Pharmaceutical Manufacturing Handbook: Production and

ORAL FORMULATION OF RECOMBINANT YEASTS 577
tures. This is particularly remarkable as the expression strategy of the model genes
had not yet been adapted to the particular constraints of the digestive environment
and promising for a future use of recombinant S. cerevisiae as host for biodrug
development.
5.5.3
ORAL FORMULATION OF RECOMBINANT YEASTS
Once the scientifi c feasibility of the new drug delivery system was established, the
development of pharmaceutical formulations allowing the oral administration of
the genetically modifi ed S. cerevisiae was considered. Ideally, these oral drug dosage
forms would improve both the survival and the heterologous activity of yeasts in
the digestive environment. The following works were carried out only with the strain
expressing the model P450. In a preliminary step, the effect of a preservation technique
(lyophilization) and an immobilization procedure (entrapment in whey
protein beads) on the survival rate and heterologous activity of the model strain
WRP45073A1 was assessed in simulated digestive conditions.
5.5.3.1
Freeze Drying of Recombinant Model Yeasts
Freeze - Drying Conditions Freeze drying is a technique of dehydration commonly
used for the formulation of drugs containing nonrecombinant Saccharomyces spp.
[48 – 50] . Standard freeze - drying conditions derived from the literature [51 – 54] and
our own experiments were applied for the lyophilization of the genetically modifi ed
model yeasts. The effect of cryoprotectants was further investigated because it
appears as one of the most important parameters during lyophilization [51, 52] .
Following galactose induction of the heterologous CYP73A1, yeasts in the beginning
of their stationary growth phase (10 9 cells/mL) were lyophilized in suspensions
of trehalose 10% w/v, maltose 10% w/v, lactose 10% w/v, or a mixture of 5% w/v
milk proteins and 10% w/v trehalose. The parameters of lyophilization are summarized
in Table 4 [55] .
Saccharomyces cerevisiae WRP45073A1 survives freeze drying and yeast survival
rates were dependent on the nature of the cryoprotectants: 13.1 ± 1.8%, 9.5 ± 6.0%,
TABLE 4 Parameters of Lyophilization Used for Recombinant Model Yeast
WRP45073A1
Recombinant yeasts Growth phase: beginning of stationnary growth phase
Cell concentration in freeze - drying fl asks: 10 9 cells/mL
Cryoprotectants 10% w/v trehalose
10% w/v maltose
10% w/v lactose
5% w/v milk proteins and 10% w/v trehalose
Control: physiological water
Freeze - drying conditions
Volume of sample: 5 mL
Cooling rate: 1 ° C/min
Condenser plate temperature: − 40 ° C
Time of lyophilization: 24 h
Heating temperature (secondary drying): 23 ° C