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Technical Briefs

# Effect of Controlled Ice Nucleation on Primary Drying Stage and Protein Recovery in Vials Cooled in a Modified Freeze-Dryer

[+] Author and Article Information
Stéphanie Passot1

UMR 782 Génie et Microbiologie des Procédés Alimentaires, AgroParisTech-INRA, 1 Avenue Lucien Brétignières, 78850 Thiverval-Grignon, Francespassot@grignon.inra.fr

Ioan Cristian Tréléa, Michèle Marin, Fernanda Fonseca

UMR 782 Génie et Microbiologie des Procédés Alimentaires, AgroParisTech-INRA, 1 Avenue Lucien Brétignières, 78850 Thiverval-Grignon, France

Miquel Galan

Telstar Industrial S.L., Josep Tapiolas 120, 08226 Terrassa, Spain

G. John Morris

Asymptote Ltd., St. John’s Innovation Centre, Cowley Road, Cambridge CB4 0WS, UK

1

Corresponding author.

J Biomech Eng 131(7), 074511 (Jun 12, 2009) (5 pages) doi:10.1115/1.3143034 History: Received November 05, 2008; Revised April 27, 2009; Published June 12, 2009

## Abstract

The freezing step influences lyophilization efficiency and protein stability. The main objective of this work was to investigate the impact on the primary drying stage of an ultrasound controlled ice nucleation technology, compared with usual freezing protocols. Lyophilization cycles involving different freezing protocols (applying a constant shelf cooling rate of $1°C/min$ or $0.2°C/min$, putting vials on a precooled shelf, and controlling nucleation by ultrasounds or by addition of a nucleating agent) were performed in a prototype freeze-dryer. Three protective media including sucrose or maltodextrin and differing by their thermal properties and their ability to preserve a model protein (catalase) were used. The visual aspect of the lyophilized cake, residual water content, and enzymatic activity recovery of catalase were assessed after each lyophilization cycle and after 1 month of storage of the lyophilized product at $4°C$ and $25°C$. The freezing protocols allowing increasing nucleation temperature (precooled shelf and controlled nucleation by using ultrasounds or a nucleating agent) induced a faster sublimation step and higher sublimation rate homogeneity. Whatever the composition of the protective medium, applying the ultrasound technology made it possible to decrease the sublimation time by 14%, compared with the freezing method involving a constant shelf cooling rate of $1°C/min$. Concerning the enzyme activity recovery, the impact of the freezing protocol was observed only for the protective medium involving maltodextrin, a less effective protective agent than sucrose. Higher activity recovery results were obtained after storage when the ultrasound technology or the precooled shelf method was applied. Controlling ice nucleation during the freezing step of the lyophilization process improved the homogeneity of the sublimation rates, which will, in turn, reduce the intervial heterogeneity. The freeze-dryer prototype including the system of controlled nucleation by ultrasounds appears to be a promising tool in accelerating sublimation and improving intrabatch homogeneity.

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Copyright © 2009 by American Society of Mechanical Engineers
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## Figures

Figure 1

Evolution of the shelf temperature and the product temperature during the freezing protocols B (constant shelf cooling rate of 1°C/min) and D (ultrasounds technology). During protocol D, only the vials with the thermocouples nucleated during the temperature holding step at −8°C. 80% of the other vials nucleated with the application of the ultrasounds at the end of the holding step.

Figure 2

Effect of the freezing protocols B (constant shelf cooling rate of 1°C/min), C (precooled shelf method), and D (ultrasounds method) on the sublimation time for the three protective media studied (S5: 5% of sucrose; S10: 10% of sucrose; Md10: 10% of maltodextrin. Standard deviation=0.5).

Figure 3

Effect of the freezing protocols B (constant shelf cooling rate of 1°C/min), C (precooled shelf method), and D (ultrasounds method) on the sublimation rates homogeneity for the three protective media studied (S5: 5% of sucrose; S10: 10% of sucrose; Md10: 10% of maltodextrin. Standard deviation=0.0004). Considering the formulation S10, the application of ultrasound did not allow control of ice nucleation in all the vials. Only 50% (instead of 80% for S5 and Md10) of the vials exhibited controlled nucleation. Consequently, the value of the absolute value of the slope is not representative of what would happen if a percentage of 80% was obtained.

Figure 4

Effect of the freezing protocols B (constant shelf cooling rate of 1°C/min), C (precooled shelf method), and D (ultrasounds method) on the enzymatic activity recovery of catalase after lyophilization and after 1 month of storage at 4°C and 25°C for the protective medium involving 10% of maltodextrin.

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