Secretion of Fc-amidated peptide fusion proteins by Chinese hamster ovary cells

BMC Biotechnology

Betty A. Eipper²  Richard E. Mains³  William Strohl¹  Michael Naso¹  Omid Vafa¹  Jiali Li⁴  Steven C. Pomerantz¹  Kristina R. Carlson³ 

[1]Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House 19477, PA, USA

[2]Department of Molecular Biology and Biophysics, UCONN Health Center, Farmington 06030, CT, USA

[3]Department of Neuroscience, UCONN Health Center, 263 Farmington Avenue, Farmington 06030-3401, CT, USA

[4]Biologics Research, Janssen Research & Development, San Diego 92121, CA, USA

关键词: Neuromedin U;    Peptide YY;    Glucagon-like peptide 1;    Mass spectrometry;    CHO cells;    Amidation;   

Others  :  1216684
DOI  :  10.1186/s12896-015-0173-5

 received in 2015-02-03, accepted in 2015-05-29,  发布年份 2015

【 摘 要 】

Background

The therapeutic use of α-amidated peptides (e.g. calcitonin, glucagon-like peptide) has increased dramatically, but there are major impediments to wider use of such peptides. Larger peptides are expensive to synthesize, and short plasma half-lives frequently limit the clinical circumstances in which the peptides would be useful. Both problems are potentially solved by producing peptides as fusions with the Fc region of human immunoglobulin.

Methods

Glucagon-like peptide 1 (GLP1), peptide YY (PYY) and neuromedin U (NMU) were expressed and purified from stable CHO lines; since the α-amide group is essential for full biological potency of many peptides, Fc-fusion peptides were expressed in CHO lines stably expressing the α-amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3). Purified fusion proteins were analyzed intact and after HRV3C rhinovirus protease cleavage, at a site in the linker separating the Fc region from the peptide, by mass spectrometry and amide-specific immunoassays.

Results

The Fc fusions were expressed at 1–2.5 μg/mg cell protein and secreted at 5-20 % of cell content per hour, in a peptide-specific manner. CHO cells express measurable endogenous PAM activity, amidating 25 % of Fc-PYY and almost 90 % of Fc-GLP1. Expression of exogenous PAM increased the level of peptide amidation to 50 % of Fc-PYY and 95 % of Fc-NMU. The Fc-GLP1 fusions were 10,000-fold less active than synthetic GLP1 in a cell-receptor cyclic AMP-based assay, as expected since the amino terminal of GLP1 is essential for full biological activity. The Fc-PYY fusions were 100-fold less active than PYY-NH ₂but 10-fold more active than non-amidated PYY-Gly.

Conclusions

This type of approach can be used for the production of stabilized α-amidated peptides aimed at clinical trials.

【 授权许可】

   
2015 Carlson et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

【 参考文献 】

• [1]Czajkowsky DM, Hu J, Shao Z, Pleass RJ. Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 2012; 4:1015-1028.
• [2]Kulathila R, Merkler KA, Merkler DJ. Enzymatic formation of C-terminal amides. Nat Prod Rep. 1999; 16:145-154.
• [3]Prigge ST, Mains RE, Eipper BA, Amzel LM. New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci. 2000; 57:1236-1259.
• [4]Czyzyk TA, Ning Y, Hsu M-S, Peng B, Mains RE, Eipper BA, Pintar JE. Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse. Dev Biol. 2005; 287:301-313.
• [5]Skulj M, Pezdirec D, Gaser D, Kreft M, Zorec R. Reduction in C-terminal amidated species of recombinant monoclonal antibodies by genetic modification of CHO cells. BMC Biotechnol. 2014; 14:76. BioMed Central Full Text
• [6]Kolhekar AS, Keutmann HT, Mains RE, Quon ASW, Eipper BA. Peptidylglycine α-hydroxylating monooxygenase: active site residues, disulfide linkages, and a Two-domain model of the catalytic core. Biochemistry. 1997; 36:10901-10909.
• [7]Kolhekar AS, Bell J, Shiozaki EN, Jin L, Keutmann HT, Hand TA, Mains RE, Eipper BA. Essential features of the catalytic core of peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Biochemistry. 2002; 41:12384-12394.
• [8]Braas KM, Stoffers DA, Eipper BA, May V. Tissue specific expression of rat peptidylglycine α-amidating monooxygenase activity and mRNA. Mol Endocrinol. 1989; 3:1387-1398.
• [9]Tsubaki M, Terashima I, Kamata K, Koga A. C-terminal modification of monoclonal antibody drugs: amidated species as a general product-related substance. Int J Biological Macromolecules. 2013; 652:139-147.
• [10]Kaschak T, Boyd D, Lu F, Derfus G, Amanullah A, Yan B. Characterization of the basic charge variants of a human IgG1: effect of copper concentration in cell culture media. MAbs. 2011; 3:577-583.
• [11]Johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem. 2007; 360:75-83.
• [12]Keire DA, Bowers CW, Solomon TE, Reeve Jr JR. Structure and receptor binding of PYY analogs. Peptides. 2002;305–321.
• [13]Brighton PJ, Szekeres PG, Willars GB. Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacol Rev. 2004; 56:231-248.
• [14]Khan S, Sur S, Newcomb CJ, Appelt EA, Stupp SI. Self-assembling glucagon-like peptide 1-mimetic peptide amphiphiles for enhanced activity and proliferation of insulin-secreting cells. Acta Biomater. 2012; 8:1685-1692.
• [15]Baker AE, Sague S, Grygiel TLR, Schmidt A, Rogers A, Jiang H, Kruszynski M, Nesspor T. The dimerization of glucagon-like peptide-2 MIMETIBODY is linked to leucine-17 in the glucagon-like peptide-2 region. J Mol Recognition. 2012; 25:155-164.
• [16]Merkler DJ. C-terminal amidated peptides: production by the in vitro enzymatic amidation of glycine-extended peptides and the importance of the amide to bioactivity. Enzyme Microb Technol. 1994, 16:450–456.
• [17]Tamburini PP, Jones BN, Consalvo AP, Young SD, Lovato SJ, Gilligan JP, Wennogle LP, Erion M, Jeng AY. Structure-activity relationships for glycine-extended peptides and the alpha-amidating enzyme derived from medullary thyroid CA-77 cells. Arch Biochem Biophys. 1988; 267:623-631.
• [18]Wulff BS, Catipovic B, Okamoto H, Gether U, Schwartz TW, Johansen TE. Efficient amidation of C-peptide deleted NPY precursors by non-endocrine cells is affected by the presence of Lys-Arg at the C-terminus. Mol Cell Endocrinol. 1993, 91:135–141.
• [19]Ciccotosto GD, Schiller MR, Eipper BA, Mains RE. Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1. J Cell Biol. 1999; 144:459-471.
• [20]Husten EJ, Tausk FA, Keutmann HT, Eipper BA. Use of endoproteases to identify catalytic domains, linker regions, and functional interactions in soluble peptidylglycine alpha-amidating monooxygenase. J Biol Chem. 1993; 268:9709-9717.
• [21]El Meskini R, Culotta VC, Mains RE, Eipper BA. Supplying copper to the cuproenzyme peptidylglycine α-amidating monooxygenase. J Biol Chem. 2003; 278:12278-12284.
• [22]Handa S, Spradling TJ, Dempsey DR, Merkler DJ. Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli. Prot Expr Purif. 2012; 84:9-13.
• [23]Siebert X, Eipper BA, Mains RE, Prigge ST, Blackburn NJ, Amzel LM. The catalytic copper of peptidylglycine α-hydroxylating monooxygenase also plays a critical structural role. Biophysical J. 2005; 89:3312-3319.
• [24]Chufan EE, De M, Eipper BA, Mains RE, Amzel LM. Amidation of bioactive peptides: the structure of the lyase domain of the amidating enzyme. Structure. 2010; 17:965-973.
• [25]Eipper BA, Stoffers DA, Mains RE. The biosynthesis of neuropeptides: peptide alpha-amidation. Annu Rev Neurosci. 1992; 15:57-85.
• [26]Guest PC, Bailyes EM, Rutherford NG, Hutton JC. Insulin secretory granule biogenesis. Biochem J. 1991; 274:73-78.
• [27]Farquhar MG, Palade GE. The Golgi Apparatus (Complex) - (1954–1981) - from Artifact to Center Stage. J Cell Biol. 1981, 91:77–103.
• [28]Gerald C, Walker MW, Criscione L, Gustafson EL, Batzl-Hartmann C, Smith KE, Vaysse P, Durkin MM, Laz TM, Weinshank RL. A receptor subtype involved in Neuropeptide-Y-induced food intake. Nature. 1996; 382:168-171.
• [29]Prigge ST, Kolhekar AS, Eipper BA, Mains RE, Amzel LM. Amidation of bioactive peptides: the structure of peptidylglycine α-hydroxylating monooxygenase. Science. 1997; 278:1300-1305.
• [30]Milgram SL, Johnson RC, Mains RE. Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules. J Cell Biol. 1992; 117:717-728.
• [31]Rajagopal C, Stone KL, Francone VP, Mains RE, Eipper BA. Secretory Granule to the Nucleus. J Biol Chem. 2009; 284:25723-25734.