MATERIALS AND METHODS 1. Chemicals and samples

Acetonitrile (ACN) and methanol (MeOH) were of analytical reagent grade from Panreac (Barcelona, Spain). Ammonium hydroxide solution, formic acid, acetone, and ethanol (EtOH) were of analytical grade from Fluka (Buchs, Switzerland). Ultrapure water was supplied by a Milli-Q purification unit from Millipore (Bedford, USA).

Somatropin (GH) and insulin (INS) were purchased from the National Institute for Biological Standards and Control (NIBSC, Potters Bar, UK). Hemoglobin (Hb) was purchased from Sigma-Aldrich (St Louis, USA). The three proteins were dissolved in a 50 mM Tris-phosphate buffer (pH 7.4) at a concentration of 2.0, 1.7, and 0.8 mg/mL for Hb, GH, and INS, respectively (stock solutions). Standard solutions of GH, Hb, and INS at desired concentrations were prepared daily by appropriate dilution of the stock solutions with water.

2.2. CE-UV instrumentation

CE experiments were performed with an HP ^3DCE System (Agilent, Waldbronn, Germany) equipped with an on-capillary diode array detector, an autosampler, and a power supply able to deliver up to 30 kV. Separation was performed in an uncoated FS capillary (BGB Analytik AG, Böckten, Switzerland) with a 50 µm I.D, a total length of 64.5 cm, and an effective length of 56 cm. Samples were hydrodynamically injected at 50 mbar for 8 s (equivalent to 1% of the effective capillary length). Prior to each sample injection, the capillary was rinsed at 2 bar for 1 min with fresh BGE. Experiments were carried out in positive polarity mode with the anode at the inlet and the cathode at the outlet. A constant voltage of 30 kV, with an initial ramping of 5000 V·s^-1 (6 s), was applied during the analysis, and the capillary temperature was maintained at 25°C. UV detection was performed at 200 nm.

New bare FS capillaries, used under basic pH conditions, were rinsed at 1 bar with MeOH, 1 M HCl, water, 0.1 M NaOH, water, and BGE for 5 min each. New bare FS capillaries, used under acidic pH conditions, were rinsed at 1 bar with MeOH, 0.1 M NaOH, water, 1 M HCl,

water, and BGE for 5 min each. When not in use, the capillary was rinsed with water and then stored dry.

For protein sizing and area recovery experiments, an ActiPix™ D100 UV Area Imaging System (Paraytec, York, United Kingdom) was coupled with CE to perform analyses with two passes through the detector [22]. Experiments were also performed in uncoated FS capillaries (BGB Analytik AG, Böckten, Switzerland) with a 50 µm I.D. Capillaries with a total length of 115 cm and effective lengths of 32 and 65.5 cm were used for area recovery experiments. Capillaries with a total length of 98 cm and effective lengths of 32 and 49.5 cm were used for protein sizing experiments. Samples were hydrodynamically injected at 50 mbar for 16 s for area recovery experiments (equivalent to 2% of the effective capillary length, taking into account the first window). Samples were hydrodynamically injected at 50 mbar for 60 s (equivalent to 8.4% of the effective capillary length, taking into account the first window) and flushed at 1 bar for 5 min for sizing experiments. Prior to each sample injection, the capillary was rinsed at 2 bar for 4 min with fresh BGE. UV detection was performed at 200 nm.

Separations were carried out with 75 mM ammonium formate buffers (pH 2.5 and 9.0). The addition of different percentages of organic modifiers was tested (5-60%, v/v, of ACN, MeOH, or EtOH) under both basic and acidic conditions.

2.3. CE-MS instrumentation

CE was coupled to an Agilent Technologies 6210 LC/MS TOF mass spectrometer (Agilent, Palo Alto, USA) via a tri-axial sheath flow ESI interface from Agilent. The coaxial sheath liquid was optimized in terms of composition (water with MeOH, isopropanol or ACN at different percentages from 25 to 75%, formic or acetic acid at different proportions, from 0.05 to 10%) and delivery rates (from 2 to 8 µL·min^-1) for each protein. The fragmentor voltage was optimized for each protein. Other MS parameters were identical for the three proteins:

ESI capillary voltage was set at +4500 V, the nebulizing gas pressure at 4 psi, the drying gas flow rate at 4 L·min^-1, and the drying gas temperature at 150°C. MS detec tion was carried out in the positive ion mode and 1 spectrum·s^-1 was acquired (9742 transients/spectrum).

CE-MS experiments involved capillaries with a length of 80 cm and an internal diameter of 50 µm. Samples were hydrodynamically injected at 50 mbar for 30 s (equivalent to 2% of the capillary length).

2.4. Measurements and calculations 2.4.1 Reversible adsorption

Relative standard deviations of migration times (MT RSDs, n=5) were calculated for successive injections.

2.4.2 Irreversible adsorption 2.4.2.1 Protein recovery

A procedure for protein recovery was adapted from one reported by Towns and Regnier [19,20]. Successive injections of protein samples were performed in the same capillary (n=5) with two passes through the detector (ActiPix™ System). The decrease in area of the protein between the first and the second pass through the detector provided a measure of the irreversible adsorption on the capillary, and is given as the area recovery percentage.

2.4.2.2 EOF

Electrosmotic flow (EOF) mobilities (n=3) were calculated with and without protein injection.

At basic pH, a strong cathodic EOF was observed and its mobility was calculated with acetone as a neutral marker. At acidic pH, acetone migrated around 110 minutes, due to the weak EOF observed at this pH. Therefore, the methodology of Williams and Vigh [17], with the assistance of pressure was used.

2.4.3 Protein sizing

The methodology for protein sizing experiments was based on the Taylor dispersion analysis (TDA) [23]. A looped capillary was used with the ActiPix™ System, providing two detection windows. A plug of the sample was hydrodynamically injected and driven by the application of external pressure along the capillary. UV absorption of the protein zone was recorded during the 1^st and 2^nd pass. Taylor dispersion induced greater width and lower amplitude of the signal recorded in the second window. Based on standard deviations associated with the solute peak broadening, the diffusion coefficient and the hydrodynamic radius of proteins could be determined with the equation 1 [21, 22, 23]:

: 4<=>?@6 6 @A 6B ()⁶?6 AB 1

where Rh = hydrodynamic radius, kB = Boltzmann constant, T = absolute temperature, t1 and t2 = peak center times at the first and the second capillary windows, τ1 and τ2 = their corresponding standard deviations, η = viscosity, and r = capillary internal radius.

2.5. Software

Buffer solutions were prepared with the help of PHoEBuS software (version 1.3, Analis, Namur, Belgium). CE ChemStation (version A.10.02, Agilent, Waldbronn, Germany) was used for CE instrument control. ActiPix™ D100 (Paraytec, York, United Kingdom) was used for ActiPix™ control and hydrodynamic radii calculations.