Habibi-Amman schreef op 13 maart 2019 10:42:
Large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows
Human lactoferrin (hLF) is a valuable protein for pharmaceutical products and functional foods, and worldwide demand for this protein has steadily increased. However, large-scale recombinant human lactoferrin (rhLF) production using current animal bioreactor techniques is limited by the low expression of foreign proteins, the use of antibiotic resistance genes and the down-regulation of endogenous milk proteins. Here, we generated a herd of marker-free, hLF bacterial artificial chromosome (BAC) transgenic cloned cows, as confirmed by Polymerase chain reaction, Southern blot and Western blot analyses. These transgenic cloned cows produced rhLF in milk at concentrations of 4.5–13.6?g/L. Moreover, the total protein content of the milk was increased. Over two hundred transgenic cloned cows were propagated by multiple ovulation and embryo transfer (MOET). A total of 400–450?g of rhLF protein, which shows similar enzymatic activity to natural hLF in iron binding and release, can be purified on a large scale from >100?L of milk per day. Our results suggested that transgenic bovine mammary bioreactors have the potential for large-scale protein production.
Human lactoferrin (hLF) is a 79?kDa multifunctional glycoprotein involved in intestinal iron absorption and the non-specific immune system1,2,3. In view of its medicinal value and the global demand for hLF, the large-scale production of functional recombinant hLF (rhLF) has become a major goal. The bovine mammary bioreactor may be an excellent system for the large-scale production of rhLF due to its large capacity for protein synthesis, efficient production, and low cost relative to those of in vitro fermentation and tissue culture systems4.
The expression level of rhLF in previous bovine mammary bioreactors has been low (0.2–2?g/L). The low efficiency of this transgenic bioreactor is attributable to the pronuclear microinjection technique used and the long waiting period required to establish transgenic animal lines5. Although our group has recently improved this technology and has generated hLF bacterial artificial chromosome (BAC) transgenic cloned cows by somatic cell nuclear transfer (SCNT)6, the expression level of rhLF in these cows was 3?g/L, which is not much higher than those of previous studies5. Thus, improving the expression levels of recombinant protein in bovine mammary bioreactors is crucial. Antibiotic-selectable marker genes have been widely used to generate transgenic animal mammary bioreactors7,8,9. However, the presence of foreign marker genes interferes with the expression of neighbouring endogenous genes and hampers phenotypic and genetic analyses10, 11. This approach may also create public concerns regarding biological safety. Thus, marker-free technology must be used to generate transgenic animals.
Importantly, previous reports have shown that recombinant proteins compete for production in the mammary glands of transgenic animals12, 13. Although exogenous ß-lactoglobulin (BLG) and rhLF proteins are highly expressed at 30?g/L in transgenic mice and goats, the endogenous milk proteins are down-regulated, and the total protein level is not increased. The ceiling effect for protein production in transgenic animal mammary glands remains to be fully investigated, especially in cattle5, 6, 13. Finally, the large-scale production of a biological protein in the animal mammary gland using a herd of a few hundred transgenic cloned cattle has thus far been poorly investigated. These disadvantages have limited the applications of transgenic animal bioreactors.
Here, we established a simple and safe method based on nucleofection14 and single-cell limited dilution15, 16 to generate marker-free hLF BAC transgenic cows that produced a high level (4.5–13.6?g/L) of functional rhLF. To the best of our knowledge, this study reports the highest levels of expression of rhLF in marker-free transgenic cows. The total protein of the milk was significantly increased. Additionally, a herd of two hundred transgenic cattle was established by multiple ovulation and embryo transfer (MOET). A total of 400–450?g of recombinant protein can be purified from >100?L of milk per day.
Was dit niet iets waar pharming in het verleden al eens mee bezig was? zelfs een patent op hebben 10 jaar geleden?
volledige link:
www.nature.com/articles/s41598-017-11...Pharming’s technology platform includes a unique, GMP-compliant, validated process for the production of pure recombinant human proteins that has proven capable of producing industrial quantities of high quality recombinant human proteins in a more economical and less immunogenetic way compared with current cell-line based methods.
Leads for enzyme replacement therapy (“ERT”) for Pompe and Fabry’s diseases are being optimized at present, with additional programs not involving ERT also being explored at an early stage at present.
Pharming has a long-term partnership with the China State Institute of Pharmaceutical Industry (“CSIPI”), a Sinopharm company, for joint global development of new products, starting with recombinant human Factor VIII for the treatment of Haemophilia A. Pre-clinical development and manufacturing will take place to global standards at CSIPI and are funded by CSIPI. Clinical development will be shared between the partners with each partner taking the costs for their territories under the partnership.
Pharming has operations in the Netherlands, Germany, France and the US, with its headquarters in Leiden (NL), with170 employees in total.
At a glance:
First product approved and marketed: RUCONEST®
Recombinant human C1-esterase inhibitor (enzyme replacement therapy)
For acute angioedema attacks in patients with hereditary angioedema (HAE)
Marketed in USA, EU, Korea and Israel with other territories coming
Platform technology makes recombinant human molecules cleanly and efficiently
New Enzyme Replacement Therapies (ERT) for other genetic conditions about to enter clinic