Characterization of a Protein Conjugate Using an Asymmetrical-Flow

Article pubs.acs.org/ac

Characterization of a Protein Conjugate Using an Asymmetrical-Flow Field-Flow Fractionation and a Size-Exclusion Chromatography with Multi-Detection System Katja Rebolj,† David Pahovnik,† and Ema Ž agar*,†,‡ †

National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia Centre of Excellence for Polymer Materials and Technologies, Tehnološki park 24, 1000 Ljubljana, Slovenia

‡

ABSTRACT: In this study we present detailed characterization of a protein-PEG conjugate using two separation techniques, that is, asymmetrical-flow field-flow fractionation (AF4) and sizeexclusion chromatography (SEC), which were online coupled to a series of successively connected detectors: an ultraviolet, a multiangle light-scattering, a quasi-elastic light-scattering, and a refractive-index detector (UV-MALS(QELS)-RI). Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a complementary characterization technique. The results of AF4 as well as SEC on two columns connected in series, with both separation techniques coupled to a multidetection system, indicate the uniform molar mass and chemical composition of the conjugate, that is, the molar ratio of protein to PEG is 1/1, the presence of minute amounts of residual unreacted protein and the aggregates with the same chemical composition as that of the conjugate. Since the portion of aggregated species is smaller in the acetate buffer solution containing 5% sorbitol than in the acetate buffer solution with 200-mM sodium chloride, the former buffer solution is more suitable for conjugate storage. The separation using only one SEC column results in poorly resolved peaks of the PEGylated protein conjugate and the aggregates, whereas MALDI-TOF MS analysis reveal the presence of the residual protein, but not the aggregates.

P

column calibration using PEG standards significantly different from the absolute molar-mass values, since the hydrodynamic volume (Vh) of a conjugate is much smaller than that of the PEG standard with comparable molar mass. In AF4, the separation of sample’s constituents is achieved within a parabolic laminar-flow, against which a perpendicular cross-flow is applied in order to drive the solutes into different velocities of laminar flow.6−9 The separation mechanism is based on the differences in mobility of sample’s constituents, which is defined by their diffusion coefficient. The separation is performed in a channel consisting of two plates separated by a spacer. The bottom channel plate is covered by a semipermeable membrane with a defined pore-size. The main parameter influencing the separation efficiency is the cross-flow velocity generated through the membrane. In a normal separation mechanism (up to a size of about 1 μm) the sample constituents are eluted from smaller to larger Vh. With the use of an efficient separation technique combined with a multidetection system (e.g., ultraviolet (UV) − multiangle light-scattering (MALS)−quasi-elastic light-scattering (QELS)−differential refractive index (RI)) the absolute molar-mass averages (Mn and Mw), molar-mass distribution (described by the dispersity, ĐM), the radius of gyration (Rg),

EGylation is a frequently used covalent modification of pharmaceutically active substances (e.g., peptides, proteins) by poly(ethylene glycol) (PEG) to improve the efficiency of drug treatment. Through PEGylation, the size of the obtained conjugate is enlarged in comparison to the protein, which results in a reduced plasma clearance of several orders of magnitude.1 Additionally, such an introduced hydrophilic surface of the conjugate lowers the opsonization and its uptake by the mononuclear phagocytic system. The first PEGylated drug launched on the market was bovine adenosine deaminase in 1990 by Enzon Inc. Since then, dozens of successful replacements have been approved as long-lasting drugs in clinical use for the treatment of severe diseases.2 PEGylation is considered to be one of the most commonly used and successful technique so far developed to enhance the therapeutic and biotechnological potential of proteins.3,4 Size-exclusion chromatography (SEC) and asymmetricalflow field-flow fractionation (AF4) are flow-assisted analytical separation techniques.5−9 SEC is an established method, nowadays routinely used for determination of the molar-mass averages and the molar-mass distribution of various synthetic and natural macromolecules.5,6 The pitfalls of SEC are well recognized, for example, the nonspecific binding of solutes to the column packing material and if not combined with a lightscattering detector, the necessity of column calibration with standards. Such obtained molar-masses are relative values, which are in the case of PEGylated protein conjugates and © 2012 American Chemical Society

Received: April 19, 2012 Accepted: August 9, 2012 Published: August 9, 2012 7374

dx.doi.org/10.1021/ac3010378 | Anal. Chem. 2012, 84, 7374−7383

Analytical Chemistry

Article

original solutions, without dilution, filtration or any other treatment. Asymmetrical-Flow Field-Flow Fractionation (AF4) with a Multidetection System. An isocratic pump (Agilent 1260, Agilent Technologies, USA) with an online vacuum degasser (Agilent 1260) and an autosampler (Agilent 1260) delivered the carrier flow and handled the sample injection onto an AF4 Eclipse3+ (Wyatt Technology Europe GmbH, Germany). A trapezoidal long channel LC 240 mm was used with a spacer height 350 μm/width 21.5 mm, and a Nadir Cellulose RC (Regenerated Cellulose) PCK5 membrane with a 10-kDa cutoff (Wyatt Technology Europe GmbH, Germany). The separations were carried out at room temperature. The running eluent prepared with MiliQ water (18.2 MΩ/ cm) was a 50-mM sodium nitrate (NaNO3) with sodium azide (NaN3) (0.02% w/v) (both from Sigma-Aldrich), which was added to prevent micro-organism growth. The eluent was filtered through a Nylon 66 membrane filter with a pore-size of 0.45 μm (Supelco Analytical 58067, USA) and sonicated for 5 min in a water-bath (Branson 2210, USA). An additional filter (0.1-μm pore size, Durapore membrane) was added to the system after the HPLC pump (PEEK Inline Filter Holder). A focusing/injection step of 2 min with a focusing-flow rate of 3 mL/min and an injection-flow rate of 0.2 mL/min was followed by a focusing/relaxation step of an additional 2 min. The sample volume injected into the channel was between 5 and 100 μL for an injected sample mass of 50−200 μg. An elution step was performed with linear gradient cross-flow rates, usually from 3 to 0 mL/min in 8 min. The detector-flow rate was 1 mL/min, except for the QELS measurements, where the detector-flow rate was reduced to 0.2 mL/min to obtain more accurate results. For the detection we used a system of successively connected detectors online: an ultraviolet (UV) detector, operating at a wavelength of 280 nm (Agilent 1260 VWD); a multiangle lightscattering (MALS) detector (DAWN-HELEOS, Wyatt Technology Corporation, USA), operating at a wavelength of 658 nm; a quasi-elastic light-scattering (QELS) module that is embedded at angle 99° of the MALS detector within the DAWN-HELEOS instrument (Wyatt Technology Corporation, USA); and a differential refractive index (RI) detector, operating at a wavelength of 658 nm (Optilab rEX, Wyatt Technology Corporation, USA). Prior to the measurement the MALS detector was calibrated using toluene, while the other detectors were normalized using bovine serum albumin (BSA). The AF4 fractograms and the SEC chromatograms were corrected for interdetector band broadening, the parameters of which (instrumental and mixing terms) were determined on monodisperse G-CSF protein sample. For the data acquisition and evaluation Astra 5.3.4.20 software (Wyatt Technology Corporation, USA) was utilized. The determination of Mw and Mn values from MALS also required the specific refractive index increments (dn/dc) of the samples, which were determined as follows; The dn/dc value of the protein was determined from the RI detector response with the exact protein injected mass calculated from the UV detector response and the known value of protein specific UV extinction coefficient at 280 nm, that is, ε = 0.815 mL/g cm, which was calculated using the PC GENE computer analysis program of protein sequences (IntelliGenetics). The dn/dc value of 0.186 mL/g was obtained for the protein. The dn/dc of PEG in 50mM NaNO3 with sodium azide (NaN3) (0.02% w/v) was determined from the RI response assuming the PEG mass

the hydrodynamic radius (Rh), and the subsequent conformation of macromolecules can be determined.8−10 Furthermore, the simultaneous use of two concentration detectors (UV and RI) enables a precise determination of the chemical composition along the molar mass distribution of the complex, two-component macromolecules (e.g., protein conjugates, copolymers etc.), if the response factors of the detectors for these two components are sufficiently different.11−15 A detailed analysis of the protein conjugates was found to be difficult in the past since conjugates can be complex mixtures, consisting of an unreacted protein and/or PEG reagent, products with a different degree of PEGylation, and in addition, the presence of aggregates in solution is possible. The methods used for the characterization of conjugates are calorimetric, radio-labeled, electrophoretic, mass spectrometric, chromatographic, etc.16−21 Chromatographic methods vary depending on the mechanism of separation, that is, interaction mechanism (HPLC), size-exclusion mechanism (SEC), and field-flow fractionation (AF4).22−25 SEC and AF4 were also coupled to various molar-mass (light-scattering detector, viscometer) and concentration detectors (UV, RI),26−33 which enable simultaneously determination of the absolute molar-masses, the size and the chemical composition of different conjugates15,31−35 as well as various synthetic copolymers11,36 and nanoparticles.37−39 The purpose of this paper is to compare two separation techniques (AF4 and SEC), both coupled to the UVMALS(QELS)-RI detection system, for the characterization of PEGylated protein conjugate. The results were compared to those obtained by MALDI-TOF MS. An efficient separation and multidetection system enables us to qualitatively and quantitatively evaluate the individual constituents of a conjugate sample stored in two different buffer solutions. The results are important for the optimization of the synthesis conditions for conjugate preparation and the selection of the proper conditions for protein-conjugate storage.

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EXPERIMENTAL SECTION Materials. The protein conjugate refers to a PEGylated granulocyte colony stimulating factor (PEG-G-CSF) and has a molecular weight of the protein component of 18,798 Da. The G-CSF protein was produced from heterologous expression in Escherichia coli. The recombinant human PEG-G-CSF was synthesized by attaching a methoxypolyethylene glycol propionaldehyde (mPEG-ald) to the N-terminal amino acid of G-CSF. The PEGylated conjugate was stored in two buffer solutions, that is, a 10-mM acetate buffer solution with 5% sorbitol at pH 3.4 (buffer solution 1) and a conjugate concentration of 16.8 mg/mL, and a 50-mM acetate buffer solution with 200-mM sodium chloride at pH 4.5 (buffer solution 2) and at a conjugate concentration of 3.4 mg/mL. The nonconjugated protein was stored in a 10-mM acetate buffer solution with 5% sorbitol at pH 4.5 and at a concentration of 4.5 mg/mL. The monofunctional methoxypolyethylene glycol propionaldehyde (mPEG-ald, NOF Corporation, Japan) with a number average molar mass of 20,664 Da and dispersity (Mw/Mn) of 1.03 was used for the protein conjugation. The mPEG-ald was dissolved in 50-mM sodium nitrate (Sigma Aldrich, USA), which was also a running eluent for the AF4 and SEC measurements. Prior to analysis the protein and the protein-conjugate were stored in refrigerator at 4 °C for four months. They were analyzed as 7375



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Figure 1. AF4-MALS fractograms (solid line: RI response, dashed line: LS response at angle 90°) of the pure protein (red), the PEG reagent (blue) and the conjugate stored in buffer solution 1 (green) and 2 (black) together with the molar mass and the hydrodynamic radius versus elution volume.

recovery from the SEC column of 100%. The value of 0.134 mL/g was obtained which match perfectly with the literature data.40 Size-Exclusion Chromatography (SEC) with a Multidetection System. SEC measurements were performed on the same apparatus as the AF4 (see previous paragraph) only that it was operating in the SEC mode. The separations were carried out on: (i) a PROTEEMA GPC analytical column with a precolumn (300 × 8 mm, Polymer Standards Service, PSS, Germany, molar mass range of 100−100 000 Da), and (ii) successively connected PROTEEMA and SUPREMA (300 × 8 mm, Polymer Standards Service, PSS, Germany, molar mass range of 100−30 000 Da) GPC columns with a precolumn. The mobile phase was in both cases an aqueous solution of 50-mM sodium nitrate with sodium azide (0.02% w/v). The eluent flow rate was 0.8 (0.6) mL/min for the separations carried out on one (two) columns. The mass of the samples injected onto the column was typically 150 μg. The data acquisition and evaluation were performed using Astra 5.3.4.20 software (Wyatt Technology Corporation, USA). Mass Spectrometry. The matrices used for the MALDITOF mass spectrometry were 2′,4′-dihydroxyacetophenone (DHAP), super DHB (9/1 mixture of 2,5-dihidroxybenzoic acid and 2-hydroxy-5-methoxy benzoic acid), and trans-2-[3-(4tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), which were supplied by Sigma-Aldrich. The trifluoroacetic acid and sodium trifluoroacetic acid were also supplied by Sigma-Aldrich. First, the solutions of protein and conjugate were dialyzed using nitrocellulose membrane filters (Milipore, 0.025 μm) for 10−15 min to remove the buffer and the preservative. After dialysis the protein solution was mixed with a 20 mg/mL solution of DHAP matrix dissolved in a 30/70 mixture of

acetonitrile/water with 0.1% TFA added. The conjugate’s solutions were mixed with a 10-mg/mL solution of super DHB dissolved in a 50/50 mixture of acetonitrile/water and 0.1% TFA. The volume ratio between the sample and the matrix solutions was 1/20. Then, 1 μL of the final solution was spotted on the target plate using the dried-droplet method. The PEG used for the protein modification was dissolved in a 50/50 mixture of acetonitrile/water with 0.1% TFA added and mixed with a 20-mg/mL solution of DCTB matrix and 0.1% NaTFA. The volume ratio between the PEG solution and the matrix solution was 1/20. Then, 1 μL of the final solution was spotted on the target plate using the dried-droplet method. The mass spectra were acquired with a Bruker UltrafleXtreme MALDI-TOF-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with a nitrogen laser (λ = 337 nm). The reflectron positive ion mode was used to acquire the mass spectrum of the PEG reagent and the linear positive ion mode was used to acquire the mass spectra of the protein and conjugate samples. The calibration was made externally with a Peptide calibration standard II (Bruker Daltonics) for the linear positive mode and a poly(methyl methacrylate) standard with a molar-mass of 27-kDa and an uniform molar-mass distribution for the reflective positive mode using the nearest-neighbor positions.

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RESULTS AND DISCUSSION The pure protein, PEG reagent and protein conjugate sample stored in two different buffer solutions were carefully analyzed by employing the AF4 separation technique combined with light-scattering based detection. The fractograms of the pure protein and the PEG reagent show significantly different elution volumes (Vel = 9.3 mL for protein and 11.1 mL for PEG) despite their comparable molar masses (Mw = 18.2 kDa, ĐM = 7376



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Table 1. Number-Average Molar-Mass (Mn), Weight-Average Molar-Mass (Mw), Dispersity (ĐM), and Hydrodynamic radius (Rh) Determined by AF4 or SEC (One or Two Columns) Coupled to a Multidetection Systema AF4-UV-MALS-RI pure protein PEG reagent PEGylated protein conjugate in buffer solution 1 conjugate component−protein conjugate protein−PEG PEGylated protein conjugate in buffer solution 2 conjugate component−protein conjugate protein−PEG a

SEC-UV-MALS-RI (one column)

SEC-UV-MALS-RI (two columns)

AF4-QELS

Mn (kDa)

Mw (kDa)

ĐM b

Mn (kDa)

Mw (kDa)

ĐM b

Mn (kDa)

Mw (kDa)

ĐM b

Rh (nm)

18.2 ± 0.3 20.9 ± 0.4 39.3 ± 0.6

18.2 ± 0.3 21.3 ± 0.5 39.7 ± 0.5

1.00 1.02 1.01

18.3 ± 0.3 21.0 ± 0.5 51.2 ± 1.4

18.3 ± 0.4 21.2 ± 0.5 52.8 ± 1.0

1.00 1.01 1.04

18.3 ± 0.3 20.8 ± 0.3 38.8 ± 0.5

18.3 ± 0.3 21.3 ± 0.3 38.9 ± 0.5

1.00 1.02 1.01

2.2 ± 0.2 4.4 ± 0.3 5.1 ± 0.3

18.2 ± 0.5 21.1 ± 0.6 39.1 ± 0.6

18.3 ± 0.5 21.4 ± 0.4 39.4 ± 0.6

1.01

24.7 ± 0.6 26.4 ± 1.9 44.1 ± 1.2

25.2 ± 1.0 27.6 ± 1.1 44.8 ± 1.1

1.02

18.1 ± 0.6 20.7 ± 0.5 39.1 ± 0.4

18.1 ± 0.5 20.8 ± 0.5 39.1 ± 0.4

1.01

4.9 ± 0.4

18.0 ± 0.7 21.1 ± 0.6

18.1 ± 0.6 21.3 ± 0.6

20.9 ± 1.2 23.2 ± 1.0

21.2 ± 0.6 23.6 ± 1.4

18.1 ± 0.5 21.0 ± 0.2

18.1 ± 0.4 21.0 ± 0.2

The results represent an average value of three measurements. bĐM = Mw/Mn.

Figure 2. AF4 fractogram (black line, RI response; blue line, UV response; red line, LS response at angle 90°) of the conjugate in buffer solution 2.

Figure 3. Enlarged RI fractograms representing the PEGylated protein conjugate in buffer solutions 1 (green) and 2 (black) together with the molar mass vs elution volume; squares for PEGylated protein conjugate, down triangles for PEG constituent of conjugate and circles for the protein constituent of the conjugate.

1.00 for protein and Mw = 21.3 kDa, ĐM = 1.02 for PEG reagent), (Figure 1, Table 1). These results indicate large differences in the hydrodynamic volumes (Vh) of the PEG and the protein. The conjugate sample in both storage solutions shows three peaks: a peak with an apex at 9.3 mL and very-lowintensity RI, UV and LS responses that corresponds to the presence of a minute amount (∼0.2 wt.%) of residual unreacted protein in the conjugate sample, a main peak due to the PEGylated protein conjugate at 11.9 mL and a peak at a high elution volume (15.6 mL) with a low-intensity UV and RI

response, but a high-intensity LS response, which is due to the presence of a small amount of aggregated species in both solutions of the conjugate sample (Figure 2). A signal that would indicate the presence of unreacted PEG reagent in the conjugate sample was not detected. The determined weight-average molar-mass (Mw) and molarmass dispersity (ĐM) of the PEGylated protein conjugate is 39.7 kDa/1.01 in buffer solution 1 and 39.4 kDa/1.01 in buffer solution 2 (Figure 1, Table 1). In order to determine the chemical composition of the conjugate, the combination of two 7377



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Figure 4. Enlarged AF4 fractogram representing aggregates of the conjugate sample stored in buffer solution 2 (black line, RI response; blue line, UV response; red line, LS response at angle 90°) together with the molar mass (bottom) and the radius of gyration (top) vs elution volume.

conjugate’s concentration was much higher in the former buffer solution (16.8 vs 3.4 mg/mL), that is, the estimated weightfraction of the aggregated species represents ∼0.1 wt.% vs ∼0.7 wt.%, respectively. The average molar ratio of protein to PEG in the aggregates was determined to be ∼1/1, which is similar to that in the conjugate. The aggregates show the distribution in molar mass as indicated by molar mass disperisity (ĐM = Mw/Mn), which is higher than 1.5 (Figure 4). We observed that the investigated protein and the protein conjugate show a very low propensity for reversible aggregation, whereas the formatted aggregates are predominantly irreversible and stable with respect to the cross-flow velocities and the solution concentration. Thus, the most probable mechanism for the formation of aggregates is through the conformational altered protein component in protein conjugate, which is, however, largely limited by the addition of 5% sorbitol to the storage buffer.42,43 The SEC measurements were carried out on the same experimental setup as the AF4, only that the separation of the samples was performed in the SEC column(s) instead of in the channel. The Mw and ĐM of the protein determined by SECMALS on two successively connected columns are 18.3 kDa and 1.00, whereas for the PEG reagent the values are 21.3 kDa and 1.02, respectively (Table 1). These values are in good agreement with the values determined by AF4-MALS (Table 1). Similar to AF4 results, also the SEC results show significantly different elution volumes for the protein and the PEG reagent although their molar masses are comparable, which again indicates the large differences in their Vh (Figures 5 and 6, top). The results of the SEC-(UV-MALS(QELS)-RI) analysis also reveal the presence of minute amounts of unreacted protein and larger amounts of aggregates in buffer solution 2 than in buffer solution 1 (Figures 5 and 6, middle and bottom).

concentration detectors is obligatory. In our system the UV and RI detectors were connected in series, and only the protein component of the conjugate was UV sensitive at 280 nm. Based on the known dn/dc values of the protein and the PEG as well as the known specific extinction coefficient of the protein, the molar masses of the individual components (PEG and protein) of conjugate and, consequently, the conjugate’s chemical composition was determined. The average molar masses of the protein and the PEG components, which were calculated across the entire conjugate peak, are in good agreement with the values determined for the nonconjugated protein and the PEG reagent (Table 1, Figure 3). These results indicate the uniform chemical composition distribution of the conjugate with a molar ratio between the PEG and the protein of 1/1. The differences between the differently stored conjugate samples are the most pronounced in the presence of highmolar-mass aggregates in solutions (Mw of the order of ∼106− 107 Da, Figures 2 and 4). The formation of soluble high-molarmass aggregates is a potential and undesirable effect during the various steps of the biopharmaceutical formulation process.41 The control and analysis of the protein conjugate aggregation is an increasing challenge to many pharmaceutical researchers. The fractograms indicate that LS detector produces high intensity signal for high molar-mass aggregated species (Figure 2). However, the molar mass characteristics of aggregates as well as their fractional amount can only be determined if the response of the concentration detector is also reliable. For this reason the data analysis of aggregates in buffer solution 1 was only possible with a sufficiently large amount of injected sample (injected mass 450 μg). The AF4-MALS results show that the conjugate drug stored in the 10-mM acetate buffer solution with 5% sorbitol at pH 3.4 (buffer solution 1) resulted in the formation of fewer aggregates in comparison to the conjugate sample stored in the 50-mM acetate buffer solution with 200mM sodium chloride at pH 4.5 (buffer solution 2), despite the 7378



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Figure 5. Top picture: SEC-MALS chromatograms (solid line: RI response, dashed line: LS response at angle 90°) of the pure protein (red), the PEG reagent (blue) and the conjugate stored in buffer solution 1 (green) together with the molar mass vs elution volume. Middle picture: SECMALS chromatogram of protein conjugate in buffer solution 1 (black line, RI response; red line, LS response at angle 90°). Bottom picture: SECMALS chromatogram of protein conjugate in buffer solution 2 (black line, RI response; red line, LS response at angle 90°). Column: PROTEEMA with a precolumn.

When using only one SEC column we determined for the PEGylated protein conjugate in both buffer solutions higher molar-mass values and a broader molar-mass distribution (Mw = 52.8/44.8 kDa, ĐM = 1.04/1.02 for buffer solutions 1 and 2, respectively) than by SEC using two columns (Mw = 38.9/39.1 kDa, ĐM = 1.01/1.01 for buffer solutions 1 and 2, respectively) or AF4-MALS technique (Mw = 39.7/39.4 kDa, ĐM = 1.01/ 1.01 for buffer solutions 1 and 2, respectively) (Table 1). The overestimated molar-mass values of the PEGylated protein conjugate obtained by SEC on one column are ascribed to the poorly separated peaks of the aggregates and the conjugate (Figure 5, middle and bottom), which is reflected in more pronounced upward curvature of the line, representing the

molar mass vs elution volume, at the left side of the conjugate peak than it is the case for the conjugate’s analysis with AF4 or SEC using two columns connected in series. These results reveal that the size-based selectivity of one SEC column is inferior, whereas that of the two SEC columns is comparable to that of AF4. The measurements of the hydrodynamic radius (Rh) by QELS and the radius of gyration (Rg) by MALS were performed online after the separation. The Rh of the PEG (4.4) is significantly higher than that of the protein (2.2) even though the molar masses of both are comparable (21.3 and 18.1 kDa, respectively), (Figure 1, Table 1). This indicates that the protein has a much more compact conformation than the PEG, 7379



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Figure 6. Top picture: SEC-MALS chromatograms (solid line, RI response; dashed line, LS response at angle 90°) of the pure protein (red), the PEG reagent (blue), and the conjugate stored in buffer solution 1 (green) together with the molar mass vs elution volume. Middle picture: SECMALS chromatogram of protein conjugate in buffer solution 1 (black line, RI response; red line, LS response at angle 90°). Bottom picture: SECMALS chromatogram of protein conjugate in buffer solution 2 (black line, RI response; red line, LS response at angle 90°). Columns: PROTEEMA and SUPREMA with a precolumn.

the MALS detector (∼10 nm). A reliable determination of the size of the aggregates present in buffer solution 1 was not possible since their weight-fraction was too small. On the other hand, the average value of Rg of the aggregates in buffer solution 2 was determined to be 38 ± 1 nm (Figure 4). The shape of the aggregates was estimated from a slope of the log− log plot, representing the radius of gyration as a function of molar mass (Figure 7).10,44 The slope of the line 0.63 suggests a relatively unperturbed PEG random coil conformation in the aggregates composed of the PEGylated protein conjugates. MALDI-TOF mass spectrometry is nowadays frequently employed in protein-conjugate analysis.45−47 The protein mass determined by MALDI TOF MS is 18.8 kDa and that of the

which is reflected also in their elution volumes in SEC chromatograms (Figures 5 and 6, top) or AF4 fractograms (Figure 1). The determined Rh value of the PEGylated protein conjugate is 5.0 ± 0.4 nm, indicating that the protein contributes only minimally to the size of the PEGylated protein conjugate, since the protein tends to simply fill in the gaps and not further extend the size of the PEG. On the other hand, the PEG attachment to the protein significantly increases the size of the conjugate and, thus, provides an extended plasma residence time for the conjugate in the treated organism, which results in an improved efficiency of the drug treatment. The Rg of the pure protein, the PEG reagent and the conjugate were not determined since their sizes are below the detection limit of 7380



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Figure 7. Plot of radius of gyration vs molar mass for the conjugate in buffer solution 2.

Figure 8. MALDI-TOF mass spectra of pure protein (left) and PEG reagent (right). The inset shows an enlarged part of the PEG mass spectrum.

PEG reagent 21.3 kDa at the peak apex with a clearly visible PEG repeating unit (44 Da) over the peak (Figure 8). These masses are in agreement with those obtained with the AF4MALS and SEC-MALS methods. The mass spectra of the conjugate sample in both buffer solutions are identical and consist of three signals. A low-intensity signal centered at 18.8 kDa is due to the presence of residual unreacted protein in the conjugate sample, whereas the signals centered at 20.2 and 40.4 kDa indicate the double-charged and the mass ion of the PEGylated protein conjugate, respectively (Figure 9). In the magnified mass spectra the repeat unit of the PEG imposed on the conjugate signal can still be observed (Figure 9, inset). In the mass spectra of the differently stored conjugate samples no residual PEG reagent was detected, which is consistent with the results of the SEC and AF4 measurements, whereas the aggregates in the conjugate samples were not detected.

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Figure 9. MALDI-TOF mass spectrum of conjugate. The inset shows an enlarged signal that corresponds to the PEGylated protein conjugate.

CONCLUSION

A conjugate sample stored in two different buffer solutions was separated into individual constituents by AF4 or SEC, both coupled to a multidetection system (UV-MALS(QELS)-RI). Both techniques detected three conjugate constituents: a PEGylated protein conjugate as the main constituent and the traces of unreacted protein and high-molar-mass aggregated species. The size-based selectivity for the aggregates and the PEGylated protein conjugate is inferior when using SEC with one column, whereas the size-based selectivity is comparable for SEC with two columns connected in series and AF4 using long channel and linear cross-flow gradient. By MALDI-TOF mass spectrometry we confirmed the constituents present in the conjugate samples, except the high-molar-mass aggregates that were not detected. Using a combination of MALS (QELS) with two concentration detectors (UV and RI), it was possible to

determine the molar-mass and the size characteristics as well as the chemical composition of the individual constituents of the conjugate sample. The PEGylated protein conjugate shows the uniform molar-mass and the chemical composition with 1/1 relationship between the protein and the PEG. The aggregates show a distribution in molar mass, whereas the average molar ratio of protein to PEG is similar to that in conjugate, indicating that they are composed of the conjugate macromolecules with conformational altered protein component. The amount of aggregates in the conjugate drug stored in the 10-mM acetate buffer solution with 5% sorbitol at pH 3.4 and a concentration of 16.8 mg/mL is much lower than in the conjugate drug stored in the 50-mM acetate buffer solution with 200-mM sodium chloride at pH 4.5 and a concentration of 3.4 mg/mL, 7381



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indicating that the addition of 5% sorbitol to the storage buffer largely prevents sample aggregation. Our results demonstrate the applicability and potential of the AF4 separation technique coupled to a multidetection system for the reliable analysis of protein conjugate samples according to the presence of unreacted protein or PEG reagent and highmolar-mass aggregated species, as well as the molar mass, chemical composition and the size of individual sample constituents.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +38614760203. Fax: +38614760300. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia through (a) the Slovenian Research Agency (Program P2-0145) and (b) the Centre of Excellence, Polymer Materials and Technologies for MALDI-TOF MS analysis.

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ABBREVATIONS PEG poly(ethylene glycol) AF4 asymmetrical-flow field-flow-fractionation SEC size-exclusion chromatography UV ultraviolet MALS multiangle light-scattering QELS quasi-elastic light-scattering RI refractive index MALDI-TOF MS matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry Rh hydrodynamic radius Vh hydrodynamic volume Rg radius of gyration ε extinction coefficient dn/dc refractive-index increment Dh diffusion coefficient Vr retention volume ĐM dispersity Mn number-average molar mass Mw weight-average molar mass

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Characterization of a Protein Conjugate Using an Asymmetrical-Flow

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