Antibody selection method

Abstract

Herein is reported a method for selecting an antibody with a systematic clearance in cynomolgus monkeys of less than 8 mL/kg/day comprising the steps of measuring the retention time of the antibody on performing an FcRn affinity chromatography with a positive linear pH gradient and on a heparin affinity chromatography with a positive linear conductivity/salt gradient, and selecting an antibody that has a relative retention time on the FcRn affinity chromatography column is less than 1.78 times the retention time difference between peaks 2 and 3 the retention time of preparation of an oxidized anti-Her3 antibody of SEQ ID NO: 03 and 04, and a relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of an anti-pTau antibody of SEQ ID NO: 01 and 02.

Claims

1. A method for selecting an antibody with a systemic clearance in cynomolgus monkeys of less than 8 mL/kg/day comprising the following steps: a) measuring the retention time of the antibody on an FcRn affinity chromatography column with a positive linear pH gradient and on a heparin affinity chromatography column with a positive linear conductivity/salt gradient, wherein the pH gradient is from a first pH value to a second pH value whereby the first pH value is from pH 3.5 to pH 6.4 and the second pH value is from pH 7.4 to pH 9.5, and wherein the conductivity/salt gradient is performed with buffer A of 50 mM TRIS pH 7.4 and buffer B of 50 mM TRIS pH 7.4, 1000 mM NaCl, and b) selecting the antibody i) if the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the retention time difference between peaks 2 and 3 of a preparation of an oxidized anti-Her3 antibody of SEQ ID NO: 3 and 4, and ii) if the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of an anti-pTau antibody of SEQ ID NO: 1 and 2.

2. The method according to claim 1, wherein the antibody is selected from the group consisting of a full length antibody, a CrossMab, a 2:1 heterodimeric T cell bispecific antibody, an antibody-cytokine fusion polypeptide, an Fc-region-cytokine fusion polypeptide, and an antibody-Fab fusion polypeptide.

3. The method according to claims 1 or 2, wherein the antibody comprises an Fc-region selected from the group consisting of a human IgG1 Fc-region, a human IgG1 Fc-region with the mutations L234A, L235A and P329G, a human IgG1 Fc-region with the knob-into-hole mutations, and combinations thereof.

4. The method according to claims 1 or 2, wherein an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as affinity chromatography ligand is used in the FcRn affinity chromatography with a positive linear pH gradient, wherein the non-covalent complex of the neonatal Fc receptor and beta-2-microglobulin is bound to a chromatography material and the non-covalent complex is conjugated to the solid phase via a specific binding pair, and wherein the non-covalent complex of the neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is mono-biotinylated and the solid phase is derivatized with streptavidin.

5. The method according to claim 4, wherein the pH gradient is from a first pH value to a second pH value whereby the first pH value is pH 5.5 and the second pH value is pH 8.8.

6. The method according to claims 1 or 2, wherein the relative retention time on the FcRn affinity chromatography column is calculated according to the following equation: $t_{\mathrm{rel},i}=\frac{t_i-t_{peak2}}{t_{peak3}-t_{peak2}}$ based on the peak definition according to t.sub.rel,i: relative retention time of peak i; t.sub.i; retention time of peak i; t.sub.peak2; retention time of the peak of the partially oxidized anti-Her3 antibody; t.sub.peak3: retention time of the peak of the non-oxidized anti-Her3 antibody.

7. The method according to claims 1 or 2, wherein the relative retention time on the heparin affinity chromatography column is calculated according to the following formula: $t_{\mathrm{rel},i}=\frac{t_i}{t_{pTau}}$ (t.sub.rel,i: relative retention time of peak i; t.sub.i: retention time of peak i; t.sub.pTau: retention time of the anti-pTau antibody peak).

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Peak definition for the calculation of relative retention times on the FcRn column.

(2) FIG. 2 FcRn relative retention plotted vs. heparin column relative retention. cross: clearance >12 mL/kg/day (“fast”); filled square: clearance between 8 and 12 mL/kg/day (“borderline”); filled circle: clearance more than 2.5 mL/kg/day but less than 8 mL/kg/day; filled star: clearance of 2.5 mL/kg/day or less.

(3) FIG. 3 FcRn relative retention plotted vs. heparin column relative retention. cross: clearance >12 mL/kg/day (“fast”); filled square: clearance between 8 and 12 mL/kg/day (“borderline”); filled circle: clearance more than 2.5 mL/kg/day but less than 8 mL/kg/day; filled star: clearance of 2.5 mL/kg/day or less; vertical lines mark the retention time ranges for therapeutically suitable clearance (lower-left quadrant, FcRn <1.78; Heparin: <0.87).

(4) FIG. 4 FcRn relative retention plotted vs. heparin column relative retention of IVIG (intravenous immunoglobulin). 2: fraction with highest heparin binding; 1: fraction with lowest heparin binding.

(5) FIG. 5 FcRn relative retention plotted vs. heparin column relative retention of antibody no. 5 and variants thereof. 1: wild-type antibody; 20: negatively patched HC variant; 27: positively patched HC variant; 112: positively patched LC variant; 183: positively patched HC variant.

(6) FIG. 6 Clearance of antibody no. 5 and variants thereof in FcRn knock-out mice. 1: wild-type antibody; 20: negatively patched HC variant; 27: positively patched HC variant; 112: positively patched LC variant; 183: positively patched HC variant.

(7) FIG. 7 Time dependent serum concentration of a non-patchy and a patchy variant of antibody no. 5 in FcRn knock-out mice.

(8) FIG. 8 FcRn relative retention plotted vs. heparin column relative retention of antibody no. 5 and histidine variants thereof.

(9) FIG. 9 Time dependent serum concentration of antibody no. 5 and a HC histidine variant thereof in FcRn knock-out mice.

(10) FIG. 10 Clearance of antibody no. 5 and a HC histidine variant thereof in FcRn knock-out mice.

(11) The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

MATERIALS AND METHODS

Antibodies

(12) 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.

(13) Synthetic genes were produced at Geneart (Life technologies GmbH, Carlsbad, Calif., USA).

(14) 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).

(15) 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

(16) 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.

(17) Beside the antibody expression cassette, the plasmids contained: an origin of replication which allows replication of this plasmid in E. coli, a ß-lactamase gene which confers ampicillin resistance in E. coli, and the dihydrofolate reductase gene from Mus musculus as a selectable marker in eukaryotic cells.

(18) The transcription unit of the antibody gene was composed of the following elements: unique restriction site(s) at the 5′ end the immediate early enhancer and promoter from the human cytomegalovirus, followed by the Intron A sequence in the case of the cDNA organization, a 5′-untranslated region of a human antibody gene, an immunoglobulin heavy chain signal sequence, the human antibody chain either as cDNA or as genomic organization with the immunoglobulin exon-intron organization a 3′ non-translated region with a polyadenylation signal sequence, and unique restriction site(s) at the 3′ end.

(19) 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

(20) 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

(21) 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. The following antibodies have been produced accordingly:

(22) TABLE-US-00031 antibody antigen no format Fc-region no. 1 IgG1, bivalent, monospecific, LALAPG  1 reference heparin 2 IgG1, bivalent, monospecific wild-type  2 3 IgG1, bivalent, monospecific wild-type  3 4 CrossMab, bivalent, bispecific KiH 4 + 5 5 IgG1, bivalent, monospecific wild-type  6 6 IgG1, bivalent, monospecific wild-type  7 7 IgG1, bivalent, monospecific wild-type  8 8 IgG1, bivalent, monospecific wild-type  9 9 IgG1, bivalent, monospecific wild-type 10 10 IgG1, bivalent, monospecific, wild-type 11 reference FcRn 11 IgG1, bivalent, monospecific wild-type 12 12 IgG4, bivalent, monospecific wild-type 13 13 IgG1, bivalent, monospecific wild-type  9 14 IgG1, bivalent, monospecific wild-type 14 15 IgG1, bivalent, monospecific wild-type 15 16 Fc-region-cytokine fusion LALAPG 16 17 2:1 heterodimeric T cell KiH 17 + 18 bispecific 18 2:1 heterodimeric T cell KiH  9 + 18 bispecific 19 IgG1, bivalent, monospecific - KiH LALAPG 16 + 19 cytokine fusion 20 IgG1, bivalent, monospecific - KiH LALAPG 16 + 17 cytokine fusion 21 IgG1, bivalent, monospecific wild-type 20 + 21 22 IgG1, bivalent, monospecific wild-type 20 + 21 23 IgG1, bivalent, monospecific wild-type 22 24 IgG1, bivalent, monospecific wild-type 23 25 IgG1, bivalent, monospecific wild-type 24 26 IgG1, bivalent, monospecific wild-type 25 27 IgG1, bivalent, monospecific wild-type 26 28 IgG1, bivalent, monospecific wild-type 27 29 IgG1, bivalent, monospecific wild-type 28 30 2:1 heterodimeric T cell KiH LALAPG 29 + 18 bispecific 31 IgG1, bivalent, monospecific wild-type  9 32 2:1 heterodimeric T cell KiH LALAPG 17 + 18 bispecific 33 IgG1, bivalent, monospecific LALAPG  9 34 IgG1-Fab fusion, trivalent, KiH LALAPG  9 + 30 bispecific 35 IgG1, bivalent, monospecific wild-type 31

Purification

(23) The 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 Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.

(24) Briefly, sterile filtered cell culture supernatants were captured on a MabSelectSuRe resin equilibrated with PBS buffer (10 mM Na.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.

(25) 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

(26) B6. Cg-Fcgrt.sup.tmlDcr 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

(27) A single dose of antibody was injected i.v. via the lateral tail vein at a dose level of 10 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

(28) Concentrations of Ustekinumab, Briakinumab, mAb 8 and mAb 9 in murine serum were determined by specific enzyme-linked immunoassays. Biotinylated Interleukin 12 specific to the antibodies and digoxigenin-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 antibody 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 human antibodies were detected by subsequent incubation with detection antibody, followed by an anti-digoxigenin 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 (Männedorf, Switzerland).

(29) All serum samples, positive and negative control samples were analyzed in duplicates and calibrated against reference standard.

Pharmacokinetic (PK) Analysis

(30) The pharmacokinetic parameters were calculated by non-compartmental analysis using WinNonlin™ 1.1.1 (Pharsight, Calif., USA).

(31) Briefly, area under the curve (AUCo.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.

(32) Plasma clearance was calculated as Dose rate (D) divided by AUCo.sub.0-inf. The apparent terminal half-life (T1/2) was derived from the equation T1/2=ln2/λz.

EXAMPLE 1

Preparation of FcRn Affinity Column

Expression of FcRn in HEK293 Cells

(33) 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.

(34) For transient expression, a 14 l stainless steel bioreactor was started with a culture volume of 8 liters 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 293 fectin (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 1 1 buckets: 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

(35) 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.

(36) 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

(37) 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 2

Chromatography Using FcRn Affinity Column and pH Gradient

Conditions

(38) column dimensions: 50 mm×4.6 mm loading: 30 μg sample buffer A: 20 mM MES, with 140 mM NaCl, adjusted to pH 5.5 buffer B: 20 mM Tris/HC1, with 140 mM NaCl, adjusted to pH 8.8

(39) 30 μl 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.

(40) 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.

(41) Briefly, the antibody (at 9 mg/mL) in 10 mM sodium phosphate pH 7.0 was mixed with H2O2 to a final concentration of 0.02% and incubated at room temperature for 18 h. To quench the reaction, the samples were thoroughly dialyzed into pre-cooled 10 mM sodium acetate buffer pH 5.0.

(42) Relative retention times were calculated according to the following equation:

(43) $t_{\mathrm{rel},i}=\frac{t_i-t_{peak2}}{t_{peak3}-t_{peak2}}$
For peak definition see FIG. 1.

EXAMPLE 3

Chromatography Using Heparin Affinity Column and pH Gradient

Conditions

(44) 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 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. Every injection sequence started with a retention time standard (anti-pTau antibody) which was used to calculate relative retention times according to the following formula:

(45) $t_{\mathrm{rel},i}=\frac{t_i}{t_{pTau}}$
(t.sub.rel,i: relative retention time of peak i; t.sub.i: retention time of peak i; t.sub.pTau: retention time of the anti-pTau antibody peak).

EXAMPLE 4

Cynomolgus SDPK Studies

(46) 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

(47) 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.

Results

(48) TABLE-US-00032 heparin affinity FcRn affinity column rel. column retention antibody rel. retention (vs. (vs. Her3 Pre-peak 1 and clearance* no pTau) Main Peak) [mL/kg/day] 1 1 0.9 61.1 2 1.01 3.92 5.01 3 0.72 0.61 3.34 4 0.59 0.88 3.97 5 0.67 0.36 3.47 6 0.61 0.59 5.13 7 0.64 0.22 6.41 8 0.56 2.52 4.1 9 0.66 0.4 5.28 10 0.66 1.03 4.1 11 0.67 0.43 5.52 12 0.63 −0.22 1.6 13 0.65 1.21 <8 14 0.44 0.29 4.73 15 0.26 −0.46 2.4 16 1.05 0.52 >12 17 1 1.42 24.9 18 0.8 3.29 90 19 0.99 0.78 17 20 1.09 1.39 16.4 21 0.9 3.36 >8 22 0.66 0.39 >8 23 0.53 0.61 5.37 24 0.13 −0.199 2.5 25 1.01 1.78 8.16 26 0.26 −0.16 4.81 27 0.53 0.37 2.45 28 0.59 0.17 4.8 29 0.52 2.43 4.87 30 0.79 0.58 6.3 31 0.55 2.24 31.92 32 0.77 0.42 7.5 33 0.52 1.91 10.08 34 0.55 1.86 31.92 35 0.84 0.01 4.63