Precise and comparative pegylation analysis by microfluidics and mass spectrometry

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 359 (2006) 54–62 www.elsevier.com/locate/yabio

Precise and comparative pegylation analysis by microXuidics and mass spectrometry Tao Yu, Joseph A. Traina, Erno Pungor Jr., Michael McCaman ¤ Berlex Biosciences, Richmond, CA 94804, USA Received 5 July 2006 Available online 5 September 2006

Abstract Standard SDS–PAGE analysis of a pegylated protein was able to conWrm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modiWed protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microXuidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1–12 PEGs per polypeptide. ConWrmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent conWrmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins. © 2006 Elsevier Inc. All rights reserved. Keywords: Pegylation; MicroXuidics; Protein characterization; Capillary electrophoresis

Protein pegylation has been gaining broad acceptance as a means to modify key performance properties of recombinant proteins, most often as a means for increasing circulatory half-life and reducing in vivo immunogenicity [1–3]. A complete characterization of a pegylated protein would serve in understanding its biochemical and physiological performance in a research setting. As such modiWed proteins enter human clinical trials or commercial applications, the expectations for complete and consistent characterization become more demanding [4]. Characterizing the degree of pegylation, especially for polyethylene glycol (PEG)1 compounds targeting the epsilon amino group of lysine residues, has been deter-

*

Corresponding author. E-mail address: [email protected] (M. McCaman). 1 Abbreviations used: PEG, polyethylene glycol; TNBS, trinitrobenzene sulfonic acid; MS, mass spectrometry; CE, capillary electrophoresis; RP, recombinant protein; Mr, relative molecular mass; DTT, dithiothreitol; SOD, superoxide dismutase; ACN, acetonitrile; TFA, triXuoroacetic acid; MALDI–TOF, matrix-assisted laser desorption/ionization time-of-Xight. 0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.08.018

mined indirectly by titrating the remaining free (lysine) amino groups in a PEG–protein conjugate compared with those in the native protein. Previous literature often has described such titrations using chemical agents such as trinitrobenzene sulfonic acid (TNBS) [5]. However, this colorimetric method detects only modiWed lysine reactivity and not actual numbers of PEG in the protein. Mass spectrometry (MS) and capillary electrophoresis (CE) have also been used to analyze the extent of pegylation [6–14]. We have investigated a simple CE-based methodology (with chip microXuidics) to separate pegylated conjugates and quantitate the separated species. Each protein 200 LabChip contains an interconnected set of microchannels in which proteins (mixed with a Xuorescent dye and SDS) are sieved by size as they are pulled through a polymer matrix by electrical current. At the end of the channel, separated proteins are mixed with a destaining solution and quantitated with a sensitive Xuorescence detector that is connected to a PC for run control and automated data analysis. The principle of separation in this CE system is similar to that in traditional SDS–PAGE in that the proteins are

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separated based on relative molecular size in the presence of SDS. Each species of pegylated protein has its size and relative concentration reported. The method is consistent and fast (90 s/sample analysis), and it consumes a limited sample (up to 4 l). Even more advantageous, this method does not require postelectrophoretic handling such as the staining and destaining of PAGE. It can provide data output in either an electropherogram or a simulated slab gel picture format, and it is well suited for electronic record storage.

SDS–PAGE with silver stain

Materials and methods

We used an Agilent model 2100 Bioanalyzer equipped with Chip Priming Station, Protein 200 Plus LabChip kit, and the Agilent software (Agilent Technologies, Palo Alto, CA, USA). Samples were prepared according to the manufacturer’s instructions. Typically, concentrated protein RP–PEG5000 samples were diluted to 0.5–1 mg/ml in phosphate-buVered saline and free RP was diluted to 0.1 mg/ml to acquire peaks distinguishable from the baseline. Standard sample preparation involved mixing 2 l of 100 g/ml RP or 1000 g/ml RP–PEG5000 with 4 l reduced sample buVer (from Agilent Protein 200 Plus LabChip kit). Samples were incubated at 95 °C for 5 min, and then 84 l of distilled water was added and 6 l of the mixture was loaded onto the Protein 200 Chip (along with other reagents) to the appropriate wells following the manufacturer’s instructions from the Agilent Protein 200 Plus LabChip kit. Two internal markers are mixed with each test sample and with a molecular weight reference “ladder”. The quantitation is calculated by comparing corrected peak area with either an upper internal marker or a standard reference.

Materials The recombinant protein (RP) in this pegylation study, an amino acid-modifying enzyme, has a predicted relative molecular mass (Mr) of 46,000 and was expressed in an Escherichia coli system. The protein was recovered as an inclusion body following bacterial cell lysis and then was solubilized in 50 mM sodium hydroxide. Dithiothreitol (DTT, 50 mM) was added immediately postsolubilization, bringing the pH down to approximately 9.0, and the mixture was titrated to pH 7.0 with phosphoric acid and allowed to refold overnight at room temperature. The RP was puriWed through Fractogel EMD TMAE HiCap (cat. no. 1.10316, EMD Chemicals, Gibbstown, NJ, USA) anion exchange chromatography. Refolding success was demonstrated when more than 90% of the enzyme activity of the refolded recombinant RP was recovered when assayed in parallel with the natural source of the enzyme. According to DNA sequence of the gene encoding RP, each protein molecule contains 28 lysines. Other reagents used included superoxide dismutase (SOD)–PEG (cat. no. S9549), catalase–PEG (cat. no. C4963), protease–PEG (cat. no. P-1459), asparaginase– PEG (cat. no. A5336), and high-purity sinapinic acid (>97%), all from Sigma–Aldrich (St. Louis, MO, USA). The acetonitrile (ACN), triXuoroacetic acid (TFA), and H2O used all were of HPLC grade. MS calibration standards were also obtained from Sigma–Aldrich and included yeast enolase (cat. no. E6126), lysozyme, L6876, and carbonic anhydrase (cat. no. C-3934). Pegylation of RP Routine protein pegylation was performed by mixing 1 mg/ml RP with 10 mg/ml “activated” PEG5000 containing a single reactive succinyl group (Shearwater Polymers, Huntsville, AL, USA) in PBS buVer (pH 8.3) for 1 h. Conjugated RP was separated from remaining unconjugated PEG reagent by chromatography of the mixture on Fractogel TMAE anion exchange resin. Partial pegylation of RP was achieved by incubating active PEG5000 and the RP for 30 min rather than 60 min at room temperature. Chemical depegylation of RP–PEG5000 was performed by incubating it at 50 °C at pH 8.0 with samples analyzed at days 0, 6, 8, 16, and 26.

Electrophoresis was performed with 4 to 12% acrylamide NuPage Bis–Tris gels (cat. no. NP0302) with MES SDS Running BuVer (cat. no. NP0002) obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). The slab was run at 200 V for 35 min. Silver staining was accomplished using the kit reagents from Pierce Chemical (Rockville, IL, USA). MicroXuidics assay

TNBS assay The number of PEG molecules attached to the primary amines of RP was determined in a colorimetric reaction with TNBS (Sigma–Aldrich). BrieXy, serial dilutions of the native RP and RP–PEG5000 were made in 100 mM sodium phosphate (pH 8.3). TNBS reagent was then added (10 mg/ ml), and the reactions were heated to 40 °C for 2 h. The absorbance was determined (A330 nm), the absorbance versus the protein concentration was plotted, and a slope of the line was determined: the number of PEG molecules attached to each molecule of RP D 1–(slope of native RP/ slope of RP–PEG) £ 29, where 29 represents the number of primary amines present in RP (28 lysines + 1N terminal). Mass spectrometry Samples were analyzed using a Voyager DERP matrixassisted laser desorption/ionization time-of-Xight (MALDI– TOF) mass spectrometer from Applied Biosystems (Foster City, CA, USA). The instrument was operated in the positive, linear, and delayed extraction mode. The accelerating voltage was maintained at 25 kV. Other parameters were adjusted to give the best signal/noise ratio for the sample being analyzed. Spectra from multiple laser shots were

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summed. Data analysis was performed using the manufacturer’s software package (Data Explorer version 4.0.0.0). A Gaussian smoothing algorithm was applied to both sample and standard spectra. The Wlter width used varied between 9 and 101 data points, depending on the noise level in the spectrum. Samples either were diluted with matrix and then spotted on the MALDI sample plate and air-dried or were mixed directly on the plate and allowed to air-dry. Samples with interfering buVers were “droplet dialyzed” against water by placing a 1-l aliquot of sample on a 0.025-m membrane Wlter (mixed cellulose) that was Xoated over water. The matrix (sinapinic acid, 10 mg/ml in ACN:H2O:TFA) was prepared fresh daily and centrifuged prior to use. Calibration was performed using the “close external standard” method. The reference proteins used were a combination of lysozyme, carbonic anhydrase, and yeast enolase that bracketed the sample mass range. Both (M + H)+ and (M + 2H)2+ ions were used. The calibration ions (M + H)+ used were 14,307, 29,023, and 46,672 for lysozyme, carbonic anhydrase, and yeast enolase, respectively. Only the reference proteins spotted close to a particular sample were used to mass calibrate that sample spectrum. Peak area quantitation by MS should be considered an estimate only because ionization eYciencies of protein versus pegylated protein have not yet been studied. Results and discussion Characterization of the pegylated protein by SDS–PAGE and TNBS Expression of the RP in an E. coli system generated a polypeptide with an expected Mr of 46,174. The puriWed RP is resolved as a single band by conventional SDS–PAGE analysis with an Mr of 45,000 (Fig. 1, second lane). When this RP was conjugated with a PEG polymer with an average Mr of 5000, hereafter identiWed as RP–PEG5000, it clearly had increased in molecular mass but was a poorly resolved broad smear by SDS–PAGE (Fig. 1, third lane), rendering this method not useful for the characterization of the product. From its predicted sequence, each RP molecule contains 28 lysines plus the N-terminal primary amine and in theory could have a maximum of 29 PEG groups added if all amines were freely available for reaction. However, one might expect that internal salt bridges and steric hindrance to the pegylation reagent would reduce the actual number of surface accessible amine groups. Following the routine pegylation reaction (see Materials and methods), an average of 17 modiWed lysines were detected in a typical RP–PEG5000 product batch using the TNBS method for quantitating the loss of free amino groups (assumed to be a direct measure of PEG groups). Bioanalyzer separation of RP–PEG5000 product species The CE separation performed by the Bioanalyzer was able to detect the puriWed RP as a single sharp peak.

Fig. 1. SDS–PAGE of RP samples: Silver-stained SDS–PAGE of 1-g samples of reduced RP and RP-PEG5000, as described in Materials and methods. SDS–PAGE standards were from Invitrogen (Marker12, cat. no. LC5677). The Mr values of these standards are 200,000, 116,000, 97,000, 66,000, 55,000, 36,000, 31,000, 21,000, 14,000, 6000, 3500, and 2500. MW Stds, molecular weight standards.

More important, this method resolved the RP–PEG5000 product into at least nine apparent peaks, based on diVerences in apparent molecular weight (Fig. 2). The Bioanalyzer software reports migration time, corrected peak area, predicted Mr, and percentage of total peak area for each peak. As shown in Table 1, the Wrst signiWcant species of RP–PEG5000 (with the smallest Mr) that comprised more than 2% of the total sample had an apparent Mr of 121,000. Thereafter, a series of peaks were observed with estimated Mr values of 132,000, 142,000, 153,000, 162,000, and 171,000. For the example shown, the predominant RP–PEG5000 adduct had an apparent Mr of 153,000. Typically, this pegylated mixture had only 40% of the enzymatic activity of the unpegylated starting material. In this series of RP–PEG5000 adducts, the Wrst resolvable species of RP larger than the unconjugated form was observed with an Mr of 69,000. This could represent a form of RP with, at the least, a single PEG added (increasing the apparent Mr of the molecule by nearly 26,000), or this could be a form of RP with several PEG 5000 molecules added per protein. Although it seems unlikely that a single PEG could have such a dramatic impact on the hydrodynamic radius as to cause a change of 26,000 in the apparent Mr, it is also clear that further analysis was necessary to identify the actual number of PEG chains present in these resolved protein peaks. If all 29 amino groups of the RP molecule reacted with the pegylating reagent, this would add a minimum of 145,000 to the 45,000 Mr of the starting protein. Because the largest detectable RP–PEG adduct is in the range of 172,000 Mr (by Bioanalyzer analysis, Fig. 2), it would appear that not all amine groups are available for PEG attachment.

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Fig. 2. Typical samples of RP and RP–PEG5000 analyzed on the Bioanalyzer. The left panels are the primary electropherograms reported by the Bioanalyzer. The right panel is an electronic reconstruction of the same data into a stained slab gel format. Table 1 Analysis of pegylated RP Peak number

Migration time (s)

Size (kDa)

Corrected area

% Total

1 2 3 4 5 6 7 8 9

29.55 31.70 33.00 34.15 35.20 36.15 37.00 37.90 38.70

68.7 94.7 108.0 120.5 131.7 142.3 152.9 162.3 171.2

0.79 1.17 3.18 11.72 39.84 92.21 118.74 112.99 66.74

0.17 0.25 0.68 2.50 8.60 19.80 25.50 24.30 14.00

Reduced pegylation of RP The Bioanalyzer Mr determinations alone were insuYcient to determine whether a species of RP with a single PEG addition was among the molecules detected. If the routine pegylation procedure produced only multiple PEG additions, identiWcation of a single PEG adduct would require modiWcation of the procedure to reduce total PEG content. For this, we performed the pegylation of protein RP using a lower ratio of PEG/protein and reduced the reaction time from 60 to 30 min (see Materials and methods) with a resulting Bioanalyzer pattern summarized in Table 2. The resulting RP–PEG5000 showed a shift of the Wrst major peak from a migration time 32.8 s to less than 28 s (cf. Figs. 2 and 3).

DeWnitive conWrmation of the PEG number assignments would best be achieved if it were possible to isolate each single species of RP–PEG5000 and then repeat the Bioanalyzer analysis. However, attempts at such reWned protein puriWcation were not successful. We then chose to apply MS analysis as an independent and orthogonal approach to conWrming Mr and PEG number. The MS analysis of the RP with reduced PEG content showed a series of Wve pegylated adducts as well as unreacted RP (Fig. 3, bottom panel). By MS, the unmodiWed RP has a measured Mr of 46,200, in close agreement with its predicted Mr. Sequentially larger adducts showed an average Mr of approximately 5500 (consistent with the average Mr of 5000 expected for the PEG polymer). The Bioanalyzer detects these adducts somewhat diVerently, with an apparent incremental increase in Mr of 11,000–19,000 for each PEG added to the RP with a low degree of pegylation Table 2 Analysis of RP with a low degree of pegylation RP–PEG5000 species

Apparent molecular weight (kDa)

% Total

RP RP–(PEG5000)1 RP–(PEG5000)2 RP–(PEG5000)3 RP–(PEG5000)4 RP–(PEG5000)5

43 58 69 88 104 118

7 24 35 25 7 <1

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Fig. 3. Partially pegylated RP–PEG5000 analyzed by Bioanalyzer and MS. Partial pegylated RP (achieved by a shorter incubation time of RP with active PEG5000) was analyzed by Bioanalyzer (top panel) and MS (bottom panel). In calculating PEG adducts on RP, the average mass of the PEG adduct (PEGavg) was determined by calculating the average mass diVerence between adjacent sample peaks in the spectrum (Peak Massn+1 ¡ Peak Massn). The number of PEG adducts (# PEG) was calculated for each peak as follows: # PEG D (Peak Mass ¡ 46174.6)/PEGavg, where 46174.6 is the calculated average (M + H)+ for RP.

(Table 3). This allowed us to conWrm that the smallest pegylated RP peak shown in Fig. 2 and in Table 1 with an Mr of 69,000 is most likely a 2-PEG5000 adduct and allows a preliminary assignment of PEG numbers to the subsequent series of RP–PEG peaks. Assignment of a PEG number to each molecular weight was based on identifying a single PEG adduct as described above and then assuming that each larger peak represented the addition of one additional PEG. By simply dividing the molecular weight increase noted for each successive adduct by the assigned PEG number, we see a trend of diminishing impact on the apparent Mr contribution for each subsequent PEG added. The apparent Mr contribution of the Wrst PEG groups can be somewhat variable and range as high as 19,000. As the PEG number approaches 10, the Mr contribution per PEG

appears to gradually stabilize and is reduced to 8000, much closer to the 5000 value assigned by the PEG reagent manufacturer. It is believed that this pattern is consistent with the idea that initial PEG additions have the most impact on protein migration in the matrix during electrophoresis. As the number of attached PEGs increases, each subsequent PEG has a less dramatic eVect on the hydrodynamic radius (and the corresponding calculated Mr) of the RP–PEG5000 molecule. Based on the above, we would surmise that in Table 1 the dominant conjugated species following standard pegylation would have 8 or 9 PEG chains attached. This is in excellent agreement with the MS analysis of the sample (data not shown), giving the dominant peaks with approximate Mr values of 85,100 and 91,000, consistent with the approxi-

Pegylation analysis by microXuidics and MS / T. Yu et al. / Anal. Biochem. 359 (2006) 54–62 Table 3 Correlation between Bioanalyzer molecular weight and pegylation number for RP–PEG5000 PEG adduct number

Estimated MW (kDa)

Estimated MW of each PEG (kDa)

RP RP–(PEG5000)1 RP–(PEG5000)2 RP–(PEG5000)3 RP–(PEG5000)4 RP–(PEG5000)5 RP–(PEG5000)6 RP–(PEG5000)7 RP–(PEG5000)8 RP–(PEG5000)9 RP–(PEG5000)10 RP–(PEG5000)11

43 58 69 88 105 118 129 140 150 160 169 177

— 15 11 19 17 13 11 11 10 10 9 8

Note. MW, molecular weight.

mate theoretical values (i.e., 46,000 + [8 £ 5000] D 86,000 and 46,000 + [9 £ 5000] D 91,000). Robustness of the Bioanalyzer for pegylation analysis By intentionally varying conditions for sample preparation, we became satisWed regarding the robustness of the Bioanalyzer for pegylation sample analysis. Despite varying denaturing heating conditions (time and temperature) and various sample loading concentrations or dilution matrices (water to phosphate-buVered saline), we saw no signiWcant changes in the electropherogram. Analytical reproducibility was excellent, with a relative standard deviation of the reported apparent Mr values ranging from 2 to 4% for a series of eight batches of RP–PEG 5000. We also noted that free PEG does not generate a signal in the Bioanalyzer even at a PEG molar concentration equivalent to the range present in the RP–PEG adduct mixture. Thus, the hydrophobic Xuorescent dye shows no interaction with the hydrophilic PEG5000 (data not shown). We conclude that the signal detected in the Bioanalyzer is from the protein itself, and although some degree of signal quenching by added PEG groups may occur, this cannot be assessed directly with the Bioanalyzer. Thus, for practical purposes, we have assumed that the Bioanalyzer quantitative analysis is correct and that values measured for “relative concentration” and the “percentage of total” are credible. Application of the Bioanalyzer to stability studies of pegylation It is known that the ester linker bridge between PEG5000 and protein is not stable at high pH (>8.0) [15]. Our experience has indicated that PEG5000 is released from RP during alkaline pH storage, but this release leaves the succinate linker (from the original activated PEG) on the RP polypeptide. To explore the feasibility of the Bioanalyzer to

59

monitor instability (depegylation), RP–PEG5000 was incubated at pH 8.0 at 60 °C for up to 26 days for accelerated depegylation. Fig. 4 shows the results of the depegylation analysis of RP–PEG5000 indicating that the predominant peaks of RP–PEG5000 contained 9 and 10 PEG chains at day 0, but the number of PEGs dropped to 3 at day 6, to 2 for RP by day 8, and to 1 by day 26. In a diVerent stability study, aliquots of RP–PEG5000, stored at ¡70 °C for either 1 month or 13 months, were compared. Pegylated product stability was preserved during the extended storage period. Both of the predominant peaks after 1 month and 13 months contained 9 and 10 PEGs. Further MS data with these same samples conWrmed the stability of frozen pegylated RP (data not shown). In this manner, the utility of the Bioanalyzer for assessing PEG stability on the RP–PEG5000 molecule was demonstrated. Method application to other pegylated proteins Several commercially available pegylated proteins were also analyzed on the Bioanalyzer to investigate the usefulness of this method for the characterization of a broader spectrum of pegylated proteins. In its native form, SOD is a dimer, where each subunit has a predicted Mr of 15,000. A commercial pegylated version, SOD–PEG5000, contains 12 mol PEG5000 per mole of dimeric protein according to the manufacturer (as assessed with the chemical TNBS method). By Bioanalyzer analysis, an apparently unpegylated peak is seen with an Mr of 17,000, whereas the dominant peak (33% of total) appears to have an Mr of 26,000 (Fig. 5A). This additional Mr of 9000 may represent 2 PEG molecules or perhaps just 1 PEG given the similar observation made for a single PEG addition to RP noted above. Analysis of this same material by MS also identiWed a peak with an Mr of 15,800 (likely representing unpegylated SOD) as well as several likely pegylated adducts whose incremental Mr increase ranged from 4700 to 5400, suggesting that each peak represented a single PEG addition from the preceding one (Fig. 5B). The largest mass PEG adduct detected by either Bioanalyzer or MS appeared to contain 6 PEGs. The electropherogram and the MS data would suggest that the supplier’s data on PEG content may be incorrect, with much of the SOD appearing to be unpegylated and only a few PEGs per SOD being detectable. It is possible that such unexpected observations might be caused by depegylation during storage in either the lyophilized or frozen state, although the magnitude of disparity is not consistent with the observations we made on the RP seen above. It is also possible that the manufacturer’s product analysis (reported as 12 lysines modiWed per molecule of dimer) does not reXect the true PEG content. This would be consistent with our observations on the disharmony between the TNBS assay and the electrophoresis as described above. We analyzed several other commercially pegylated proteins by Bioanalyzer and MS (Table 4) for PEG adduct

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Fig. 4. Analysis of chemically depegylated RP–PEG5000. Chemical depegylation of RP–PEG5000 was performed by incubating RP–PEG5000 at 50 °C at pH 8.0 with sample aliquots analyzed at days 0, 6, 8, and 26. The test sample concentration was 1 mg/ml. The Bioanalyzer running conditions are described in Materials and methods.

quantitation. These data report on individual protein peaks that comprise more than 20% of the total signal measured. A signiWcant amount of nonpegylated protein was seen for each commercial product. There was no indication of proteins with high numbers (>3) of PEG adduct. In summary, all of these pegylated products appear to have signiWcantly less than is indicated in supplier product literature (estimated indirectly by the TNBS method). Conclusion As more pegylated proteins Wnd use in clinical investigations, there is an urgent need for precise chemical analysis to ensure product consistency and safety. The reaction of pegylated protein with TNBS is one means of assessing occupancy of amino groups targeted by the activated PEG reagent. However, this result may be misleading because the absence of a free amine does not prove the presence of a PEG chain. Unstable PEG attachment, such as ester bonds, is subject to hydrolysis releasing the PEG group without forming free amines. Thus, site occupancy may reXect initial accessibility of the amine group to the pegylation compound but not the continued presence of PEG itself. In this regard, the TNBS assay would also be a poor indicator of the stability of the PEG adduct. It is conceivable that, under conditions where all available amino groups are modiWed but are then subject to follow-up ester hydrolysis,

the resultant modiWed protein might have no PEG chains attached despite TNBS results suggesting full occupancy. The analysis of the commercial pegylated proteins described here suggests the presence of upward of 30% nonpegylated protein. If the absence of PEG signiWcantly alters its performance (e.g., activity, half-life), experiments that assume complete occupancy and signiWcant PEG content could be seriously misinterpreted. Therefore, a molecular sizing-based assessment of the pegylated protein would appear to be a much more accurate and useful tool for its characterization. Although methods such as MS, proton NMR, and CE are useful for analyzing pegylated recombinant proteins, the complexity and instrumentation expense of these methods make them unattractive as robust, routine quality assurance tools. SDS–PAGE and size exclusion chromatography HPLC can also serve as pegylation analysis tools but work eVectively only at the lower degrees of pegylation (e.g., 1–3 PEGs/protein molecule). We encourage laboratories working with pegylated proteins, especially multiply pegylated adducts, to consider using the Bioanalyzer as a product characterization tool. The cost, training, and maintenance of the device are modest, whereas the quality, reproducibility, and robustness of this technology are impressive, as we have tried to demonstrate here. Although the molecular mass measurements obtained by MS certainly exceed the precision of the Bioanalyzer, there is

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Fig. 5. Bioanalyzer and MS results for SOD–PEG5000. Commercially available SOD–PEG5000 was analyzed by Bioanalyzer (top panel) and MS (bottom panel). Peaks are labeled with PEG adduct numbers as estimated from Mr values. Table 4 Bioanalyzer and MS analysis of commercial pegylated proteins Sample name

MW of subunit # PEG/subunit with no Supplier Bioanalyzer MS pegylation (kDa) data

SOD–PEG5 16.0 Asparaginase–PEG5 35.5 Protease–PEG5 27.0

6 10 6

0 and 1 0 and 1 0 and 1

0 and 1 0 and 1 0 and 1

reason to treat the peak intensity distribution with caution. MALDI mass spectra of proteins with a low degree of pegylation have been observed to be biased toward the lower PEG adduct species. It was speculated that this bias results from diVerential desorption, ionization, and detection eYciencies among the pegylated species [6,12]. It should also be pointed out that the MALDI mass spectra of pegylated

proteins typically contain doubly charged ions and protonbound dimer ions in addition to the protonated molecule. These ions sometimes can interfere with the quantitation of some PEG adduct ions, depending on the mass range of the protein and PEG. Quantitation with the Bioanalyzer does not suVer from the bias and interferences described above. In our laboratory, the Bioanalyzer has provided a robust and easy-to-use means to generate quantitative and relevant molecular size-based characterization data. This device was one of the Wrst chip-based technologies designed for semiautomated separation and analysis of protein, DNA, or RNA. We have previously shown its robust utility for analysis of nucleic acids in a viral gene therapy application [16]. Our results with pegylated proteins demonstrate the further role of microXuidics instrumentation in the modern biotechnology laboratory.

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