Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 29;18(1):82.
doi: 10.1186/s12896-018-0495-1.

A chicken bioreactor for efficient production of functional cytokines

Lissa R Herron  1   2 , Clare Pridans  3   4 , Matthew L Turnbull  3   5 , Nikki Smith  3 , Simon Lillico  3 , Adrian Sherman  3 , Hazel J Gilhooley  3 , Martin Wear  6 , Dominic Kurian  3 , Grigorios Papadakos  7 , Paul Digard  3 , David A Hume  3   4   8 , Andrew C Gill  3   9 , Helen M Sang  10
Affiliations

A chicken bioreactor for efficient production of functional cytokines

Lissa R Herron et al. BMC Biotechnol. .

Abstract

Background: The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications.

Results: Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1.

Conclusion: Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications.

Keywords: Animal biotechnology; Biologics; Cytokines; Recombinant proteins; Transgenic animals.

PubMed Disclaimer

Conflict of interest statement

Ethics approval

Approval for animal experiments was obtained from The Roslin Institute’s and The University of Edinburgh’s Protocols and Ethics Committees. The experiments were carried out under the authority of a UK Home Office Project License under the regulations of the Animals (Scientific Procedures) Act 1986.

Consent for publication

Not applicable.

Competing interests

D.A.H. holds a patent position on the applications of CSF1-Fc in liver disease, and of CSF1 in kidney regeneration. Part of this work was performed on a collaborative grant part-funded by Zoetis, Inc. L.R.H. is now employed by Roslin Technologies, who will be commercializing the chicken platform, as well as the current proteins for use as research reagents.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Expression of biologically-active human interferon α2a by transgenic hens a Schematic representation of the interferon α2a transgene; ovalbumin regulatory elements including the estrogen response element (ERE) and the steroid-dependent regulatory element (SDRE); lysozyme signal peptide (LSP) for secretion; the coding sequence of human interferon α2a; EIAV long terminal repeats (LTR). b Western blot of dilutions of transgenic egg white protein (lanes 1 and 2) and 5 μg of commercially-available interferon α2a protein (lane 3), stained with antibody for human interferon α2a. c Activity of whole egg white assayed for activation of the interferon-stimulated response element (ISRE) by measurement of luciferase. Egg white from transgenic hens was compared with egg white with a range of concentrations of control protein added, and wild type egg white as a negative control. Measurements were taken in triplicate. Graph shows mean + SEM. d Interferon α2a was purified from egg white and confirmed by reducing SDS-PAGE Instant Blue staining. e SDS-PAGE of 5 μL samples of egg white before ovomucin precipitation (lane 1), pellet after first centrifugation (lane 2), supernatant after first centrifugation (lane 3), pellet after pH adjustment and second centrifugation (lane 4), and supernatant after pH adjustment and second centrifugation (lane 5); and western blot of 10 μL samples from each stage of purification: Blue Sepharose load (lane 6), Blue Sepharose flow-through (lane 7), Blue Sepharose wash (lane 8), Blue Sepharose eluate (lane 9) and final pure protein (lane 10). f Purified interferon α2a from egg white and a purchased control protein were tested in a viral dose-inhibition assay against influenza A virus. Measurements were taken in triplicate. Graph shows mean ± SEM
Fig. 2
Fig. 2
Expression of biologically-active porcine CSF1-Fc by transgenic hens. a Schematic representation of the pCSF1-Fc transgene, with HIV LTRs; a modified ovalbumin promoter containing the ERE and SDRE; LSP for secretion; the coding sequence of porcine CSF1; the coding sequence of porcine Fc; oPRE. b Egg white from wild type and transgenic hens were homogenized and diluted 1:50 in PBS (lanes 1 and 2) and separated under reducing conditions by SDS-PAGE with CHO-cell produced pure protein (lane 3) and transferred to nitrocellulose membrane and stained with anti-porcine Fc to confirm expression. c Activity of whole egg white was tested with a MTT cell survival assay. Whole egg white from transgenic hens was compared with wild type egg white with four concentrations of control protein added, while wild type egg white was used as a negative control. Measurements were taken in triplicate. Graph shows mean + SEM. d SDS-PAGE and western blot of 5 μL samples of egg white before ovomucin precipitation (lanes 1 and 4), supernatant after first centrifugation (lanes 2 and 5), and supernatant after pH adjustment and second centrifugation (lanes 3 and 6). e pCSF1-Fc dimer was purified from egg white as confirmed by non-reducing SDS-PAGE stained with Instant Blue. f Western blot of 10 μL samples from each stage of purification: MabSelect SuRe load (lane 1), MabSelect SuRe flow-through (lane 2), MabSelect SuRe wash (lane 3), MabSelect SuRe eluate (lane 4) and final pure protein (lane 5). g Activity of purified pCSF1-Fc from fresh egg white, egg white that had been frozen at -20 °C and -80 °C for at least 1 month before purification, and CHO-cell produced pCSF1-Fc was tested in a MTT Ba/F3-CSF1R cell-survival assay. Measurements were taken in triplicate. Graph shows mean + SEM. h Bone marrow from pigs was cultured in CHO-cell-produced pCSF1-Fc, pCSF1-Fc or without growth factor (cells only) for 7 days. MTT was used to assess cell viability. Measurements were taken in triplicate. Graph shows mean + SEM
Fig. 3
Fig. 3
The effect of egg purified pCSF1-Fc treatment in mice is identical to that of pCSF1-Fc from CHO cells. Mice were injected with PBS, 1 μg/g pCSF1-Fc or 1 μg/g egg purified pCSF1-Fc for 4 days prior to sacrifice on day 5. a EDTA-blood was collected via cardiac bleeds and the percentage of F4/80+CD11b+ myeloid cells were determined by flow cytometry. Graph shows the mean + SEM. Significance is indicated by *p = 0.0163 and **p = 0.0037 using a t-test; n = 4. b Formalin-fixed paraffin-embedded livers were stained with an antibody against F4/80. The percentage of F4/80 staining was determined using ImageJ. Graph shows the mean + SEM. Significance is indicated by ***p = 0.0004 and ****p < 0.0001 using a test-test; n = 4. c Livers were weighed and percent body weight calculated. Graphs show the mean + SEM. Significance is indicated by ***p = 0.0004 using a t-test. There was no significant difference between the two pCSF1-Fc populations (p = 0.2482). d Spleens were weighed and percent body weight calculated. Graphs show the mean + SEM. Significance is indicated by ****p < 0.0001 and ***p = 0.0009 using a t-test. There was no significant difference between the two pCSF1-Fc populations (p = 0.1122)
Fig. 4
Fig. 4
Expression of biologically-active human CSF1-Fc by transgenic hens. a Purity of 10 μL samples from 15 mL size exclusion fractions of hCSF1-Fc from egg white demonstrated by non-reducing SDS-PAGE stained with Instant Blue. Identity was confirmed by western blot transfer and staining with mouse anti-human CSF1 antibody and LICOR IRDye 680RD Donkey anti-Mouse IgG (H + L) secondary antibody. b Western blot of 10 μL samples from each stage of purification: MabSelect SuRe load (lane 1), MabSelect SuRe flow-through (lane 2), MabSelect SuRe wash (lane 3), MabSelect SuRe eluate (lane 4) and final pure protein (lane 5). c Bone marrow from pigs was cultured in purified pCSF1-Fc from egg white, purified hCSF1-Fc from egg white, and the same protein after vacuum drying and reconstitution, or without growth factor (cells only) for 7 days. MTT was used to assess cell viability. Measurements were taken in triplicate. Graph shows mean + SEM
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kintzing JR, Filsinger Interrante MV, Cochran JR. Emerging strategies for developing next-generation protein therapeutics for cancer treatment. Trends Pharmacol Sci. 2016;37:993–1008. doi: 10.1016/j.tips.2016.10.005. - DOI - PMC - PubMed
    1. Pavlou AK, Reichert JM. Recombinant protein therapeutics--success rates, market trends and values to 2010. Nat Biotechnol. 2004;22:1513–1519. doi: 10.1038/nbt1204-1513. - DOI - PubMed
    1. Lagassé HAD, Alexaki A, Simhadri VL, Katagiri NH, Jankowski W, Sauna ZE, et al. Recent advances in (therapeutic protein) drug development. F1000Res. 2017;6:113. doi: 10.12688/f1000research.9970.1. - DOI - PMC - PubMed
    1. Mauro VP. Codon optimization in the production of recombinant biotherapeutics: potential risks and considerations. BioDrugs. 2018;32:69–81. doi: 10.1007/s40259-018-0261-x. - DOI - PubMed
    1. National Academies of Sciences, Engineering, and Medicine, Health and Medicine Division, Board on Health Care Services, Committee on Ensuring Patient Access to Affordable Drug Therapies . Making medicines affordable: a national imperative. Washington (DC): National Academies Press (US); 2017. - PubMed

Publication types

MeSH terms