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. 2015 Jun 27:15:61.
doi: 10.1186/s12896-015-0173-5.

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

Kristina R Carlson  1 , Steven C Pomerantz  2 , Jiali Li  3 , Omid Vafa  4 , Michael Naso  5 , William Strohl  6 , Richard E Mains  7 , Betty A Eipper  8   9
Affiliations

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

Kristina R Carlson et al. BMC Biotechnol. .

Abstract

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-NH2 but 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.

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Figures

Fig. 1
Fig. 1
Design of vectors encoding Fc-peptidylglycine precursors. a Key features of the Fc-peptidylglycine precursors are illustrated. b The amino acid sequences of human GLP1-Gly, PYY-Gly and NMU-Gly are shown (http://www.bachem.com/research-products/). c Eadie-Hofstee plots illustrating the effects of synthetic PYY-Gly and GLP1-Gly on conversion of [125I]-Acetyl-Tyr-Val-Gly into [125I]-Acetyl-Try-Val-NH2 by purified PAM 820 s (0.085 ng/40 μl ≈ 1 fmol enzyme/tube). d Calculated kinetic parameters for PYY-Gly and GLP1-Gly. e pEAK Rapid cells were transiently transfected with vectors encoding Fc-GS-GLP1 (calculated mass discounting oligosaccharides; 31,720), Fc-AP-GLP1 (mass; 32,020), Fc-GS-PYY (mass; 32,730) and Fc-AP-PYY (mass; 33,090); control non-transfected cells (non) were analyzed at the same time. Aliquots of spent medium (20 μl) were fractionated by SDS-PAGE and visualized using antibody to human Fc; 18 ng human IgG (hIgG) was analyzed at the same time
Fig. 2
Fig. 2
Characterization of endogenous CHO cell PAM. a Five isoforms of Cricetulus griseus PAM are included in the primary RefSeq assembly of the Chinese hamster genome; these isoforms closely resemble those observed in rat, mouse and human: XP_003505817.1, isoform 1; ERE82825.1, isoform 2; XP_003505819.1, isoform 3; XP_003505820.1, isoform 4; XP_003505821.1, isoform 5. b The specific activity of endogenous PHM in the soluble (TM) and solubilized (TMT) crude particulate fractions is indicated. c The solubilized particulate fraction from two separate preparations of wildtype CHO cells (224 μg protein) and from CHO cells stably expressing PAM1 (20 μg protein) were immunoprecipitated using an affinity-purified antibody to the C-terminus of PAM, eluted and fractionated by SDS-PAGE, transferred to PVDF membranes and visualized using affinity-purified antibody to the PHM domain (JH1761); molecular weights of marker proteins analyzed along with PAM1 are indicated. A lighter exposure is shown for the other Wt sample so that the doublet of 92 and 97 kDa bands is visible. Apparent molecular weights calculated from three separate analyses are shown ± Std Dev. d PHM and PAL assays were performed on lysates prepared from wildtype CHO cells and from CHO cells stably expressing rat PAM1 or rat PAM 820 s; lysates were prepared in 1 % TX-100
Fig. 3
Fig. 3
Characterization of Fc-fusion protein expression and secretion in wildtype CHO lines. Samples of cell extract (CE) and spent medium (16 h collection) prepared from CHO lines expressing Fc-AP-GLP1 (a) [15 μg (7 %) of CE; 0.9 % of spent medium], Fc-GS-PYY (b) [10 μg (8.5 %) of CE; 1.3 % of spent medium) and Fc-AP-NMU (c) [10 μg (5.3 %) of CE; 1.3 % of spent medium) were fractionated by SDS-PAGE; samples and a human IgG standard were visualized using antibody to human Fc. d Using GeneTools, data from several similar analyses (n = 3–4) were quantified to determine μg Fc/mg cell protein. e The secretion rate for Fc was calculated for several experiments by quantifying the amount of Fc recovered from the cell extract (CE) and from the medium using GeneTools; for each cell line, secretion rate was expressed as % cell content secreted per hour
Fig. 4
Fig. 4
Characterization of Fc-fusion protein expression in CHO lines expressing PAM. Fc-AP-GLP1 (a) and Fc-GS-PYY (b) were expressed in PAM1 CHO cells; Fc-AP-NMU (c) was expressed in PAM820s CHO cells. Aliquots of cell extract (a, 5 %; b, 3 %; c, 6 % of total cell extract) and spent medium (a, 0.7 %; b, 1.3 %; c, 2.3 % of total medium volume) were prepared as described for Fig. 3. d Using GeneTools, data from several similar analyses were quantified to determine μg Fc/mg cell protein (d) and Fc secretion rate (e)
Fig. 5
Fig. 5
Purification and mass spectroscopic analysis of Fc-fusion proteins. a After concentration using a Vivaflow flip flow filtration system and a Centricon, secreted Fc-fusion proteins were purified by binding to HiTrap Protein A cartridges; bound protein was eluted by application of low pH buffer and aliquots (E1-E4) were subjected to SDS-PAGE; purified Fc was analyzed at the same time. Comparison of glycan heterogeneity by intact mass analysis of Fc-AP-GLP1 (b) and Fc from manufacturing cell line (c). Peak labels are shorthand notation for glycosylation state. All molecules exhibited two bi-antennary core fucosylated glycans (BiF/BiF) with an additional Gn (n = 1-4) galactose and Sn (=1–2) sialic acid (N-acetylneuraminic acid) moieties distributed between the two glycans
Fig. 6
Fig. 6
Reversed-phase HPLC-MS analysis of HRV3C cleavage products. a Total ion chromatogram (TIC); b single-charge deconvoluted mass spectrum (MaxEnt3) from the m/z data under the peptide peak. c Purified Fc-fusion proteins were cleaved with HRV3C protease and subjected to mass spectroscopic analysis as described in Methods. Each sample was analyzed in triplicate; standard errors are shown
Fig. 7
Fig. 7
Bioassay of purified Fc-GLP1 fusion proteins. HEK cells expressing the GLP-1R were used to assess the ability of Fc-GLP1 fusion proteins purified from the spent medium of different CHO cell lines to stimulate cAMP production. a The activity of GLP1, GP-GLP1-NH2 and GP-GLP1was compared; as expected, amidation did not affect bioactivity while appending the GP dipeptide to its amino-terminus reduced the potency of GLP1-NH2. b The activity of GLP1 was compared to that of Fc-AP-GLP1 fusion proteins purified from the spent media of WT CHO cells, CHO cells expressing PAM1 and WT CHO cells treated with 10 μM bathocuproine disulfonate to inhibit endogenous PHM throughout the entire collection period. Parallel dose–response curves were observed for all of the peptides and proteins tested; Fc-GLP1 was at least 10,000-fold less active than GLP1. The y-axis shows the TR-FRET signal at 665 nm; the signal obtained by cells in the absence of any added peptide is indicated
Fig. 8
Fig. 8
Bioassay of purified Fc-PYY fusion proteins. CHO cells expressing the NPY-2R were used to assess the ability of Fc-PYY fusion proteins purified from the spent medium of different CHO lines to inhibit forskolin-stimulated cAMP production; PYY binding to NPY-2R activates Gαi, decreasing cAMP production. Synthetic PYY(3–36)NH2 was more potent than synthetic GP-PYY-NH2; synthetic GP-PYY-Gly was at least 10,000-fold less potent than the corresponding amidated peptide. Although the Fc-PYY fusion protein produced by PAM1 cells was about 200-fold less active than synthetic GP-PYY-NH2, it was 10-fold more active than synthetic GP-PYY-Gly. Parallel dose–response curves were observed for all of the peptides and proteins tested
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