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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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• ongoing supply of product is guaranteed (by breeding);
• milk is biochemically well characterized, and the physicochemical properties of the major native milk proteins of various species are well known. This helps rational development of appropriate downstream processing protocols (Table 3.15).
Despite the attractiveness of this system, a number of issues remain to be resolved before it is
broadly accepted by the industry. These include:
• variability of expression levels. While in many cases expression levels of heterologous proteins exceed 1 g/l, in some instances expression levels as low as 1.0mg/l have been obtained;
• characterization of the exact nature of the post-translational modifications the mammary system is capable of undertaking, e.g. the carbohydrate composition of tPA produced in this system differs from the recombinant enzyme produced in murine cell culture systems;
• significant time lag between the generation of a transgenic embryo and commencement of routine product manufacture. Once a viable embryo containing the inserted desired gene is generated, it must firstly be brought to term. This gestation period ranges from 1 month for rabbits to 9 months for cows. The transgenic animal must then reach sexual maturity before breeding (5 months for rabbits, 15 months for cows). Before they begin to lactate (i.e. produce the recombinant product), they must breed successfully and bring their offspring to term. The overall time lag to routine manufacture can, therefore, be almost 3 years in the case of cows or 7 months in the case of rabbits. Furthermore, if the original transgenic embryo turns out to be male, a further delay is encountered as this male must breed in order to pass
< Upstream ' processing
Figure 3.8. The production and purification of tPA from the milk of transgenic goats (WAP = murine whey acid). The downstream processing procedure yielded in excess of an 8000-fold purification factor with an overall product yield of 25%. The product was greater than 98% pure, as judged by SDS-electrophoresis
on the desired gene to daughter animals—who will then eventually produce the desired product in their milk.
Another general disadvantage of this approach relates to the use of the micro-injection technique to introduce the desired gene into the pronucleus of the fertilized egg. This approach
Table 3.14. Typical annual milk yields (litres) as well as time lapse between generation of the transgene embryo and first product harvest (first lactation) of indicated species
Species Annual milk yield (l) Time to first production batch (months)
Cow 6000-9000 33-36
Goat 700-800 18-20
Sheep 400-500 18-20
Pig 250-300 16-17
Rabbit 4-5 7
Table 3.15. Some physicochemical properties of the major (bovine) milk proteins
Protein Caseins b-Lactoglobulin a-Lactalbumin Serum albumin IgG
Concentration (g/l) 25 (Total) 2-4 0.5-1.5 0.4 0.5-1.0
Mass (kDa) 20-25 18 14 66 150
Phosphorylated? Yes No No No No
Isoelectric point Vary 5.2 4.2-4.8 4.7-4.9 5.5-8.3
is inefficient and time-consuming. There is no control over issues such as if/where in the host genomes the injected gene will integrate. Overall, only a modest proportion of manipulated embryos will culminate in the generation of a healthy biopharmaceutical-producing animal.
A number of alternative approaches are being developed which may overcome some of these issues. Replication-defective retroviral vectors are available which will more consistently (a) deliver a chosen gene into cells and (b) ensure chromosomal integration of the gene. A second innovation is the application of nuclear transfer technology.
Nuclear transfer entails substituting the genetic information present in an unfertilized egg with donor genetic information. The best-known product of this technology is ‘Dolly’ the sheep, produced by substituting the nucleus of a sheep egg with a nucleus obtained from an adult sheep cell (genetically, therefore, Dolly was a clone of the original ‘donor’ sheep). An extension of this technology applicable to biopharmaceutical manufacture entails using a donor cell nucleus previously genetically manipulated so as to harbour a gene coding for the biopharmaceutical of choice. The technical viability of this approach was proved in the late 1990s upon the birth of two transgenic sheep, ‘Polly’ and ‘Molly’. The donor nucleus used to generate these sheep harboured an inserted (human) blood factor IX gene under the control of a milk protein promoter. Both now produce significant quantities of human factor IX in their milk.
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