Bioconjugate Chem. 2008, 19, 2163–2170
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Sites of Modification of Hemospan, a Poly(ethylene glycol)-Modified Human Hemoglobin for Use As an Oxygen Therapeutic Kim D. Vandegriff,*,† Ashok Malavalli,† Gnel M. Mkrtchyan,† Stephanie N. Spann,† Dale A. Baker,†,‡ and Robert M. Winslow†,‡ Sangart Inc., 6175 Lusk Boulevard, San Diego, California, 92121, and Department of Bioengineering, University of California, San Diego, La Jolla, California 92093. Received June 30, 2008; Revised Manuscript Received September 2, 2008
Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW ∼5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of ∼8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual R and β globins was made using reverse-phase HPLC, showing a series of R globins conjugated with 0 to 3 PEGs and a series of β globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on β globins at β8, β17, β59, β66, β93, β95, and β132 and three sites identified on R globins at R7, R16, and R40.
INTRODUCTION Hemospan is a new class of hemoglobin-based oxygen therapeutic, designed using site-specific, maleimide poly(ethylene glycol) (PEG) conjugation chemistry with oxyhemoglobin (Hb) (1, 2). Hemospan, also referred to as MP4 or MalPEGHb in the literature, has been the subject of extensive biochemical (2-6) and physiologic studies (7-10). The product has completed phase I (11) and phase II clinical trials in Sweden (12) and the U.S., with phase III trials just finished in Europe. Several general benefits have been attributed to PEG conjugation to proteins, including decreased immunogenicity, prolonged intravascular retention time, and increased colloidal osmotic pressure as a function of PEG mass and protein concentration (3). A recent study using small-angle X-ray scattering to obtain the three-dimensional structure of Hemospan in solution (P5K6 in that study) showed that PEGylation increases the macromolecular dimensions of hemoglobin from a maximum particle dimension of 70 Å for Hb to 130 Å for P5K6; in addition, PEGylation introduces an intermolecular repulsive effect that increases with PEG mass (6). Even though this PEGylated Hb molecule is not cross-linked intramolecularly and, therefore, remains capable of tetramer-dimer dissociation upon dilution (13), the PEG surface shielding and intermolecular repulsion prolong the circulatory half-life of Hemospan to approximately 20 h in surgical patients (12). In the present study, we describe the number and distribution of PEG polymers on the individual R and β globin subunits in Hemospan. Because the maleimide-PEG raw material has a relatively wide mass distribution (∼5500 ( 750 Da), sites of modification, under reaction conditions specified for Hemospan, were identified by mass analysis using a small molecular weight model, i.e., N-ethyl maleimide (NEM) in the place of * Corresponding author.
[email protected]; 858-450-2414, Telephone; 858-450-2499, Fax. † Sangart Inc. ‡ University of California.
maleimide-PEG. NEM as a model compound was validated recently in a study of modification sites in a similar product made using thiolation/maleimidation reactions with deoxyHb (14). Thiolation/maleimidation under Hemospan reaction conditions with oxyHb shows both similar and dissimilar sites compared to the same reaction conditions carried out on deoxyHb reported by Iafelice (14). A primary difference is that the β93Cys is modified in Hemospan, leading to higher O2 affinity (2, 4).
EXPERIMENTAL SECTION Hemospan Production. Hemospan was prepared using the process described earlier (2), with a small increase in processing temperature from 6° ( 2 to 10 °C, which does not affect the final reaction product. Briefly, stroma-free Hb was purified from human red blood cells and diluted to 1 mM (in tetramer) in phosphate-buffered saline (PBS), pH 7.4. Hb thiolation was carried out using a 10-fold molar excess (over Hb tetramer) of 2-iminothiolane (2-IT, Sigma Aldrich, St. Louis, MO) for 4 h at 10 °C. This was followed by PEG conjugation using a 20fold molar excess (over Hb tetramer) of maleimide-activated PEG (MW ∼5500 Da) (NOF Corp., Tokyo, Japan) for 2 h at 10 °C. The PEG-modified hemoglobin was diafiltered against lactated Ringer’s solution using 70 kD tangential flow filtration to remove both unreacted Hb and maleimide-activated PEG. The final product was concentrated to a final Hb concentration of 4.2 g/dL. NEM-Hb Production. NEM-Hb was prepared using the same reaction conditions described above for PEG conjugation. Hb was first reacted with a 10-fold molar excess of 2-IT for 4 h at 10 °C, followed by reaction with 20-fold molar excess of N-ethyl maleimide (NEM) (in the place of maleimideactivated PEG) for 2 h at 10 °C. NEM-modified Hb was dialyzed against lactated Ringer’s solution to remove unreacted NEM. Degree of Hemoglobin Modification. The degree of thiolation and maleimidation (or PEGylation) was evaluated from the number of reactive thiols on Hb after the thiolation and
10.1021/bc8002666 CCC: $40.75 2008 American Chemical Society Published on Web 10/07/2008
2164 Bioconjugate Chem., Vol. 19, No. 11, 2008
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Figure 1. SDS-PAGE of Hb and Hemospan. (A) Coomassie blue staining, specific for protein; (B) iodine staining, specific for PEG. Lanes: (1) Hb; (2) MW markers; (3) Hemospan. Densitometry maps for Hb (C) and Hemospan (D) are shown for protein staining; the densitometry peaks for Hemospan are identified on the right side of (B).
maleimidation reactions. Free thiols were measured by disulfide exchange with the 4,4′-dipyridinedisulfide to produce the corresponding 4-thiopyridone (4-TP), measured at 324 nm (ε ) 1.98 × 104 M-1 cm-1) (15). The difference between the number of free thiols immediately following thiolation versus post-maleimidation corresponds to the number of PEG or NEM molecules conjugated to the hemoglobin tetramer. Two additional test methods were used to evaluate the degree of PEGylation: (1) 1H NMR spectroscopy (Varian 400 MHz; Inova; performed at Spectral Data Services, Inc., Champaign, IL), using D2O as the solvent, (trimethylsilyl)propionic acidD4 as internal standard, and comparing spectra of Hemospan with samples of pure PEG (10 mg) dissolved in D2O following the method of Jackson (16). The NMR signal at 3.5-3.9 ppm represents the chemical shift for the oxyethylene groups in PEG, and the integrated signal for pure PEG was used to calculate the amount of PEG in the hemoglobin conjugate. (2) A dry weight method was performed using Hb and Hemospan at equal concentrations in heme by subtracting the dry weight of Hb from the dry weight of PEG-conjugated Hb to calculate the number of ∼5 kD PEGs conjugated per Hb molecule. Dry
Table 1. Percent of Total for the Protein-Stained Gels for Hb and Hemospan (Figure 1A) According to the Densitometry Maps Shown in Figure 1C,D, Ranked from the Top to the Bottom of the Gels, and Percent of total for the Hemospan PEG-Stained Gel from Figure 1B, Ranked from the Top to the Bottom of the Gel Hb (protein stain)
Hemospan (protein stain)
peak
density (%)
1 2
9.36 90.64
a
ND ) not detected.
Hemospan (PEG stain)
peak
density (%)
peak
density (%)
PEGs
1-2 3-4 5 6 7 8 9
2.30 8.16 12.55 22.49 28.09 20.94 5.47
1-2 3-4 5 6 7 8 9
2.83 12.01 17.06 26.65 27.87 13.57 NDa
>4 >4 4 3 2 1 0
weights were made following lyophilization (Virtis, Gardiner, NY) and oven-drying at 130 °C until weights no longer changed. Degree of PEGylation by SDS-PAGE and MALDITOF Mass Spectrometry (MS). Hemospan was analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis (SDS-
Sites of Modification of Hemospan
PAGE) on Bis-Tris precast gels (Invitrogen), with a 4-12% gradient. Experiments were carried out under conditions to avoid removing conjugated PEG from the globins, and so heating and β-mercaptoethanol treatments were not employed in the SDS process. To confirm dissociation of globins, control experiments were performed on Hb both with and without heating and β-mercaptoethanol treatment in which no differences in band separation were observed. Gels were stained differentially using Coomassie blue for protein and iodine for PEG (17, 18), and bands were analyzed by densitometry (Image Scanner, PowerLook 1120). Protein and PEG-conjugated globin bands were electroeluted for MALDI-TOF analysis by mixing samples with sinapinic acid in 50% acetonitrile/0.1% formic acid. An intact Hemospan solution sample was submitted independently for MALDI-TOF mass analysis. Samples were air-dried on the MALDI sample plate and analyzed on an Applied Biosystems Voyager DE mass spectrophotometer. Degree of PEGylation by Reverse-Phase HPLC. Reversephase high-performance chromatography (RP HPLC) was used for analysis of modified globins. The modified hemoglobin solution was eluted on a C3 column (4.6 × 50 mm) (Agilent 1100 series) at a flow rate of 2 mL/min at 50 °C in 35-40% acetonitrile with 0.1% trifluoroacetic acid (TFA) over 1 min, followed by 40-51% acetonitrile with 0.1% TFA over 11 min. Mass Analysis by Electrospray Mass Spectrometry (ESITOF MS). Hb and NEM-Hb were desalted on a PD-10 column containing Sephadex G-25 medium (GE) and submitted for ESITOF analysis on an Agilent ESI-TOF mass spectrometer. Samples were electrosprayed into the TOF reflectron analyzer at an ESI voltage of 4000 V and a flow rate of 200 µL/min (50:50 acetonitrile/water/0.1% formic acid v/v) using flow injection with an Agilent microwell autosampler. The averaged mass spectrum was converted to a neutral charge state spectrum using a maximum entropy deconvolution algorithm (MaxEnt) as part of the Analyst data analysis software (Agilent). Sites of Hemoglobin Maleimidation. Sites of thiolation/ maleimidation in Hemospan were identified using NEM-Hb as a model compound. To evaluate potential preferences of Hb residues to react with 2-IT, Hb was subjected to thiolation under identical reaction conditions at four different ratios of Hb to 2-IT (1:3, 1:5, 1:7, and 1:10). NEM was added subsequently at a Hb/NEM molar ratio of 1:20 in each case. NEM-Hb samples prepared at the molar reaction ratio of 1:10:20 were run in triplicate. The same sites were found to be modified for all ratios tested, and samples prepared at the lower reaction ratios were run only once. NEM-Hb samples were subjected to SDS-PAGE on Bis-Tris precast gels, with a 4-12% gradient. Gels were stained with Coomassie blue. After staining, NEM-Hb bands were excised and pretreated prior to in-gel trypsin digestion. Reduction of disulfide linkages was performed using 10 mM dithiothreitol, followed by alkylation with 55 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO). In-gel digestion with trypsin (Promega) was performed overnight at 37 °C at an estimated (1:30) enzyme to substrate ratio in 50 mM ammonium bicarbonate. Tryptic peptides were extracted from the gel using acetonitrile and formic acid and subjected to analysis by LC/ MS/MS (Scripps Research Institute, MS Lab, San Diego, CA). Separation was performed on a laser-pulled, 100 µm ID C18 column with a tip of
