Poly(ethylene glycol) - ACS Publications - American Chemical Society

Chapter 15

Downloaded by UNIV OF OKLAHOMA on September 3, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch015

Isolation of Positional Isomers of Mono -poly(ethylene glycol)ylated Interferon/α-2a and the Determination of Their Biochemical and Biological Characteristics Seth P. Monkarsh, Cheryl Spence, Jill E. Porter, Alicia Palleroni, Carlo Nalin, Perry Rosen, and Pascal Bailon 1

Departments of Biopharmaceuticals and Oncology, Hoffmann-La Roche, Inc., Nutley, ΝJ 07110 MonoPEGylated interferon α-2a (monoPEG-IFN) is produced by conjugating interferon α-2a to a chemically reactive form of polyethylene glycol-5000 in 1:1 ratio, via a urea linkage. Potentially, all 11 lysine residues and the N-terminal amino group in interferon α-2a are available for PEGylation. Thus monoPEG -IFN is a mixture of individual species, each having a unique site of PEGylation. A method involving sulfopropyl-high performance liquid chromatography (SP-HPLC) which exploits the minute local charge differences between the individual monoPEG-IFN α-2a species (positional isomers) is used to separate them. Eleven positional isomers were isolated by this method, each having a specific lysine as their PEGylation site indicating all eleven lysines of interferon α-2a were PEGylated. However, N-terminal amino group was not derivatized. All isolated species exhibit antiviral and antiproliferative activities. In recent years pharmaceutically active proteins have been conjugated with polyethylene glycol (PEG) to improve circulating half-life, bioavailability, solubility, stability, as well as to reduce immunogenicity and toxicity. For example interferon cc-2a which is an anti-viral and anti-tumor agent has been conjugated to PEG resulting in improved pharmacokinetic properties and retention of biological activity (I). Typically, conjugation of interferon a-2a (IFN) to chemically activated PEG results in a mixture of mono, di, tri and oligo PEGylated protein conjugates. These individual species could possibly be separated using a combination of conventional methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), high performance size exclusion chromatography (HPSEC), high performance ion exchange chromatography (HPIEC), reversed-phase high performance liquid chromatography (RP-HPLC) and high performance hydrophobic interaction chromatography (HPHIC), among others (2-7). Each of these individual species 1

Corresponding author.

© 1997 American Chemical Society

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

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208

POLY(ETHYLENE GLYCOL)

are comprised of positional isomers which has PEG attached to a different lysine of interferon a-2a. Here we report the isolation and characterization of the positional isomers of monoPEG-IFN. Main focus of this chapter is on separation methods for monoPEG-IFN isomers. The biochemical and biological characteristics of the separated isomers will be reviewed using data from another publication (Monkarsh et al., J. Anal Biochem., in press). A method (8), originally developed for the separation of deamidated and amidated forms of human interleukin-lct, as well as for isoforms of human interleukin-ΐβ with and without N-terminal methionine, has been modified and adapted for the separation of monoPEG-IFN positional isomers. The theoretical number of positional isomers in a PEGylated molecule can be calculated from the following equation: For Ν PEGylation sites, taken Κ at a time; Ν! = P; wherein k is the number of sites actually (N-k)! · k! occupied by PEG and Ρ is the possible number of positional isomers. For monoPEG-IFN, k=l and the possible number of positional isomers, Ρ = 12. Similarly for diPEG interferon ct-2a, k=2 and P= 66; for triPEG interferon a-2a, k = 3 and Ρ = 220, and soon. Materials and Methods Preparation of mono-PEGylated interferon ct-2a (PEG-IFN). MonoPEG-IFN is produced by conjugating interferon cc-2a to a chemically reactive form of polyethylene glycol 5000 (PEG-5000, average M.W. 5300 daltons) in 1:1 molar ratio via a urea linkage (Figure 1). The aforementioned PEG reagent was obtained from Roche Kilo Lab 9). The PEGylation reaction was performed by mixing interferon (~6mg/ml) and the PEG reagent at a protein :reagent molar ratio of 1:3 in 5 m M sodium acetate, pH 10 buffer. After lhour of mixing at room temperature, the reaction was stopped by adding one-twentieth volume of 1M glycine, pH 7.5. The pH of the reaction mixture was adjusted to 5-6 by the addition of onetwentieth volume of 1M sodium acetate, pH 4. The reaction mixture was diluted 4-fold with 40mM ammonium acetate pH 4.5 and applied onto a Whatman CM-52 cellulose column (2mg protein/mL resin), previously equilibrated with the same buffer. The unabsorbed materials were washed away and the adsorbed PEG-IFN was eluted using a 0-0.5M linear sodium chloride gradient in the equilibration buffer. The column effluents were monitored by U V absorbance at 280nm. Fractions containing monoPEG-IFN was identified by SDS-PAGE. Alternatively, the PEG-IFN was made using a solid-phase method (JO). In the solid-phase method IFN was bound to a strong anion exchange resin and the PEGylation reagent in the appropriate buffer and concentration was recirculated through the column for 30 minutes. The excess reagent and reaction byproducts were washed away from the column and the PEGylated IFN was eluted with increasing salt concentrations. Elution conditions were chosen such that the unmodified IFN remained on the column. After elution was complete, the column was reequilibrated with the buffer. Then an amount of IFN equal to the amount PEGylated (eluted) was applied onto the column and the whole PEGylation process was repeated. This continuous PEGylation process could be repeated as many times as needed. Fractions containing the monoPEG-IFN from both the solution and solid-phase methods was pooled, concentrated by ultrafiltration and stored in 40 mM ammonium acetate, pH 5.0 buffer at a concentration of 9 mg/ml.



15.

209

MONKARSH ETAL. Mon-PEGylatedInterferon-o2a

JOO

RO(CH,CH-yO) -CH,CH-,-N ON* A ""H n

R = CH

3

ΝΗ-,-Procein

H N RO(CH2CH 0) -CH CH 2

n

2

Nhi-Protein

Β

/ 2

Figure 1. Reaction scheme of the formation of urea linkage between protein and the PEG reagent. A . PEG reagent and B. urea-linked PEG-protein conjugate.


210



Separation of PEG-IFN Positional Isomers Analytical Scale. A Waters 626 biocompatible, analytical high-performance liquid chromatography system (Millipore, Milford, M A ) was used. This HPLC system consisted of Model 626 L C pump, Model 600S powerline control module, model 486 variable-wavelength absorbance detector, a model 717 Plus autosampler and data was collected and processed using the Millenium software package version 2.00. A commercially available stainless-steel HPLC column containing TSK-Gel SP-5PW type, hydrophilic SP-strong cation exchange support (10 μπι, 7.5 mm I.D. χ 7.5 cm L.; TOSO H A A S Mahwah, NJ) was equilibrated with 3.7 m M acetic acid, pH 4.3 (pH adjusted very carefully with ION sodium hydroxide; buffer A) at a flow rate of 1 ml/min. The stock monoPEG-IFN was diluted 10-fold with buffer A , pH adjusted to 4.3 with 1 Ν hydrochloric acid (Fisher Scientific) and 70 μg protein was applied to the column via autosampler. The column was washed with buffer A for 20 minutes, followed by a linear ascending pH gradient (pH 4.3-6.4) was performed from 0-100% of 10 m M dibasic potassium phosphate, pH adjusted to 6.4 with 6N hydrochloric acid (Pierce, Rockford, IL); buffer B). The pH gradient was completed in 220 minutes. The column effluents were monitored by U V absorbance at 280 nm. The column was regenerated by washing with 0.2 M dibasic potassium phosphate containing 0.5 M sodium chloride pH 8.0 for 15 minutes at 2.0 ml/min. The column was reequilibrated with buffer A for 40 minutes at 2 ml/min to attain the starting pH conditions for the next run. A l l operations were carried out at ambient temperature (23-25°C) and all buffers were sparged continuously with ultra-high-purityhelium (99.999% pure; JWS Technologies, Piscataway, NJ). Preparative Scale. A Waters Delta Prep 3000 preparative HPLC system equipped with a preparative TSK-gel SP-5PW HPLC column (13μπι, 21.5 mm I.D. χ 15cm L; TOSO HAAS) was used under the same buffer and gradient conditions as in the analytical scale except that the flow rate was 6 ml/min. Data were collected and analyzed using the Dynamax PC HPLC data system (Rainin Instruments, Woburn, MA). The PEG-IFN stock (9 mg/ml) was diluted 10-fold with buffer A and pH adjusted to 4.3, as before. Ten milliliters of the diluted PEG-IFN stock equivalent to 9 mg was applied onto the column. As each of the monoPEG-IFN species were eluted individual pool fractions were collected manually and rechromatographed on the same column to determine any changes in elution profile. Protein in each of the pooled samples was individually concentrated in a Centriplus-10 concentrator (Amicon, Beverly, M A , USA). Protein Determination. Protein concentrations were determined by the amino acid composition analyses performed on a post-column amino acid analyzer with reaction with fluorescamine (11). SDS-PAGE Analysis. Protein from the individual pools was subjected to SDSPAGE analysis using non-reducing conditions according to the methods of Laemmli (12) . Antiviral Assay. The antiviral activity of both IFN cc-2a and the isolated PEGIFN species were determined by the reduction of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney (MDBK) cells (13) Antiproliferative Assay. Human Daudi cells (Burkitt's lymphoma) were maintained as stationary suspension cultures in RPMI 1640 media supplemented by 10% fetal bovine serum and 2mM glutamine (Grand Island Biologicals, Grand


15.

MONKARSH ETAL.

Mon-PEGylatedInterferon-a-2a

111

Island, NY). The cells were screened and found to be free of mycoplasma. The assay was performed as previously described (14). Antiproliferative activity of monoPEG-IFN and its separated positional isomers was determined by its inhibition of cell proliferation in human Daudi cells (a Burkitt's lymphoma) as measured by the decrease in incorporation of 3-H-thymidine into cellular DNA. The results were expressed as % inhibition calculated from the following formula: % Inhibition = [(A - B)/A ] χ 100, where; A = cpm in control culture (cells incubated in medium alone) Β = cpm in experimental culture The 50% inhibition or IC value was caculated from % inhibition of of cell growth based upon 15.6-1000 pg/ml protein concentrations. Downloaded by UNIV OF OKLAHOMA on September 3, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch015

50

Results and Discussion Application of the analytical SP-HPLC method to both the solution-phase and solid-phase derived monoPEG-IFN resulted in separation of monoPEG-IFN into at least 9 species for each application (Figure 2). These peaks elute between pH 5.6 and pH 6.3 and appear to have similar relative peak heights and retention time for both monoPEG-IFN preparations. Unmodified interferon a-2a elutes at pH 6.4, around 245 minutes (data not shown). In general, we have noted minor variations in the profiles of the 3 major peaks mainly due to column-to-column differences. However, the results show that the monoPEG-IFN from the solid-phase and solution-phase methods display similar fingerprint patterns indicating that the same species are present in the two preparations. The preparative scale elution profiles obtained with 9 mg monoPEG-IFN are illustrated in Figure 3. Both analytical and preparative elution profiles have similar finger prints except that the major peaks 5, 6 and 7 are broader and poorly resolved in the preparative mode. In addition, the preparative-scale separation of the monoPEG-IFN resulted in eleven species for each application. Elution profiles of the samples did not change upon rechromatography (data not shown). Amino acid composition analyses performed on the rechromatographed samples indicate agreement between the amino acid compositions of unmodified and modified IFN samples. SDS-PAGE analysis indicated that each preparative peak pool contained monoPEG-IFN, free of aggregates or degradation products (data not shown). Recovery of monoPEG-IFN from the preparative SP-HPLC was fairly high. When 9mg PEG-IFN was applied to the column, the total protein recovery was 73-78%, as determined by amino acid composition analysis. An aliquot of the pooled samples (all peaks collected as a single pool) was rechromatographed by analytical SP-HPLC and the resulting fingerprint was unchanged demonstrating that there is no selective loss of any of the species. MonoPEG-IFN is interferon a-2a monoPEgylated with polyethylene glycol (PEG-5000), in a 1:1 molar ratio, via a urea linkage. This conjugation results in the modification of primary amino groups of IFN ct-2a. When the separated PEG-IFN species were subjected to peptide mapping, sequencing and mass spectrometric analyses, they demonstrated that the individual species are positional isomers which differ only in their site of PEGylation (Mokarsh, S. P. et al., Anal. Biochem., in press)). There are 12 primary amino groups (N-terminus plus 11 lysine side chains) in interferon ct-2a, all of which are potentially available for PEGylation. Eleven out of the 12 possible positional isomers were identified, all of which involved lysines as their PEGylation sites (Table 1). However, no positional isomer involving the N-terminus was detected with the SP-HPLC method (data not shown). Since the N-terminal amino acid of interferon a-2a is a cysteine which is involved in a disulfide bond, it is presumed that the primary amino group of the N terminus may not be readily available for PEGylation and may also be due to the reaction pH.


212


o.cos —· J


0.004

-C

—J

0.002

-J

Figure 2. Analytical scale SP-HPLC profiles of PEG-IFN produced by the solidphase (A) and solution-phase (B) methods. Seventy μg PEG-IFN were used in each analysis.


15.

MONKARSH E T A L .

213

Mon-PEGylatedInterferon-a-2a


A

Figure 3. Preparative SP-HPLC profiles of PEG-IFN produced by the solid-phase (A) and solution-phase (B) methods. Ten sample pools were collected as indicated by the vertical bars on the peaks in the profile. The peaks were numbered 1-7, 8A, 8B,9and 10.


214


Table I. Antiviral and Antiproliferative Activities of MonoPEG-IFN Species produced by the solution-phase method. Downloaded by UNIV OF OKLAHOMA on September 3, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch015

Samples

Peak No. Antiviral

Bioactivities **PEG Sites * Antiproliferative

lO^xIU/mg

ICso (pg/mL)

IFN

NA

21.0

33

NA

MonoPEG-IFN

NA

7.2

180

NA

Species-1

1

6.7

227

κ

31

Species-2

2

8.4

213

Species-3

3

6.4

113

Species-4

4

4.3

273

κ

23

Species-5

5

4.0

200

Species-6

6

3.0

267 K , ,

Species-7

7

4.5

340

Species-8

8A

5.9

170

Species-9

8B

6.3

293

κ

83

Species-10

9

6.7

250

κ

49

Species-11

10

1.2

361

κ"

131

K

121

164

131

Κ 121 K

164

70

2

NA = Not Applicable The % inhibition of cell growth based upon the concentrations of PEG-IFN (15.6-1000 pg/mL) was utilized to calculate 50% inhibition values ( I C 5 0 ) . **Data taken from Monkarsh et al., Anal. Biochem. (in press) and Κ is the symbol for lysine.



15.

MONKARSH E T AL.

Mon-PEGylated

Interferon-α-2a

215

MonoPEG-IFN is a mixture of positional isomers, each being conjugated with a PEG-5000 by a urea linker at a different site. The sites (mainly lysines) and the extent to which each of the lysines become PEGylated are dependent upon individual pKa and steric accessibility, as well as the chemical process itself. The pKa of each of the 11 lysine residues is expected to be slightly different due to the location on the polypeptide chain and the local microenvironment. Therefore, all lysines of interferon a-2a are not equally active. Modification of the basic lysines not only lowers the local pi, but also changes the overall pi of the protein. Each positional isomers may have a slightly different pi and a unique position of attachment of a single PEG molecule. The end-result is heterogeneity in charge, site of PEGylation and distribution of positional isomers in the PEG-IFN preparation. The SP-HPLC method takes advantage of minute local charge differences to separate and isolate the PEG-IFN positional isomers into 10 peaks or fractions. The results listed in Table 1 (last column) indicate that each peak except 6 and 7, corresponds to a single positional isomer with a unique PEGylation site, whereas peaks 6 and 7 are mixtures containing 2-3 positional isomers. MonoPEG attachment at various sites in interferon a-2a may produce physicochemical differences (e.g., changes in conformation, electrostatic binding properties, hydrophobicity, local lysine pKa, pi etc.) in the individual monoPEGIFN conjugate species. These physicochemical differences could influence the binding affinity to the IFN-receptor and consequently affect their bioactivities. However, each of the 11 monoPEG-IFN positional isomers exhibited antiviral and antiproliferative activities. The sum of activities of individual species appears to be slightly lower than that of the parent monoPEG-IFN mixture which was produced by the solution-phase method (see Table I). It is also possible that some loss of activity might have occured during additional handling of the samples, as well as due to non-ideal storage (buffer) conditions. However, the differences in activity observed in both assays fall within the range of variations usually associated with these types of bioassay. The species derived from the solid-phase method also exhibited similar biological activities (data not shown). A l l monoPEG-IFN species exhibited bioactivity, some more than others, indicative of the effect of site of PEGylation on IFN/receptor interactions and the resulting biological activity. The results listed in Table I indicate a substantial loss in in vitro bioactivities upon PEGylation of interferon cc-2a. In contrast, the unfractionated monoPEG-IFN exhibited significant enhancements in circulating half-life and in vivo biological activity (Palleroni, A , Hoffmann-La Roche, Nutley, NJ, unpublished data). The positional isomers were not tested individually for in vivo biological activities. In summary, PEG-IFN is a mixture of positional isomers in which each PEG moiety is attached at a unique site on interferon via a urea linkage. A method involving SP-HPLC which exploits minute local charge differences between the positional isomers is used to separate them. A l l isolated PEG-IFN positional isomers exhibit antiviral and antproliferative activities. Acknowledgments We want to thank Dr. Wen-Jian Fung for her critical review and Ms. Audrey Faronea for preparing the manuscript. Literature Cited 1. 2.

Gilbert, C.W. 1995, International patent publication number WO95/13090 Abuchowski, Α.; van Es, T.; Palczuk, N.C.; Davis, F.F. J.Biol.Chem. 1977, 252, 3578.


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3. 4. 5. 6.

7. Downloaded by UNIV OF OKLAHOMA on September 3, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch015

8. 9. 10. 11. 12. 13. 14.

Sharp, K.A.; Yalpani,M.;Howard, S.J.; Brooks, D.E. Anal. Biochem. 1986, 154, 110. McGoff, P.; Baziotis, A.C.; Maskiewicz, R. Chem. Pharm. Bull. 1988, 36, 3079. Snider, J.; Neville,C.;Yuan, L.-C.; Bullock, J. J. Chrom. 1992, 599, 141. Vestling, M.M.; Murphy, C.M.; Fenselau,C.;Dedinas, J.; Doleman, M.S.; Harrsch, P.B.; Kutney, R.; Ladd, D.L.; Olsen, M.A. In: Techniques in Protein Chemistry III; Angeletti, R.H., Ed.; Academic Press: NY, NY, 1992, pp. 477-485. Vestling, M.M.; Murphy, C.M.; Keller, D.A.; Fenselau,C.;Dedinas, J.; Ladd, D.L.; Olsen, M.A. Drug Metabolism and Disposition 1993, 21, 911. Monkarsh, S.P.; Russoman, E.A.; Roy, S.K. J. Chromatog. 1993,. 631, 277. Karasiewcz, R.; Nalin,C.;Rosen, P. 1995, U.S. Patent No. 5,382,657. Porter, J.; Cavilhas,C.;Prinzo, K.; Schaffer, C.A.; Monkarsh, S.; Bailon, P. 1996, "Continuous solid-phase PEGylation of proteins", Recovery of Biological Products VIII Symposium, October 20-25, Tuscon, AZ. Pan, Y.-C.; Stein, S. In Methods of Protein Characterization, Shively,J. Ed.; Humana Press: Clifton, NJ, 1986, pp 105-119. Laemmli, U.K. Nature 1970, 227,.680. Rubinstein, S.; Familletti, P.; Pestka, S. J. Virology 1988, 37, 755. Borden, E.C.; Hogan, T.F.; Voelkel, J.G. Cancer Research 1982, 42, 4948.


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