly, a GE AKTA Explorer chromatography system was used for all runs. A 10 × 600 mm GE Tricorn column was packed with GE Source 15S cation resin to a bed height of 585 mm and all processes were performed at ambient temperature. In general the antibody starting material (at pH 6.1) was loaded on to the column to a load density of ~30 mg/mL. The loaded column was then washed with equilibration buffer (30 mM MES, 10 mM NaCl, pH 5.85) to remove any non-binding molecules and adjust the pH of the bound antibody and column. Next, 10 column volumes (CV) of the displacer (5 mM Sachem SP1, 27 mM MES, 9 mM NaCl, pH 5.75) was loaded at 30 cm/hr to displace the bound mAb. The displacement train was fractionated at the column outlet. Every fifth fraction was then analyzed using an ion exchange column on an HPLC (IEC method described below) to determine the fraction purity.
Fractions of charge variants with >90% purity were pooled and transferred into an Amicon Ultra-15 (30,000 MWCO from Millipore, Bedford, MA), concentrated and buffer exchanged with 20 mM histidine acetate buffer, pH 6.0. The appropriate amounts of a concentrated sucrose and polysorbate 20 solutions were added to the individual pools to obtain a final concentration of 30 mg/ml charge variant, 120 mM sucrose and 0.02% polysorbate 20. The samples were sterile-filtered with 0.22 µm PVDF membrane filters (50 mL Durapore Vacuum-Driven Filter Device, Millipore, Bedford, MA, USA). The individual charge variants in the above formulation were tested using size-exclusion and ion-exchange chromatography and checked for protein content, pH, osmolality and endotoxin levels using the methods described below.
Protein concentration measurements. The protein concentration of isolated charge variant fractions was determined by measurement of the UV-absorbance on an Agilent 8453 spectrophotometer (Sunnyvale, CA, USA) via volumetric sample preparation. The samples were blanked against the formulation buffer and the absorbance was measured at Amax at 278–279 nm.
Osmolality. The osmolality was measured by freezing point method using an Advanced Instrument Model 3320 and calibrated on the same day with 100 and 500 mOsm/kg Clinitrol Controline osmolality standards (Advanced Instrument, Norwood, MA, USA).
Size exclusion chromatography. Size variant distribution was determined by size exclusion chromatography (SEC) using a TosoHaas column G3000 SWXL (Tosoh Biosep, LLC, Montgomeryville, PA, USA) at ambient temperature on an Agilent 1200 HPLC (Santa Clara, CA, USA). Samples were eluted over 30 minutes with a 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.2 mobile phase monitored by a UV detector at 280 nm. Chromatograms were integrated using Dionex Chromeleon Software (Sunnyvale, CA, USA) and relative percent peak areas obtained.
Circular dichroism. Far and near-UV circular dichroism (CD) spectroscopy was used to ensure that no major structural change resulted in the tertiary structure of mAbs upon modification of different residues. Experiments were conducted using a Jasco 815 spectropolarimeter (Easton, MD, USA). Solution ellipticities for the near UV and the far UV were measured from 340-240 nm and from 190–250 nm in potassium 10 mM phosphate buffer (pH 6.0) respectively. Studies were conducted at 0.8 mg/ml mAb concentration using 10 mm path length cell for measuring the CD spectra in the near UV region and 0.5 mg/ml mAb concentration in a 1 mm path length cell for measuring the far UV region. Multiple scans, at a resolution of 0.1 nm and a scan rate of 10 nm/min, were accumulated and averaged in order to improve signal to noise ratio. Buffer spectra obtained with the same acquisition parameters were subtracted from the averaged scans for IgG1 solutions.
Limulus amoebocyte lysate assay for endotoxin determination. Endotoxin levels were measured to confirm comparability and remained within predetermined study criteria post-formulation of the charge variants from the starting material. All samples were endotoxin tested by the chromogenic LAL method using a Kinetic-QCL assay kit with a BioWhittaker Kinetic-QCL reader (BioWhittaker, Wokingham, UK), according to manufacturer's instructions. This assay is a quantitative, kinetic assay for the detection of Gram-negative bacterial endotoxin. Gram-negative bacteria catalyze the activation of a pro-enzyme in the LAL. The activated enzyme catalyzes the splitting of p-nitroaniline (pNA) from the colorless substrate Ac-Ile-Glu-Ala-Arg-pNA. The concentration of the endotoxin in a sample is calculated from its reaction time by comparison to the reaction time of the standard curve.
Capillary isoelectric focusing for pI determination.
Imaged capillary isoelectric focusing (icIEF) is known to be an effective tool with high separation resolution in biomolecular analysis. This technique has been confirmed to be suitable for fast determination of pI
of proteins and is able to distinguish subtle differences in charge.65,66
Briefly, the pI
of each charge variant fraction was determined by icIEF using an iCE280 analyzer (Convergent Bioscience) with a fluorocarbon-coated capillary cartridge (100 µm × 5 cm). Capillary isoelectric focusing was achieved using a mixture of 0.35% methyl cellulose, 5% total carrier ampholytes (3–10 Pharmalyte) and 0.1% pI
markers varying from 5.8 to 9.8 (5.8, 6.6, 7.0, 7.5, 8.2, 9.5 and 9.8) in purified water. The anolyte was 80 mM phosphoric acid and the catholyte was 100 mM sodium hydroxide, both in 0.1% methyl cellulose. All samples were diluted, mixed with the ampholyte solution and then focused by introducing a potential of 1,500 volts for 1 min, followed by a potential of 3,000 volts for 5 min. The final protein concentration in the ampholyte and sample mixture was 0.25 mg/mL. The separation was monitored at 280 nm.
Ion exchange chromatography. Charge variants were separated on a 4.0 × 250 mm Dionex ProPac cation exchange (IEC) column WCX-10. The mobile phase was a MES buffer at pH 6.0 at a flow rate of 1.0 mL/min. Bound forms were eluted with a sodium chloride gradient. The column temperature was 42°C and detection was at 280 nm.
To quantify the amount of acidic forms due to sialic acid, the acidic variant fraction was analyzed with and without treatment with neuraminidase, which removes sialic acid. The difference in the percent peak area between treated and untreated samples enabled quantitation of acidic forms containing sialic acid.
HPLC-MS of peptide digests. Acidic variants were sulfitolyzed, digested with endoproteinase Asp-N and analyzed by reverse phase HPLC on a Zorbax 300SB-C8 column. Mobile phase A was 0.1% TFA in deionized water and mobile phase B was 0.08% TFA in isopropyl alcohol. Absorbance was monitored at 214 nm and masses were determined by electrospray mass spectrometery using a Thermo Fisher LTQ.
Basic variants were reduced, carboxymethylated, digested with trypsin and analyzed by reverse phase HPLC on a VYDAC C-18 column. Solvent A was water, solvent B was acetonitrile and both had 0.1% trifluoroacetic acid (TFA). A 0–60% gradient was used to separate the peptides. Absorbance was monitored at 214 nm and masses determined by electrospray mass spectrometry using an API3000.
Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Acidic variant fractions were analyzed by both reduced and non-reduced CE-SDS. Samples were labeled with 5-carboxytetramethylrhodamine succinimidyl ester. After removal of excess dye, the samples were either treated with iodoacetamide (non-reduced sample) at 70°C for 5 min or DTT (reduced sample) at 70°C for 20 min. The samples were analyzed on a Beckman PA 800 capillary electrophoresis system equipped with a 50 um diameter uncoated fused-silica capillary. Samples were injected electrokinetically and separation was performed at a constant voltage of 15 kV over a 40 min period. The sieving matrix was SDS-MW gel buffer (Beckman Coulter) and the capillary maintained at a temperature of 20°C. The migration of labeled components was monitored by laser induced fluorescence detection using excitation at 488 nm and the emission at 560 nm.
Boronate affinity chromatography. A TSK-Gel boronate-5PW column (Tosoh), 7.5 × 75 mm, 10 µm particle size was used to determine glycation levels. The affinity-based separation occurred such that the non-glycated molecules flow through the column equilibrated in 0.1 M HEPES, 0.2 M NaCl, 25 mM Tris, pH 8.6, then the glycated molecules are single-step eluted from the resin with the same buffer that also contained 0.5 M sorbitol. Column temperature was 40°C and the mobile phase flow-rate was 1.0 mL/minute. Each sample was run in triplicate with 100 µL injections of a 1:1 dilution in equilibration buffer (50 µg loads). Eluent absorbance was monitored at 280 nm to determine the percentage of glycated mAb in each charge variant fraction.
Affinity measurements binding to rat FcRn.
The coding regions of the rat FcRn α-chain ectodomain (Met1-Ser309) and the full-length β2
-microglobulin light chain (rβ2
m) were generated by gene synthesis (Blue Heron). The coding regions of rat FcRn and r β2
m were subcloned into a previously described pRK mammalian cell expression vector.67
For expression and purification of rat FcRn, human embryonic kidney (HEK293) cells were transfected using FUGENE (Roche Applied Science) according to the manufacturer's protocol. After 24 h of incubation with transfection complexes, cells were switched to serum-free PSO4 medium [Genentech; 1 g/L Pluronic F-68, 5.5 g/L combination nonselect medium (Life Technologies), 4.3 g/L glucose, 1.22 g/L sodium bicarbonate, 0.1 g/L gentamicin sulfate (pH 7.1); 350 milliosmolar] supplemented with 5 mg/L recombinant bovine insulin and trace elements and grown for 7 days. Cells were pelleted by centrifugation and rat FcRn was purified from the culture supernatants by pH-dependent binding to a human IgG-Sepharose (Amersham) column. Briefly, supernatants were acidified to pH 5.8 with 50 mM MES and flowed over a 4 mL hIgG-Sepharose column at ~1.5 mL/min. After washing with >10 column volumes of wash buffer (20 mM MES, 150 mM NaCl, pH 5.8), bound rat FcRn was eluted with 20 mM HEPES, 150 mM NaCl, pH 8.0. Eluted proteins were then concentrated and further purified by size exclusion chromatography on a Superdex 200 column (Amersham) with PBS pH 6.0 as the running buffer. Fractions containing monomeric FcRn were pooled, and the concentration was determined on a Nanodrop 8000 spectrometer (Thermo Scientific) using a mass extinction coefficient of 1.95 Lcm−1
at 280 nm.
All binding and kinetics studies were performed on recombinant rat FcRn, by surface plasmon resonance technology using a Biacore T-100™ instrument (GE Healthcare, Piscataway, NJ, USA). All experiments were carried out at 37°C. Rat FcRn (5 µg/mL) was immobilized onto two of the four flow cells (FCs) of a Series S CM5 sensor chip (GE Healthcare), FC2 and FC4, using a standard amine coupling procedure according to the manufacturer's protocol. The immobilization levels were approximately 40 response units (RU) per flow cell. The other two FCs, FC1 and FC3, were subjected to the same immobilization protocol in the absence of coupling ligand (rat FcRn) to serve as in-line background reference flow cells for FC2 and FC4, respectively, in subsequent experiments. All mAb samples were diluted to 25 nM and 250 nM in running buffer (PBS, 0.05% polysorbate, pH 6.0) and were injected for 2 min at a flow rate of 50 µL/minute. Surfaces were regenerated between cycles by two sequential injections of PBS, pH 7.4 (35 seconds per injection at 50 µL/min). To ensure that any differences seen between different charge variants were beyond assay variation, a statistical experimental design was used in which eight independent runs for each sample were carried out in random order using two separate sensor chips. The reference-corrected binding responses were reported 5 seconds before the end of each injection.
Experiments measuring the kinetics of the interaction between rat FcRn and mAb charge variants were performed essentially as described above, except that each injection consisted of a 5 min association phase and a 6 min dissociation phase. The first dissociation rate constant (kd1), first association rate constant (ka1) and the first dissociation equilibrium constant (KD1) from kinetic analyses were calculated with a bivalent analyte model using the BIAevaluation Software (version 3.2; GE Healthcare). Four independent experimental runs were carried out using two separate sensor chips and the results were averaged.
To assess the in vitro potency of mAb charge variants, a cell culture assay was used to measure their ability to inhibit the proliferation of cells expressing the receptor. Briefly, cells were seeded in 96-well tissue culture microtiter plates and incubated overnight at 37°C under 5% CO2 to allow cell attachment. The following day, the culture medium was removed and serial dilutions of each mAb fraction were added to the plates. The plates were then incubated for four days at 37°C under 5% CO2 and the relative number of viable cells was quantified indirectly using a redox dye, alamarBlue® according to the manufacturer's protocol. The control materials were also tested using the same assay parameters and conditions. Each mAb concentration was assayed in triplicate and the changes in color as measured by fluorescence were directly proportional to the number living cells in the culture. The absorbance of each well was then measured on a fluorescence 96-well plate reader. The results, expressed in relative fluorescence units (RFU), were plotted against the antibody concentration and a parallel line analysis program was used to estimate the anti-proliferative activity of the antibody relative to the reference material.
Intravenous and subcutaneous pharmacokinetic studies in normal rats.
The PK studies were conducted in two parts using Sprague-Dawley male rats. The first was an IV study that evaluated statistically the acidic charge variant, main peak and starting material. In this study, a total of 36 male rats, weighing 279–317 g and 8 weeks in age, were assigned to three different groups, each consisting of 12 rats per group. All animals received a single IV bolus dose of 10 mg/kg via jugular cannula. Blood samples (approximately 0.2 mL) from the jugular vein of each animal were collected pre-dose and post-dose at 5 min; 1, 4, 8, 24, 32 h and 2, 4, 7, 10, 14, 21, 28 and 35 days. Blood samples were collected in serum separators. Diluted serum sample were assayed for antibody concentrations using a receptor-binding direct Enzyme Linked ImmunoSorbent Assay (ELISA). In this particular assay, a recombinant antigen receptor specific for this mAb molecule was used as the capture reagent and mouse anti-human IgG Fc conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., Catalog # 209-035-098) was used as the detection reagent. The minimum dilution of sample for the ELISA was 1/100 and the minimum quantifiable concentration was 400 ng/mL. In brief, 96-well polystyrene microtiter plates (Nunc, Thermo Fisher Scientific, catalog # 439454) were coated with 2 mg/mL of the antigen with 0.05 M sodium carbonate buffer and incubated for 2–6 h at 2°C–8°C. The coat solution was then removed from the wells and the wells were blocked with approximately 200 µL of assay buffer (phosphate buffer saline/0.5% bovine serum albumin/0.05% polysorbate 20/0.033% proclin 300) for 1–2 h at room temperature with agitation. The wells were then washed with 400 µL × 6 of PBS Buffer (phosphate buffer saline/0.05% Polysorbate 20). Standards (in duplicate), controls (in duplicate) and serially diluted samples were added to the plate at 100 µL and incubated at room temperature for 2 h ± 5 min with agitation. The wells were then washed with 400 µL of PBS buffer six times. Mouse anti-human IgG Fc conjugated to horseradish peroxidase was diluted 1:1,500 in assay buffer and added to each well at 100 µL. The plates were incubated at room temperature for 1 h ± 5 min with agitation and wells were washed six times with 400 µL of PBS Buffer, before 100 µL of a 1:1 mixture of tetramethyl benzidine (TMB) peroxidase substrate (0.4 g/L TMB) [Kirkegaard & Perry Labs (KPL) 50-76-0] and peroxidase solution B, 0.02% hydrogen peroxide (KPL 50-65-00) was added to each well. After approximately 15 min of incubation, the reaction was stopped with the addition of 100 µL of 1 M phosphoric acid to each well. The yellow color developed by conversion of the substrate was measured on a Biotek ELX800 plate reader using two filters, 450 nm for detection absorbance and 630 nm for reference absorbance.
The second was a subcutaneously administered study conducted with 48 male rats that evaluated statistically the acidic and basic charge variant, main peak and starting material. The animals (12 per group), weighing 279–317 grams and 8 weeks of age were assigned to four different groups. Each rat was administered a 0.1 mL subcutaneous dose in the right lateral flank at 10 mg/kg. Approximately 0.2 mL of blood was collected from the tail vein for each animal at pre-dose and post-dose at 1, 3, 6 and 24 h and days 2, 3, 4, 7, 10 and 14. The concentrations of the antibody in serum were determined using a receptor-binding ELISA as described above.
Pharmacokinetic data analysis.
The serum mAb time concentration data for all groups in both studies were analyzed using non-compartmental analysis (Model 200, WinNonlin Pro, Version 5.0.1; Pharsight Corporation; Mountain View, CA, USA). For plotting purposes, all LTR values were converted to ½*LTR, with a value of 0.2 µg/mL. Based on prior experience with this mAb, AUC0–14 was chosen as the primary endpoint for this study.