Pharmaceutical Manufacturing Handbook: Production and

576 IN VITRO VALIDATION AND ORAL FORMULATION
dard batch cultures. Therefore, the low secretion levels of GST – V 5 H 6 may be only
imputed to the large size of the protein and/or the genetic construction, but not to
the particular digestive conditions. This hypothesis is consistent with the results of
Zsebo et al. [44] , who showed that S. cerevisiae is an effi cient host for small polypeptide
secretion, but not for larger proteins, which accumulate in the periplasmic
place and cell wall of the yeast. Improved secretion levels might be obtained with
another heterologous protein or a different expression vector. In the overall in vitro
system, the total amount of GST – V 5 H 6 regularly increased during digestion to reach
3.3 ± 0.7 μ g ( n = 4) after 270 min digestion (Figure 4 b ).
To check that the GST – V 5 H 6 recovered in the TIM was truly secreted by living
recombinant yeasts and did not result from cell lysis, a control strain producing an
intracellular form of the model protein was contructed (without the leader sequence)
and tested in similar experimental conditions. Some GST – V 5 H 6 was found in the
ileum showing that cell lysis occurred in this compartment. At the end of digestion,
the total amount of GST – V 5 H 6 released by the control strain represented 30% (1
± 0.2 μ g, n = 2) of the protein produced by WppGSTV 5 H 6 , showing a signifi cant
contribution of cell lysis to the release of the protein in the TIM.
For the fi rst time, the amount of heterologous proteins secreted by recombinant
S. cerevisiae was evaluated throughout the upper digestive tract. Until now, the
secretion effi ciency of recombinant microorganisms had never been directly quantifi
ed throughout the digestive tract. The ability of recombinant strains to produce
heterologous proteins in the digestive environment had been mainly demonstrated
indirectly, following the biological effect of the protein: immune response (antibody
production) [7, 15 – 17, 19, 20] , growth improvement [45] , or reduction in colitis
symptoms [5] . Nevertheless, the ability of recombinant bacteria to initiate protein
synthesis in situ has been reported in a few studies. Oozeer et al. [46] have shown
that engineered Lactobacillus casei was able to initiate the synthesis of luciferase
during its transit in the digestive tract of a human fl ora - associated mouse model.
Steidler et al. [5] have demonstrated in mice the in situ synthesis of mouse IL - 10 by
recombinant L. lactis , the viability of these microorganisms being required to achieve
their therapeutic effect (see Section 5.5.1.2 ). They have further documented this
result by showing the de novo synthesis of IL - 10 in the colon of IL - 10 − / − mice.
Moreover, these authors have quantifi ed the amount of IL - 10 produced by the
engineered L. lactis in two animal models, but only in a limited part of their digestive
tract : (i) 7 ng of mouse IL - 10 was recovered in the colon of IL - 10 − / − mice, but
the interleukin was not detectable in other areas of the gastrointestinal tract [5] and
(ii) about 470 pg/mL of human IL - 10 was found in an ileal loop of a pig 4 h after
injection of the recombinant bacteria [47] . Unlike what was previously obtained in
the TIM with GST – V 5 H 6 , the concentrations of IL - 10 found in the digestive tract
of the animals were much lower than that recovered in batch cultures [5, 47] .
5.5.2.4
Conclusion
For the fi rst time, the ability of engineered S. cerevisiae to carry out a bioconversion
reaction [29] and initiate the synthesis and secrete protein compounds of various
sizes [30] was shown throughout the upper gastrointestinal tract in human simulated
digestive conditions. The CA4H specifi c activity of WRP45073A1 and the secretion
level of GST – V 5 H 6 were surprisingly similar to that obtained in classical batch cul-