Preparation and Characterization of Poly(ethylene glycol)ylated

Chapter 12

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 29, 2016 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch012

Preparation and Characterization of Poly(ethylene glycol)ylated Human Growth Hormone Antagonist 1

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Kenneth Olson , Richard Gehant , Venkat Mukku , Kathy O'Connell , Brandon Tomlinson , Klara Totpal , and Marjorie Winkler 4

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Departments of Recovery Sciences, QC Biochemistry, and Protein Chemistry and Analytical Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080 4

In order to prepare long-lasting human growth hormone antagonist (GHA), the protein was conjugated with polyethylene glycol (PEG) to increase its molecular size. Following conjugation, the preparation was fractionated to select the optimal molecule for biological assessment. The molecules were characterized to assist in defining the molecules as drug substances for the purpose of receiving approval for clinical studies and eventually approval as therapeutic drugs. Tryptic peptide analysis was found to be an extremely useful tool to identify the PEGylation sites. Analysis revealed the pattern of PEGylation as well as the consistency of PEGylation from lot to lot. The optimal molecules were found to be between 40-50,000 kD in molecular mass. The results obtained by tryptic analysis were confirmed by MALDI -TOF mass spectrometry. In all cases, the PEGylated proteins were active with a sustained half-life. Modification of proteins with polyethylene glycol (PEG) to improve their therapeutic properties is becoming an option for the preparation of protein pharmaceuticals which demonstrate a rapid clearance from the body or a neutralizing or toxic immunological response. For a recent review see reference 1. PEGylated human growth hormone (hGH) has been shown to have a sustained biological response in animal models (2). Human growth hormone antagonist (GHA) is a mutant of h G H which has been designed to block hGH-induced receptor dimerization. This is achieved by mutating glycine 120, preventing interaction with the receptor at one site, and enhancing receptor binding at the second receptor binding site through the use of several other site specific mutations (3,4). This potential pharmaceutical will also benefit from PEGylation by exhibiting a sustained antagonistic response (data not shown). There are 9 potential amine PEGylation sites (3,4) in G H A including the ocamino group and the 8 ε-amino groups of lysyl residues. The propensity of these 170

© 1997 American Chemical Society

In Poly(ethylene glycol); Harris, J. Milton, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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PEGylated Human Growth Hormone Antagonist

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amines to be PEGylated depends upon their pKa values as well as their exposure on the surface of the molecule. Other than the α-amino group, it is diffucult to selectively PEGylate specific sites since a number of the exposed lysines have similar pKa values. Proteins with an effective molecular weight above approximately 70,000 daltons are not cleared by the glomerular filtration system of the kidney (5). In the case of hGH, M W 22,100 daltons, it was observed that a minimum of 4-5 P E G moieties with an average molecular weight of 5,000 (PEG5000) need to be incorporated into the protein. Excess PEGylation of a protein may decrease its activity. For example, it could block an active site on the protein preventing interaction with a receptor, or decrease the protein-receptor interaction by altering the diffusion of the molecule in solution. It was found that the selectivity process to obtain a specific molecular mass range for hGH could be achieved by cationexchange chromatography (2). Fractionation of PEGylated proteins by cationexchange chromatography is related to the number of lysines which are PEGylated. By incorporating a cation-exchange chromatographic step after PEGylation, molecules containing more or less than the desired number of polyethylene glycol groups can be selectively removed from the preparation. During preclinical development of a PEGylated protein, it is not only necessary to make sure that the protein can be purified in a reproducible manner before PEGylation, but also to make sure that the PEGylation can be controlled to produce a product with the desired characteristics. This can be achieved by controlling the PEGylation reaction and by fractionation of the PEGylated product. Since pharmaceutical development is a lengthy and expensive process, it is critical to select the desired PEGylation product early in the development cycle. One characterization tool that we have applied to PEGylated proteins is tryptic hydrolysis. Trypsin is a protease which cleaves a protein at the carboxyl side of lysyl or arginyl residue creating a number of peptides. These peptides can be fractionated by high pressure liquid chromatography (HPLC) creating a "tryptic map" of the protein (2). It was found that PEGylated lysine residues are resistant to trypsin hydrolysis. Analysis of the tryptic maps before and after PEGylation revealed which lysines were conjugated. Experimental PEG Derivatization. Purified G H A ( molecular weight 22kD) was obtained from the Recovery Science Dept. at Genentech. The reaction of the succinimidyl ester of carboxymethylated methoxy-PEG or the succinimidyl derivative of PEG propionic acid (SPA, Shearwater Polymers, Inc, Huntsville, AL)(Figurel) with G H A was studied and optimized to produce a majority of the species with a molecular mass between 40,000-50,000 daltons. Approximately an equal molar amount of PEG5000 reagent for each potential reactive site was found to be optimal. A protein concentration of 10 mg/ml was used in 50mM sodium phosphate, pH 7.5. The components were mixed and incubated at room temperature for approximately one hr. Column Chromatography. The reaction mixture was first fractionated on a Phenyl Toyopearl 650M (TosoHaas, Montgomeryville, PA) column to remove small amounts of high molecular weight cross-linked species. Sodium citrate was added to



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the reaction mixture, to a final concentration of 0.35M, before application to the column. The preparation was loaded at a protein concentration of up to 3.7 grams per liter of resin onto the Phenyl Toyopearl column equilibrated in 0.35M sodium citrate/0.05M Tris, pH 7.6. The PEGylated protein was eluted with a 6-column volume (CV) linear gradient from 0.35M sodium citrate/0.05M Tris, pH 7.6 to 0.05M Tris buffer, pH 7.6. The main protein peak was pooled and desalted either by G-25 Sephadex (Pharmacia) or by diafiltration into 25mM sodium acetate, pH 4.0. The desalted preparation was applied (7 grams protein/liter of resin) to an S Sepharose Fast Flow column (Pharmacia, Piscataway, NJ) equilibrated in 25mM sodium acetate, pH 4.0, and eluted with a gradient generated using a 7-column volume linear gradient from 25mM sodium acetate, pH 4.0 to 25mM sodium acetate/250mM NaCl, pH 4.0. A l l chromatographic steps were performed at room temperature, and protein was detected by U V absorbance at 280nm. Fractions were collected and characterized. SDS Gel Electrophoresis. Samples were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE)(6). Gradient polyacrylamide gels of 10-20% or 2-15% polyacrylamide were purchased from Integrated Separation Systems, Natick, M A . Samples were loaded onto a SDS polyacrylamide gel and run for approximately 50 minutes at 55mA. The protein bands were stained with Coomassie Blue. MALDI-TOF Mass Spectrometry. Fractions or pools from the S Sepharose Fast Flow chromatography were analyzed by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) mass spectrometry (9). The analyte was mixed with alpha cyano-4-hydroxy-iratt,s-cinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid and dried on the sample holder. A Voyager Elite M A L D I - T O F mass spectrometer (PerSeptive Biosystems, Framingham, M A ) was operated in the linear mode. Fractions from the S Sepharose Fast Flow column elution which contained PEGylated protein in the 40kD-50kD molecular mass range were pooled. Tryptic Map Analysis. The sites of PEGylation were determined by comparing the tryptic maps of the protein before and after PEGylation. PEGylated G H A was digested with bovine trypsin (Worthington, Freehold, NJ) 1:20 (w/w) trypsinrprotein for 8 hr at 37°C in ImM calcium chloride, 0.1M sodium acetate, and lOmM Tris at pH 8.3. The reaction was stopped with the addition of phosphoric acid to pH 2. The digested protein was loaded onto a C-18 Nucleosil column and eluted according to Reference 2. The peaks of the chromatogram (tryptic map) were integrated and normalized for comparative purposes using the peptides which did not contain lysyl residues. The reduction in the area of the peaks was used to determine the sites and degree of PEGylation. Bioassays. The biological activity of PEGylated species was determined by a modified, non-radioactive version of the previously described cell-based biological assay (3,8). The cells are transfected with the gene for the hGH receptor and, therefore, grow upon interaction with hGH. The assay was run in the presence of 15ng/ml hGH to measure the inhibitory activity of growth hormone antagonists.


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Results and Discussion Preparation of PEGylated Proteins. PEGylation conditions were optimized to produce PEGylated G H A with a mass of primarily 40-50 kilodaltons. We were able to achieve similar results with either 5kD, lOkD, or 20kD PEG (data not shown). The fractionation of the PEGylated protein was achieved by cation-exchange chromatography (2). With increasing PEGylation of a protein, there is a reduction in the number of primary amino groups, resulting in decreased ionic interaction with the cation-exchange resin. Fractions were collected and analyzed by SDS-PAGE (Figure 2). Proteins of higher molecular weight, i.e., containing more PEGylated sites, elute first from the column consistent with their reduced basicity and decreased affinity for the cation-exchange column. This method can be scaled to prepare large quantities of PEGylated proteins with a specific molecular mass range. Characterization by MALDI-TOF. MALDI-TOF mass spectrometry was used as an analytical tool to determine the actual molecular mass of fractionated PEGylated proteins. It was found that not only is gel filtration not very effective in the separation of various PEGylated species, but also their apparent molecular weight did not correlate with the molecular weight of protein standards. The effective molecular weight as determined by gel filtration is greatly influenced by the conformation and hydration of the PEGylated protein rather than the molecular mass. In 1988, Karas and Hillenkamp published a benchmark paper determining the molecular weight of proteins greater than lOkD using laser desorption ionization with nicotinic acid as a matrix (9). This technique has now become widely adopted and is used extensively for the mass analysis of proteins including PEGylated conjugates. The protein solution is mixed with an UV-absorbing matrix and deposited on the probe. The energy from a laser is adsorbed primarily by the matrix, vaporizing the matrix which carries the ionized protein polymer into the vapor phase generating a gas-phase polymer. The molecular mass is then determined by a time-of-flight mass analyzer. This method is semi-quantitative and has allowed the determination of the presence of small amounts of more highly PEGylated species, in addition to those with lower levels of conjugation. The SDS-PAGE results, which showed that the more highlyPEGylated proteins eluted first from the cation-exchange column, followed by fractions that had a lower level of PEGylation, were confirmed by MALDI-TOF mass spectrometry. Figure 3 shows the mass spectral result of a G H A preparation with primarily 4-5 PEG5000 molecules per protein molecule. In order to see any signal in mass spectroscopy, the protein needs to be ionized. The first signal in the 20,000 m/z range represents the doubly ionized species. The second signal (singly ionized) reveals the presence of 4 and 5 PEG5000 moieties conjugated to G H A and a small amount with 6 PEG5000. Now that PEGylated proteins can be fractionated and characterized, it is possible to correlate the mass of the molecule with the biological activity (2). Additional benefits of mass spectral analysis include verification of the reproducibility of the manufacturing process with regard to the molecular mass and determination of molecular mass limits for lot certification. Characterization by Tryptic Map Analysis. Tryptic maps are an important tool in the determination of the site and extent of PEGylation. The peptides generated for


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Figure 2. Chromatogram of S Sepharose Fast Flow fractionation of a G H A PEG preparation with SDS-PAGE analysis of some selected fractions. Lane 1, 143 μg starting material; lanes 2-7, 20 μΐ of fractions 10, 20, 37, 53, 70, and 90, respectively.


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G H A are shown in Table I. Figure 4 shows the tryptic map of G H A before and after PEGylation. Tryptic peptides of G H A were identified by amino acid sequence analysis and mass spectrometry (data not shown). Elimination or reduction in a tryptic peptide peak is indicative of PEGylation. For each complete PEGylation of a lysyl residue, two peptides disappear from their original positions in the tryptic map since cleavage after the PEGylated lysyl residue does not occur. Peptides containing PEGylated residues are more highly retained by the CI8 Nucleosil column and elute as broad peaks at the end of the map. The results are summarized in Table II. Since two peptides are affected by PEGylation, a peptide which does not contain PEGylation sites adjacent to a peptide which can be PEGylated may be used to quantitate the extent of PEGylation. In the case of G H A only the N-terminal amine in F l is 100% PEGylated. Lysines at positions 38, 145, 158 and 140 were PEGylated in approximately 80% of the protein molecules, while lysine in position 120 was PEGylated in 40% of the molecules. Lysine at position 158 was only PEGylated in 26% of the molecules. The tryptic peptides on either side of T15 are PEGylated so the extent of PEGylation on Κ145 can only be approximated. The average for this preparation was 4-5 PEG5000V GHA. Table 1. Sequence and Tryptic Fragments of G H A Peak Name Fragment Sequence Tl 1-8 FPTIPLSR Tic 1-6 FPTIPL T2 9-16 LFDNAMLR T3 17-19 ADR T4 LNQLAFDTYQEFEEAYIPK 20-38 T5 39-41 EQK T6 YSFLQNPQTSLCFSESIPTPSNR 42-64 T7 65-70 EETQQK T8 71-77 SNLELLR T9 78-94 ISLLIQSWLEPVQFLR T10 SVFANSLVYGASDSNVYDLLK 95-115 TlOcl 95-99 SVFAN T10c2 SLVYGASDSNVYDLLK 100-115 T10-11 SVFANSLVYGASDSNVYDLLKDLEEK 95-120 Til 116-120 DLEEK T12 121-127 IQTLMGR T13 128-134 LEDGSPR T14 135-140 TGQIFK T15 141-145 QTYSK T16 146-158 FDTNSHNDDALLK T6-17* 42-64 + YSFLQNPQTSLCFSESIPTPSNR159-172 NYGLLYCFNADMSR T18 173-178 VSTFLR T19-20* 179TVQCR183+ 184-L91 SVEGSCGF * Disulfide-bonded peptides


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Retention time (min) Figure 4. High performance reverse-phase liquid chromatogram of tryptic peptides from unmodified G H A and 4-5 PEG5000 G H A . The second T15 peptide reflects the cyclization of the N-terminal glutamine to pyroglutamic acid. The arrows identify the peptides which are reduced.


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There are several buried lysines which are not conjugated even in the presence of excess PEG reagent. The reactivity of a lysine can be estimated by the extent that the peptide is reduced in the tryptic map. Those peptides that are completely missing in the map contain or are adjacent to the most reactive sites and those peptides that are reduced very little or not at all contain the least reactive sites. The order of PEGylation can also be determined by limited PEGylation followed by cation exchange chromatography and tryptic analysis. For example, the PEGylation site in the monoPEGylated protein can be analyzed to determine which residue was conjugated. This analysis can then be conducted on the two PEG molecule, three PEG molecule, etc. For GHA the most reactive site is the N-terminal phenylalanine due to its lower pKa value. Although tryptic analysis is not absolute due to incomplete tryptic digestion and the difficulties associated with the analysis of multiple PEGylation sites, it gives a good approximation of the level of PEGylation as well as the sites of conjugation. The results correlate well with mass spectrometry.

Table II. Tryptic analysis of PEGylated GHA Tryptic Peptide Amino Acid % Reduction Analyzed Residue of PEGylated peptide Tl T4 T14,T15,T16 T6-17 T14 T12 T8

Fl K38 K145 K158 K140 K120 K70

100 81 -80 79 78 39 26

In Vitro Biological Activity. A cell-based bioassay exists for hGH in which the fulllength hGH receptor is stably transfected into a premyeloid cell line (FDC-P1 cells) which can then be induced to proliferate in the presence of hGH (3,8). Thymidine incorporation into the FDC-P1 cells was directly correlated with the ability of hGH to cause dimerization of the receptor (10). The results of PEGylated hGH in the cellbased assay were previously published (2). Potency decreased with the extent of PEGylation and a linear correlation was found to exist between the log of the increase in EC50 (the concentration of hormone required for 50% maximal stimulation of cell proliferation) and the number of PEG groups conjugated to hGH. The loss of activity with increased PEGylation appears to be substantial. This can be misleading, however, as it was found that the increase in the half-life of h G H more than compensates for the loss of binding to the receptor in the bioassay (2). An adaptation of the hGH bioassay (5,77) was made to convert it into a useful assay for growth hormone antagonists. By maintaining a constant hGH concentration in the assay to effect a near maximal stimulation, the inhibition of activity can be measured in the presence of antagonists. The IC50 (concentration of antagonist



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where a 50% decrease in cell proliferation is seen) is a measure of the extent of inhibition of hGH receptor binding in the cell-based assay. Figure 5 shows the effect of G H A and PEGylated G H A on the bioassay. Inhibition occurs at a relatively low dose of GHA. The PEGylated molecule (MW 40-50kD) showed an 65-fold lower activity in the bioassay than the unPEGylated GHA. The traditional rat weight gain bioassay, designed to measure the growth effects of hGH, cannot be used for G H A since there is no constitutive exposure to growth hormone in these hypophysectomized animals. The cell-based bioassay is useful for measuring the bioactivity of hGH, the effect of PEG conjugation on receptor binding, and the reduction of activity of PEGylated G H A relative to GHA. Conclusion Considerable progress has been made in the PEGylation, fractionation, and analytical characterization of a product such as PEGylated recombinant G H A . Cation-exchange chromatography is a powerful tool to fractionate PEGylated proteins based on their extent of PEGylation (2). The desired PEG species can be produced through control of the pH and the stoichiometry of the components in the PEGylation reaction followed by cation-exchange chromatography. Tryptic map analysis and mass spectrometry have been applied to PEGylated proteins to assist in the characterization of the therapeutic protein produced. Tryptic map analysis provides a useful technique to determine the sites and extent of PEGylation in a conjugated protein which has been PEGylated through lysyl moieties and the N-terminal amine. It was found that certain sites are PEGylated more than other sites. The N-terminus was always 100% conjugated, while others were about 80%. Several lysines were conjugated in about 30% of the molecules. This tryptic map analysis is useful in that it provides an opportunity to measure the consistency of the PEGylation process from lot to lot. A well-characterized therapeutic protein should include data on the sites of PEGylation. This has not typically been done for complex PEGylated proteins. It is not only useful but critical that biological activity be measured on PEGylated proteins prior to clinical studies in humans. The cell-based hGH bioassay allows a correlation of the extent of PEGylation to bioactivity. Studies in animals can demonstrate the combined effects of altered bioactivity and clearance. PEGylation must be controlled since over-PEGylation can decrease biological activity or create a molecule with a longer half-life than desired, while a protein with limited PEGylation may be cleared faster than desired. PEGylation had a similar effect on G H A as it did to hGH. The in vitro activity decreased as the extent of PEGylation increased. This is not expected to have an adverse effect on in vivo activity, however. In the case of hGH, the PEGylated protein had as much or more activity than the authentic protein (2). Methods are described in this report to not only determine the molecular weight of PEGylated proteins but to also measure the relative abundance of various PEGylated species. In addition, MALDI-TOF mass spectrometry is a useful tool to determine the size of the conjugated proteins during the development of the PEGylation process. Other methods, such as gel permeation chromatography can provide an approximate molecular size, but the large hydrodynamic radius of PEG groups results in an apparent molecular size of the PEGylated protein which is



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Figure 5. Cell proliferation assay of G H A and PEGylated GHA. The samples were incubated in the presence of 15 ng/ml of hGH. Cell growth was measured by fluorescence, measuring the uptake of alamar blue. R F U = Relative Fluorescence Units


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considerably higher than the actual mass. For instance, a PEGylated protein with a mass of approximately 50,000 daltons can have an apparent molecular mass of 300,000 daltons when compared with globular proteins on a gel permeation column. M A L D I - T O F mass spectrometry provides an accurate molecular mass of a PEGylated protein. In addition, it can see the relative abundance of other PEGylated species in the preparation providing semi-qualitative analysis and assigning to them a molecular mass.

Acknowledgments This work was sponsored by Sensus Corporation, Austin, Texas. Development of tryptic map analysis of PEGylated proteins was pioneered by Rosanne Chloupek, Glen Teshima, and Eleanor Canova-Davis.

Literature Cited 1.

Zalipsky, S.; Lee, C. In PEG Chemistry: Biotechnological and Biomedical Applications; Harris, J.M., Ed.; Plenum Publishing Corp.: New York City, NY, 1992, pp 347-370. 2. Clark, R.; Olson, K.; Fuh, G.; Marian, M.; Mortensen, D.; Teshima, G.; Chang, S.; Chu, H.; Mukku, V.; Canova-Davis, E.; Somers, T.; Cronin, M.; Winkler, M.; Wells, J. Journal Biol. Chem. 1996, 271, 21969-21977. 3. Fuh, G.; Cunningham, B.; Fukunaga, R.; Nagata, S.; Goeddel, D.; Wells, J. Science, 1992, 256, 1677-1680 4. Cunningham, B.; Wells, J. Proc. Natl.Acad.Sci. 1991, 88, 3407-3411. 5. Venkatachalam, M.; Rennke, H. Circ. Res. 1978, 43, 337-347. 6. Laemmli, U.K. Nature. 1970, 227, 680-685. 7. Watson, E.; Shah, B.; DePrince, R.; Hendren, R.W.; Nelson, R. B. BioTechniques . 1994, 16, 278-281. 8. Colose, P.; Wong, K.; Leong, S.; Wood, W.I. J. Biol. Chem. 1993, 268, 12617-12623. 9. Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. 10. Spencer, S.; Hammonds, R.G.; Henzel, W.J.; Rodriquez, H.; Waters, M.J.; Wood, W.I. J. Biol Chem. 1988, 263, 7862-7867. 11. Roswall, E.C.; Mukku, V.R.; Chen, A.B.; Hoff, E.H.; Chu, H.; McKay, P.A.; Olson, K.C.; Battersby, J.E.; Gehant, R.L.; Meunier, Α.; Garnick, R.L. Biologicals, 1996, 24, 25-29


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Preparation and Characterization of Poly(ethylene glycol)ylated

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