Combined Approach to the Analysis of Recombinant Protein Drugs

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also intera...
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Anal. Chem. 2006, 78, 1085-1092

Combined Approach to the Analysis of Recombinant Protein Drugs Using Hollow-Fiber Flow Field-Flow Fractionation, Mass Spectrometry, and Chemiluminescence Detection Aldo Roda,* Daniela Parisi, and Massimo Guardigli

Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital, Via Massarenti 9, 40138 Bologna, Italy Andrea Zattoni and Pierluigi Reschiglian

Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution. In functional protein drugs obtained by recombinant cell culture expression systems, a huge variety of impurities may be present, and their origin cannot be easily predicted. These impurities may include “process-related impurities” such as components of cell culture media or host cell proteins and nucleic acids, as well as antibodies, solvents, buffers, or stationary-phase components, which are released during separation and purification processes. In addition, “product-related impurities” could be present, including molecular variants with properties different from those of the desired product, which can be obtained together with the target compound or formed during manufacture or storage. Large-scale production of recombinant protein drugs requires that * Corresponding author. Fax: +39 (0)51 343398. E-mail: [email protected]; http://www.anchem.unibo.it. 10.1021/ac0511492 CCC: $33.50 Published on Web 01/07/2006

© 2006 American Chemical Society

the expressed proteins maintain their native and active conformation (the folded state) after high-throughput refolding. The impurities present in recombinant, functional protein drugs can not only affect safety of the final product but also interact with the expressed protein and, then, alter its functionality. The ICH harmonized guidelines, which define procedures and acceptance criteria for the quality of biological and biotechnological products,1 are very restrictive since the results are often method-dependent. Choice and optimization of the analytical procedures should focus on the possibility to separate the desired product from the impurities, to identify and quantify all the possible impurities with suitable sensitivity, and to evaluate the biological activity of the product and related impurities. The analytical problem becomes particularly complicated in the case of functional protein drugs since all these goals cannot be achieved by a single reference technique but only by means of a combined approach. The approach should be able to “see” all the possible molecules present in the sample. Since protein denaturation or other factors affecting protein functionality must be controlled, a proper combination of nondegrading methods for the “gentle” sample preparation and separation might be preferred. This combination should allow us to establish whether native conditions of the expressed proteins are maintained. If different supramolecular forms of the expressed protein are found, the approach may also establish whether these forms have different functionality. Finally, to evaluate possible biological activity of impurities present at very low amount, highly sensitive methods must be used. In this work, a combined approach for the analysis of the functional protein urate oxidase (uricase, EC 1.7.3.3) is presented. Uricase in native form is a homotetramer without a prosthetic group, the relative molar mass (Mr) of which is ∼132 000.2 X-ray analysis shows that the tetramer is composed of two dimers, which form a tunnel-shaped protein. The enzyme is stable at pH 7.3(1) Q6B Guideline. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. ICH, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 1999; www.ich.org. (2) Conley, T. G.; Priest, D. G. Biochem J. 1980, 187, 727-732.

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9.5 and below 50 °C.3 A recombinant uricase drug from Aspergillus flavus expressed in Saccharomyces cerevisiae was recently commercialized as Rasburicase. Rasburicase is used in pediatric patients receiving anticancer therapy,4-7 and it is reported to have high purity and specific activity.8,9 To compare different uricase preparations, Rasburicase and a recombinant uricase from Candida sphaerica expressed in Escherichia coli were analyzed. Reversed-phase (RP) HPLC with electrospray ionization/quadrupole time-of-flight mass spectrometry (ESI/Q-TOFMS) and matrixassisted laser desorption/ionization (MALDI) TOFMS with SDS PAGE and 2D SDS PAGE were first applied as conventional methods to detect and identify the sample impurities. It is known, however, that undesired interaction between proteins and RP stationary phases can cause protein adsorption or entanglement, which reduces protein recovery and separation.10 Separation performance can be improved by using low-size particle packing and narrow-bore, long columns under high- or ultrahigh-pressure conditions.11 Under these harsh conditions, however, protein degradation may occur. Organic modifiers used in RP HPLC mobile phases can also induce protein denaturation.12 Maintenance of the three-dimensional, native structure and recovery of lowabundance protein impurities after separation in fact constitute necessary requirements for final assessment of functional protein drug purity and for the evaluation of biological activity of the drug and related impurities. Otherwise, when MALDI/TOFMS, with SDS PAGE and 2D SDS PAGE for peptide fingerprinting, is used for the identification of protein posttranslational modifications and for the identification of protein impurities, possible functional variation without changes in the protein Mr value or in the peptide sequence cannot be taken into account. To analyze the uricase samples under nondegrading conditions, an innovative separation method, the hollow-fiber (HF) channel variant of flow field-flow fractionation (FlFFF), was applied in combination with MALDI/ TOFMS, and with a chemiluminescence (CL) assay to evaluate the sample enzymatic activity. FlFFF applies to the analysis of macromolecular and supramolecular bioanalytes, including proteins.13 If compared to RP HPLC, ion-exchange or size-exclusion chromatography (SEC), and electrophoresis, FlFFF shows intrinsic advantages for the analysis of (3) Bonnete`, F.; Vivare`s, D.; Robert, Ch.; Colloc’h, N. J. Cryst. Growth 2001, 232, 330-339. (4) Bosly, A.; Sonet, A.; Pinkerton, C. R.; McCowage, G.; Bron, D.; Sanz, M. A.; Van den Berg, H. Cancer 2003, 98, 1048-1054. (5) Lee, A. C.; Li, C. H.; So, K. T.; Chan, R. Ann. Pharmacother. 2003, 37, 1614-1617. (6) Yim, B. T.; Sims-McCallum, R. P.; Chong, P. H. Ann. Pharmacother. 2003, 37, 1047-1054. (7) Liu, C. Y.; Sims-McCallum, R. P.; Schiffer, C. A. Leuk. Res. 2005, 29, 463465. (8) Bayol, A.; Capdevielle, J.; Malazzi, P.; Buzy, A.; Bonnet, M. C.; Colloc’h, N.; Mornon, J. P.; Loyaux, D.; Ferrara, P. Biotechnol. Appl. Biochem. 2002, 36, 21-31. (9) Product Information. Rasburicase (Fasturtec), New York, Sanofi-Synthelabo, October 2003. (10) Nugent, K. D. In High-Performance Chromatography of Peptides and Proteins; Mant, C. T., Hodges, R. S., Eds.; CRC Press: Boca Raton, FL, 1991; Vol. 1, pp 279-287. (11) Tolley, L.; Jorgenson, J. W.; Moseley, M. A. Anal. Chem. 2001, 73, 29852991. (12) Wilson, K.; Walker, J. Principles and techniques of practical biochemistry, 5th ed.; Cambridge University Press: Cambridge, U.K., 2000. (13) Reschiglian, P.; Zattoni, A.; Roda, B.; Michelini, E.; Roda, A. Trends Biotechnol. 2005, 23, 475-483.

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whole proteins in native form. First, in FlFFF, the separation mechanism is structured by the action of a hydrodynamic field across an empty, capillary channel rather then by the interaction with a stationary phase.14 Since the separation space is empty, there is very little (if any) mechanical or shear stress on the protein molecule. This allows for the separation of high molar mass proteins and protein complexes or aggregates without sample entanglement/adsorption on the stationary phase. Second, FlFFF is so versatile that almost any solution can be used as mobile phase. This is a key point not only to avoid possible protein degradation due to the mobile phase but also not to affect ionization if further MS analysis is performed. Neither organic modifiers nor relatively high ionic strength buffers are required as in the case of RP HPLC15 or CZE16,17 in which, moreover, high voltages can contribute to alter proteins from their native form. Third, in the case of high molar mass proteins, FlFFF selectivity is higher than in SEC, which is in fact known not to be able to detect high molar mass aggregates. Fourth, the driving force that structures separation in FlFFF is the viscous force exerted on the sample components by the cross-flow stream. Retention in FlFFF is, in principle, proportional to the analyte diffusion coefficient.18,19 Differences in FlFFF retention can then indicate not only differences in molar mass but also in protein size and conformation. Because of these key features, FlFFF shows very effective for nondegrading separation and further characterization of high molar mass proteins and protein oligomers.20 HF FlFFF is a prototype subset of FlFFF. Fundamentals of HF FlFFF were given relatively recently, 21,22 and a wide selection of currently available HF membranes today makes this technique able to fractionate either nanometer- or micrometer-sized particles of different origin.23-27 HF FlFFF was coupled with MALDI/ TOFMS and ESI/TOFMS for protein analysis,28,29 and the use of microbore HF channels most recently improved HF FlFFF performance in protein analysis to a level comparable to that of standard FlFFF.30 With respect to conventional FlFFF, HF FlFFF shows unique features for the separation of functional protein drugs under nondegrading conditions before their MALDI/ (14) Ratanathanawongs-Williams, S. In Field-Flow Fractionation Handbook; Schimpf, M. E., Caldwell, K. D., Giddings, J. C., Eds.; Wiley-Interscience: New York, 2000; Chapter 17. (15) Brenner-Weiss, G.; Kirschhofer, F.; Kuhl, B.; Nusser, M.; Obst, U. J. Chromatogr., A 2003, 1009, 147-153. (16) Cao, P.; Moini, M. J. Am. Soc. Mass Spectrom. 1999, 10, 184-186. (17) Rochu, D.; Pernet, F.; Bon, C.; Masson, P. J. Chromatogr., A 2001, 910, 347-357. (18) Liu, M. K.; Li, P.; Giddings, J. C. Protein Sci. 1993, 2, 1520-1531. (19) Fuh, C. B.; Levin, S.; Giddings, J. C. Anal. Biochem. 1993, 208, 80-87. (20) Silveira, J. R.; Raymond, G. J.; Hughson, A. G.; Race, R. E.; Sim, V. L.; Hayes, S. F.; Caughey, B. Nature 2005, 437, 257-261. (21) Carlshaf, A.; Jo ¨nsson, J. A. J. Chromatogr. 1989, 461, 89-93. (22) Jo ¨nsson, J. A.; Carlshaf, A. Anal. Chem. 1989, 61, 11-18. (23) Lee, W. J.; Min, B.-R.; Moon, M. H. Anal. Chem. 1999, 71, 3446-3452. (24) Moon, M. H.; Lee, K. H.; Min, B.-R. J. Microcolumn Sep. 1999, 11, 676681. (25) Min, B.-R.; Kim, S. J.; Ahn, K.-H.; Moon, M. H. J. Chromatogr., A 2002, 950, 175-182. (26) Reschiglian, P.; Roda, B.; Zattoni, A.; Min, B.-R.; Moon, M. H. J. Sep. Sci. 2002, 25, 490-498. (27) Reschiglian, P.; Zattoni, A.; Roda, B.; Cinque, L.; Melucci, D.; Min, B.-R.; Moon, M. H. J. Chromatogr., A 2003, 985, 519-529. (28) Reschiglian, P.; Zattoni, A.; Cinque, L.; Roda, B.; Dal Piaz, F.; Roda, A.; Moon, M. H.; Min, B.-R. Anal. Chem. 2004, 76, 2103-2111. (29) Reschiglian, P.; Zattoni, A.; Roda, B.; Cinque, L.; Parisi, D.; Roda, A.; Dal Piaz, F.; Moon, M. H.; Min, B.-R. Anal. Chem. 2005, 77, 47-56. (30) Kang, D.; Moon, M. H. Anal. Chem. 2005, 77, 4207-4212.

TOFMS characterization. Low channel volume (in the order of 100 µL, or less in the case of microbore HF FlFFF30) reduces sample dilution. This is a key point to increase detection sensitivity. Possibly disposable usage of the HF channels eliminates the risk of run-to-run sample carryover, a crucial aspect for purity analysis. The uricase samples were fractionated through HF FlFFF, and sample fractions collected at the HF FlFFF system outlet were analyzed by MALDI/TOFMS to finally assess sample purity as well as the possible presence of solution-phase aggregates and of different structural forms of uricase. Since the gentle separation mechanism of HF FlFFF does not alter the ternary and quaternary structures of proteins, the enzymatic activity of fractionated uricase was not expected to be altered either. Sample fractions were then analyzed to evaluate the enzymatic activity of their components. For this purpose, a new CL assay was specifically designed for the ultrasensitive evaluation of uricase activity.31 The assay relies on detection of the hydrogen peroxide produced by the enzymatic reaction through the CL oxidation of bis(2,4,6-trichlorophenyl)oxalate (TCPO) in the presence of the fluorescent energy acceptor dipyridamole. A key feature of this assay is its extremely high sensitivity (the limit of detection is of the order of 0.01 ng of uricase), which makes it suitable for the enzymatic activity measurement of even trace components of the sample fractions obtained by HF FlFFF. EXPERIMENTAL SECTION Samples. Rasburicase from A. flavus expressed in S. cerevisiae was obtained from Sanofi-Syntelabo (Milan, Italy). The enzyme formulation for human use consisted in a lyophilized powder of specific activity 18.2 units/mg (one enzyme activity unit converts 1 µmol of uric acid to allantoin per minute at 30 °C and pH 8.9) containing some excipients such as alanine, mannitol, NaH2PO4, and Na2HPO4. Recombinant uricase from Candida sp. expressed in E. coli (specific activity 4.5 units/mg) was from Sigma-Aldrich (St. Louis, MO, Catalog No. U0880), hereafter named Sigma uricase. It was supplied as lyophilized powder containing citrate (as stated by the manufacturer). All used reagents were of analytical grade. RP HPLC-ESI/Q-TOFMS. An HPLC Gold System (Beckman Instruments, Fullerton, CA) was employed. A 20-µL aliquot of the samples at a concentration of 1 mg/mL was injected into a C4 column (5-µm particle diameter; 150 mm long × 4.60 mm internal diameter Jupiter; Phenomenex, Torrance, CA) and eluted with a mixture of solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in ACN) using a 30-65% B linear gradient over 30 min at a flow rate of 1.0 mL/min. A model UV6000LP high-sensitivity diodearray detector (DAD) (ThermoQuest, Austin, TX) equipped with a 5-cm path-length, fiber-optic guided light-pipe cell was employed. The UV/visible signal was recorded in the wavelength range from 190 to 400 nm. A Q-TOF hybrid mass analyzer, the model Q-ToF Micro (Micromass, Manchester, U.K.), was employed with a Z-spray ion source. The HPLC effluent was split to obtain a flow rate of 200 µL/min at the ion source inlet. The source temperature was set (31) Guardigli, M.; Sestigiani, E.; Mandreoli, M.; Ramazzotti, E.; Santoro, A.; Roda, A. In V Mediterranean Basin Conference on Analytical Chemistry, Silvi Marina, Teramo, Italy, 24-28 May, 2005. Abstract Book, Caroli, S., Pino, D., Eds.; Istituto Superiore di Sanita`, Rome, Italy, 2005; p 147. ISSN 0393-5620.

at 80 °C, the capillary voltage at 3000 V, the cone voltage at 40 V, and the collision energy at 5 V. The mass spectra were acquired in an m/z range spanning from 500 to 3000, and mass calibration over the entire range was performed by means of direct injection of a mixture of horse heart myoglobin and bovine trypsinogen (both from Sigma-Aldrich). Spectra were processed by MassLynx (Waters, Milford, MA). MALDI/TOFMS. MALDI/TOFMS analysis of the whole sample was performed using a Voyager DE Pro (Applied Biosystems, Foster City, CA) equipped with a pulsed N2 laser operating at 337 nm. Positive ion spectra were acquired in linear mode over an m/z range from 10 000 to 150 000 using a 25 000-V accelerating voltage, a 22 500-V grid voltage, and a delay extraction time of 150 ns. The spectrum for each spot was obtained by averaging the result of 120 laser shots. External mass calibration was performed using the single- and double-charged ions of bovine serum albumin (Sigma-Aldrich). The analysis was performed by spotting on the target plate 1 µL of the sample, corresponding to ∼1 µg of enzyme, mixed with an equal volume of the matrix solution. Solutions of (A) 50 mg/mL sinapinic acid or of (B) 10 mg/mL R-cyano-4-hydroxycinnamic acid in 1:1 v/v ACN/H2O containing 0.1% v/v TFA were used as matrix. Trypsin digest from PAGE was analyzed in reflector, positive detection mode over an m/z range from 500 to 5000 using a 20 000V accelerating voltage, a 15 200-V grid voltage, and an extraction delay time of 40 ns. Matrix B was used. Sinapinic acid and R-cyano-4-hydroxycinnamic acid were purchased from Sigma-Aldrich, ACN from J. T. Baker (Phillipsburg, NJ), and TFA from Carlo Erba (Milan, Italy). Water was obtained from a Milli-Q Plus purification system (Millipore, Bedford, MA). SDS PAGE. The enzyme samples (10 µg in 20 µL of H2O) were analyzed by 12% polyacrylamide (w/v) gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS) buffer (25 mM Tris, 198 mM glycine, 0.1% w/v SDS, pH 8.3). After electrophoresis, the gel slabs were stained using Coomassie Blue (Bio-Rad, Hercules, CA). The SDS PAGE lanes were excised, destained with ACN, reduced with dithithreitol (DTT, Sigma-Aldrich) and alkylated with iodoacetamide (Sigma-Aldrich). The SDS PAGE lanes were incubated overnight at 37 °C with trypsin (Gold Mass spectrometry Grade, Promega, Madison, WI) in 50 mM NH4HCO3 (pH 7.5). 2D SDS PAGE. The first dimension (isoelectric focusing, IEF) was performed on a Protean IEF Cell (Bio-Rad). Linear pH 3-10 IPG strips were rehydrated for 16 h at 50 V in rehydrating buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.8% carrier ampholite). Samples (10 µg) were applied during rehydration. Gels were equilibrated for 15 min in 2 mL of a 125 mM DTT solution in equilibration buffer (2% SDS, 6 M urea, 30% glycerol, 5 mM Tris-HCl, pH 8.6) and for 10 min with 2.5 mL of a 250 mM iodoacetamide solution in equilibration buffer. Samples from the IEF run were subsequently run on 12% (w/ v) PAGE. Strips were sealed with 0.5% agarose into a mini Protean 3 chamber (Bio-Rad), and the gels were run at a constant voltage of 120 V. The gels were stained using colloidal Coomassie Blue G-250 (Bio-Rad) for 16 h and subsequently destained using 25% MeOH in H2O. Digital images of the gels were acquired by a GS800 calibrated densitometer (Bio-Rad) and processed using the PDQuest software (Bio-Rad). Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

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Figure 1. (a) HF FlFFF channel design. (b) HF FlFFF system setup.

The 2D SDS PAGE spots were incubated overnight at 37 °C with trypsin (Gold Mass Spectrometry Grade, Promega) in 50 mM NH4HCO3 (pH 7.5). HF FlFFF. The HF FlFFF system was a prototype, whose setup and run operations were described in previous papers.26-29 The HF FlFFF channels were homemade of a piece of polysulfone HF membrane sheathed by two pieces of 1/8-in.-o.d. Teflon tube. A tee connection was positioned between the two tubes to make the radial outlet of the flow field. Two hand-tight male fittings were positioned at the channel inlet and outlet. The HF membrane had a 30 000 Mr cutoff, nominal inner radius of 0.040 cm, and length of 24 cm (dried conditions). The HF FlFFF system employed two pumps, a HPLC pump model LC-2000Plus (Jasco, Tokyo, Japan) and a syringe pump (model pump 11; Harvard Bioscience, Holliston, MA). Sample injection was made via a model 7125 injection valve (Rheodyne, Cotati, CA) equipped with a 20-µL external PEEK loop. The required sample injection/focusing/ relaxation process was set for 3 min, with the focusing point determined as described elsewhere.23 The radial flow rate (Vrad) was set at 0.4 mL/min and the longitudinal, outlet flow rate (Vout) at 0.3 mL/min. Spectrophotometric UV/visible detection was made by the UV6000LP spectrophotometer operating at 280 nm. The mobile phase was a 50 mM solution of ammonium acetate (Sigma-Aldrich) in Milli-Q water at pH 7.0. This solution is commonly employed as a nondegrading protein solvent for MS operations. We already proved that this mobile phase neither degrades proteins nor suppresses ionization.29 Proteins were 1088

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dissolved in the mobile phase in a concentration range of 0.050.2% (w/v). The HF FlFFF channel design and the HF FlFFF system diagram are reported in Figure 1a and b, respectively. CL Assay. The assay was performed in 96-well microtiter plates. When necessary, the uricase samples were diluted in Milli-Q water. Fixed sample aliquots (50 µL) were dispensed in the wells. Subsequently, 25 µL of a 1.0 mM solution of uric acid in 0.1 M imidazole buffer (pH 7.5) was added to each well, and the plate was incubated for 45 min at 37 °C. After incubation, the enzymatic reaction was stopped by adding 100 µL of a 0.1 mM dipyridamole solution in ACN, and the H2O2 produced during the incubation step was determined by CL. The measurements were performed using a microtiter plate luminometer (Luminoskan Ascent, Thermo Labsystems, Helsinki, Finland) equipped with a built-in reagent dispenser: 50 µL of a TCPO suspension in 1:10 (v/v) acetone/H2O (prepared immediately before the measurement by diluting a 5 mM TCPO solution in acetone) was added to each well, and the resulting flash-type CL emission was recorded using a 2-s integration time. The uricase activity was evaluated by interpolating the CL signal, after blank subtraction, on a linear calibration curve obtained for each experimental session. Rasburicase standard solutions in the range 0-1.0 ng/ well of enzyme (corresponding to ∼(0-2) × 10-5 unit/well) where used for the calibration. TCPO, uric acid, and dipyridamole were from Sigma-Aldrich.

Figure 2. MALDI/TOF mass spectrum of Rasburicase. (a) m/z range, 10 000-40 000; (b) m/z range, 20 000-150 000. MALDI matrix: (A) (50 mg/mL sinapinic acid).

RESULTS AND DISCUSSION The combined approach was carried out as follows: (1) preliminary analysis by RP HPLC with UV/visible DAD, ESI/QTOFMS, and MALDI/TOFMS; (2) peptide fingerprinting by SDS PAGE and 2D SDS PAGE with MALDI/TOFMS after trypsin digestion of the lanes; (3) HF FlFFF with MALDI/TOFMS and CL enzyme activity assay. Step 1. RP HPLC with UV/visible DAD allowed for a preliminary evaluation of the uricase sample purity. Rasburicase appeared to be highly pure while Sigma uricase showed peaks that could be potentially ascribed to unknown protein impurities. Assuming constant absorptivity, such peaks appeared to account for almost 30% w/w of the total protein content. RP HPLC-ESI/Q-TOFMS results for the two samples are separately reported as Supporting Information (Figures I, II). The total-ion chromatogram of Rasburicase is reported in Figure Ia. The mass spectrum of peak 1 in Figure Ia is reported in Figure Ib. It shows the presence of only one protein of Mr ) 34 153.95 ( 0.81, which agrees with the theoretical average Mr value of the uricase monomer deduced from the N-acetylated sequence (Mr ) 34 151.66).8 The presence of just the enzyme monomer may be due to the fact that the RP HPLC system can alter noncovalent interactions between the protein subunits. Peaks 2 and 3 indicated the presence of impurities, the mass spectra of which could be ascribed to a polymer with a high polydispersity degree, constituted of monomeric units of Mr ) 44 (data not shown). Such an Mr value could correspond to the -CH2CH2O- monomeric unit of poly(ethylene glycol) (PEG). PEG is often used in industrial processes of protein refolding to protect the protein surface. Blank analysis excluded that the found polymeric species had a source different from the sample. The total-ion chromatogram of Sigma uricase is reported in Figure IIa. Peak 1, which by comparison with RP HPLC DAD of Rasburicase can be ascribed to uricase, showed the mass spectrum reported in Figure IIb. This spectrum was impossible to deconvolute, likely because of the presence of posttranslational variants of the enzyme that were not resolved by RP HPLC. The mass spectrum relative to the peak 2 corresponded to a protein with Mr ) 28 425.12 ( 1.88 (data not shown). Mass spectra of the peak cluster 3 could be assigned to protein impurities of Mr values ranging from 10 000 to 13 000 (data not shown).

Figure 3. MALDI/TOF mass spectrum for Sigma uricase. MALDI matrix: (A) (50 mg/mL sinapinic acid).

MALDI/TOFMS of Rasburicase confirmed its high purity. The mass spectrum recorded in a m/z range between 10 000 and 40 000 is reported in Figure 2a. It shows the presence of three signals corresponding to the monocharged (m/z ∼34 000), doublecharged (m/z ∼17 000), and triple-charged (m/z ∼11 000) ions of the uricase monomer. At higher m/z values (Figure 2b), it is also possible to observe the presence of four intense signals at m/z values of ∼34 000, ∼68 000, ∼99 000, and ∼132 000. The m/z values of these signals fairly agree with the molar mass of the different oligomers that are known to constitute the supramolecular structure of uricase (monomer, dimer, trimer, and tetramer, respectively). Because of the significant amount of sample loaded to evaluate the possible presence of impurities, it was necessary to investigate whether the presence of uricase oligomers could be due to MALDI-induced phenomena or to their actual presence in the sample. Different sets of MALDI/TOFMS experiments were performed using the two different matrix solutions, and the effect of changing the sample/matrix ratio or of using different laser energies was considered as well. In all cases, the presence of signals ascribed to uricase oligomers was observed. The mass spectrum of Sigma uricase (Figure 3) shows a weak, broad signal corresponding to a protein with an average Mr of 35 000, which can be attributed to the uricase monomer. The signal broadness might be ascribed to the occurrence of protein modifications, of likely posttranslational origin, which can give raise to an almost continuous signal distribution. Many additional peaks, which are imputable to protein impurities with average Mr values ranging from 10 000 to 28 000, are also present. Step 2. SDS PAGE profiles of the two samples are separately reported as Supporting Information (Figure III). In both samples, it was possible to observe the presence of an intense band with Mr ranging from 25 000 to 40 000. However, the Sigma uricase showed the presence of many additional, though less intense bands at lower Mr values, which could be ascribed to protein impurities. The main lanes obtained from Sigma uricase (b1, b2) were excised and digested with trypsin, and the peptide mixture was analyzed through MALDI/TOFMS. Peptide mass lists were processed by means of the ProFound database search algorithm (http://prowl.rockefeller.edu). Lane b1 corresponded to the peptide fingerprint of urate oxidase from Candida sp., and lane b2 to the 2,3-bisphosphoglycerate-dependent phosphoglycerate Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

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mutase from E. coli, which is the host microorganism in which the DNA from Candida sp. was cloned. This protein may have not been eliminated during the industrial purification process. Other minor impurities found in the Sigma uricase sample, with Mr of ∼10 000, may also derive from E. coli, but they were not identified because of their small amount. This is an example showing that the presence of impurities that are difficult to identify can seriously affect characterization of recombinant proteins. Tryptic digest analysis of the Rasburicase excided lane confirmed the presence of only one protein in the sample, which in fact corresponded to the peptide fingerprint of uricase from A. flavus. Since SDS PAGE is characterized by a relatively poor resolution in the case of proteins of similar molar mass (e.g., due to isoforms or posttranslational modifications), bidimensional electrophoresis (2D SDS PAGE) was used to separate proteins by differences in their isoelectric point. Results are separately reported as Supporting Information (Figure IV). 2D SDS PAGE of Sigma uricase (Figure IVa) showed the presence of a high number of spots at Mr ≈ 34 000, which differed in their isoelectric point. The isoelectric point differences could be explained as posttranslational modifications on the enzyme. The presence of such a high number of isoforms might explain the highly convolute RP HPLC-ESI/TOFMS spectrum (Figure IIb, Supporting Information), as well as the broad m/z distribution of the MALDI/TOFMS signal (Figure 3) ascribed to the uricase monomer. MALDI/TOFMS of the tryptic digest of the spots followed by database search confirmed the presence of uricase from Candida sp. 2D SDS PAGE of Rasburicase (Figure IVb, Supporting Information) showed the presence of only three spots, which likely corresponded to different enzyme isoforms with different isoelectric point. Step 3. The approach described in the above sections allowed us to detect sample impurities. However, the presence in the Rasburicase MALDI/TOFMS spectrum of the monomer signal together with signals possibly ascribed to uricase oligomers required us to establish whether these different forms of uricase were present in the sample or were rather due to analysis artifacts. Since HF FlFFF does not alter the native protein structure,29 the uricase samples were run through HF FlFFF, and collected fractions were analyzed by MALDI/TOFMS. Since HF FlFFF retention is related to protein diffusion coefficient, which is related to protein Mr,32 retention times were scaled to Mr values using the calibration plot obtained in previous work with standard proteins.29 If the Mr values measured by HF FlFFF are related to the Mr values independently obtained through MALDI/TOFMS, the actual presence of protein complexes in solution could be thereby confirmed. The Rasburicase fractograms obtained in four repeated runs are reported in Figure 4a. A single, highly reproducible band is observed, the retention time of which agrees with the Mr value of the uricase tetramer. Nevertheless, the Sigma uricase fractogram (Figure 5a) shows a bimodal profile with two maximums, whose retention time values agree with what expected from the Mr values of the uricase dimer and tetramer, respectively. MALDI/ TOFMS was performed on the HF FlFFF fractions. For the (32) Li, P.; Hansen, M. In Field-Flow Fractionation Handbook; Schimpf, M. E., Caldwell, K. D., Giddings J. C., Eds.; Wiley-Interscience: New York, 2000; Chapter 28.

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Figure 4. HF FlFFF of Rasburicase. (a) UV/visible fractogram at 280 nm; four repeated runs. (b) Enzymatic activity of the collected fractions determined by the CL assay.

Figure 5. HF FlFFF of Sigma uricase. (a) UV/visible fractogram at 280 nm. (b) Enzymatic activity of the collected fractions determined by the CL assay.

Rasburicase fractions, spectra very similar to the spectrum of the unfractionated sample (Figure 2b) were obtained (data not shown). The spectra for the Sigma uricase fractions collected at retention time values that corresponded to the elution of the dimer and of the tetramer are reported in Figure 6a,b, respectively. In both cases, the spectra contain a signal at an m/z value that corresponds to the enzyme monomer. These findings confirm that MALDI could break up the uricase oligomers that HF FlFFF showed to be present in solution. In the spectrum obtained from the Sigma uricase fraction collected at the retention time corresponding to the molar mass of the dimer (Figure 6a), other signals at lower m/z values are found (∼10 400; ∼12 600; ∼13 600; ∼14 100; ∼28 400). They can be assigned to protein impurities, as suggested by PAGE analysis. Since the Mr cutoff value of the HF membrane was 30 000, such proteins of Mr