URL: https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775 TITLE: Detection of Collapse and Crystallization of Saccharide ... - PMC ERROR: None [Skip to main content](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#main-content) ![Image 1](https://cdn.ncbi.nlm.nih.gov/pmc/pd-medc-pmc-cloudpmc-viewer/production/a2b04810/var/data/static/img/us_flag.svg) An official website of the United States government Here's how you know Here's how you know ![Image 2](https://cdn.ncbi.nlm.nih.gov/pmc/pd-medc-pmc-cloudpmc-viewer/production/a2b04810/var/data/static/img/icon-dot-gov.svg) **Official websites use .gov** A **.gov** website belongs to an official government organization in the United States. ![Image 3](https://cdn.ncbi.nlm.nih.gov/pmc/pd-medc-pmc-cloudpmc-viewer/production/a2b04810/var/data/static/img/icon-https.svg) **Secure .gov websites use HTTPS** A **lock** ( ) or **https://** means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. 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Learn more: [PMC Disclaimer](https://pmc.ncbi.nlm.nih.gov/about/disclaimer/) | [PMC Copyright Notice](https://pmc.ncbi.nlm.nih.gov/about/copyright/) ![Image 14: Frontiers in Chemistry logo](https://cdn.ncbi.nlm.nih.gov/pmc/banners/logo-frontchem.png) Front Chem . 2018 Jan 26;6:4. doi: [10.3389/fchem.2018.00004](https://doi.org/10.3389/fchem.2018.00004) * [Search in PMC](https://pmc.ncbi.nlm.nih.gov/search/?term=%22Front%20Chem%22[jour]) * [Search in PubMed](https://pubmed.ncbi.nlm.nih.gov/?term=%22Front%20Chem%22[jour]) * [View in NLM Catalog](https://www.ncbi.nlm.nih.gov/nlmcatalog?term=%22Front%20Chem%22[Title%20Abbreviation]) * [Add to search](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775?term=%22Front%20Chem%22[jour]) # Detection of Collapse and Crystallization of Saccharide, Protein, and Mannitol Formulations by Optical Fibers in Lyophilization [Jacqueline Horn](https://pubmed.ncbi.nlm.nih.gov/?term=%22Horn%20J%22[Author]) ### Jacqueline Horn Find articles by [Jacqueline Horn](https://pubmed.ncbi.nlm.nih.gov/?term=%22Horn%20J%22[Author]) , [Wolfgang Friess](https://pubmed.ncbi.nlm.nih.gov/?term=%22Friess%20W%22[Author]) ### Wolfgang Friess Find articles by [Wolfgang Friess](https://pubmed.ncbi.nlm.nih.gov/?term=%22Friess%20W%22[Author]) * * Author information * Article notes * Copyright and License information Munich Edited by: Davide Fissore, Politecnico di Torino, Italy Reviewed by: Petra Foerst, Technische Universität München, Germany; Adel Mhamdi, RWTH Aachen University, Germany This article was submitted to Chemical Engineering, a section of the journal Frontiers in Chemistry ✉ *Correspondence: Wolfgang Friess wolfgang.friess@lrz.uni-muenchen.de Received 2017 Nov 06; Accepted 2018 Jan 10; Collection date 2018. Copyright © 2018 Horn and Friess. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. [PMC Copyright notice](https://pmc.ncbi.nlm.nih.gov/about/copyright/) PMCID: PMC5790775 PMID: [29435445](https://pubmed.ncbi.nlm.nih.gov/29435445/) ## [](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775)Abstract The collapse temperature (Tc) and the glass transition temperature of freeze-concentrated solutions (Tg') as well as the crystallization behavior of excipients are important physicochemical characteristics which guide the cycle development in freeze-drying. The most frequently used methods to determine these values are differential scanning calorimetry (DSC) and freeze-drying microscopy (FDM). The objective of this study was to evaluate the optical fiber system (OFS) unit as alternative tool for the analysis of Tc, Tg' and crystallization events. The OFS unit was also tested as a potential online monitoring tool during freeze-drying. Freeze/thawing and freeze-drying experiments of sucrose, trehalose, stachyose, mannitol, and highly concentrated IgG1 and lysozyme solutions were carried out and monitored by the OFS. Comparative analyses were performed by DSC and FDM. OFS and FDM results correlated well. The crystallization behavior of mannitol could be monitored by the OFS during freeze/thawing as it can be done by DSC. Online monitoring of freeze-drying runs detected collapse of amorphous saccharide matrices. The OFS unit enabled the analysis of both Tc and crystallization processes, which is usually carried out by FDM and DSC. The OFS can hence be used as novel measuring device. Additionally, detection of these events during lyophilization facilitates online-monitoring. Thus the OFS is a new beneficial tool for the development and monitoring of freeze-drying processes. **Keywords:**freeze-drying, lyophilization, optical fiber system, glass transition, collapse, crystallization, monitoring ## [](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775)Introduction Freeze-drying is commonly used for the long-term stabilization of biopharmaceuticals which cannot be stabilized adequately in the liquid state. Efficient development of freeze-drying cycles is of utmost importance as the process is time and cost consuming. Short process times without putting the protein stability at risk are desired (Oetjen and Haseley, [2004](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B29); Bosca et al., [2013](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B5); Pisano et al., [2013](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B35)). It is essential to analyze the formulation to be freeze-dried regarding critical parameters for the freeze-drying process. Typical parameters of amorphous matrices, as formed by the most frequently used saccharides for stabilization of proteins, sucrose and trehalose, are the collapse temperature (Tc) and the glass transition temperature (Tg') of the freeze-concentrated solution (Meister and Gieseler, [2009](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B27); Pansare and Patel, [2016](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B30)). They characterize temperatures at which the mobility of the system greatly increases, either at the drying front (Tc) or in the frozen state (Tg'). Tc is typically determined by freeze-drying microscopy (FDM) whereas the standard method for Tg' is differential scanning calorimetry (DSC). FDM mimics the freeze-drying process in miniature by freezing and drying small volumes of formulation under the microscope. Tc can be defined as either the onset of visible collapse or full collapse (Meister and Gieseler, [2009](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B27); Bosch, [2014](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B6)). Tg' as analyzed by DSC reflects the temperature at which the heat capacity of the freeze-concentrated formulation markedly changes (Pansare and Patel, [2016](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B30)). Both values help to define the upper limit of the product temperature (Tp) during the primary drying step. In lab scale, Tp is typically measured by thermocouples that can only represent the temperature of the surrounding environment although the local temperature of the sublimation front might be more critical for the occurrence of collapse. Tc values are usually 1–3°C higher than Tg' values and drying above Tc may result in macrocollapse of the lyophilizate. Drying above Tg' but below Tc can be utilized e.g., for highly concentrated protein formulations (Colandene et al., [2007](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B9)). It enables higher product temperatures, faster drying and thus shorter process times without loss of cake structure if protein stability is preserved (Colandene et al., [2007](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B9)). Nanoparticle suspensions were also shown to increase collapse temperatures (Beirowski et al., [2017](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B4)). Macrocollapse is not only a question of elegant cake appearance, but it can, but not necessarily has to be correlated to higher residual moisture levels after the process, destabilization of the API, longer reconstitution time or prolonged secondary drying (Chatterjee et al., [2005](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B8); Passot et al., [2007](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B31); Bosch, [2014](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B6)). Nevertheless, since it is known that an 1°C increase in Tp can shorten primary drying times by about 13%, the interest is to dry at the highest possible Tp (Pikal, [1990](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B34)). Crystalline bulking agents like mannitol not necessarily stabilize proteins but form crystalline scaffolds that provide robust and elegant cake structures (Johnson et al., [2002](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B22); Hawe and Frieß, [2006](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B16); Varshney et al., [2007](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B39); Peters et al., [2016](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B33)). Their controlled and complete crystallization during the freeze-drying process is of interest since partial crystallization might induce subsequent crystallization of the amorphous fraction during storage leading to potential loss of drug stability (Izutsu and Kojima, [2002](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B18)). Crystallization occurs mainly during thermal treatment before drying starts (Jena et al., [2017](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B19)). In order to force crystallization, an annealing step is usually conducted at temperatures above Tg' (Liao et al., [2007](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B26)). The temperature at which crystallization (Tcry) occurs can be determined by DSC measurements (Hawe and Frieß, [2006](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B16)). Thus, the correct characterization of the system is of utmost importance. FDM and DSC both provide good approximations but are based on low sample volume which is dried in a thin film by FDM or freeze/thawed in small aluminum crucibles by DSC not necessarily reflecting the several milliliters in a vial during freeze-drying. Furthermore, the high heating rates of 5–20°C/min in DSC analysis do not correspond to the typical 0.5–1°C/min during lyophilization but facilitates the Tg' analysis due to a more distinct baseline shift (Her and Nail, [1994](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B17)). The heating rates applied in FDM analysis are lower, however, may still not represent the heating rate within the vial (Meister and Gieseler, [2009](https://pmc.ncbi.nlm.nih.gov/articles/PMC5790775#B27)). The distance from heating source (shelf) to the product combined with the larger sample volume does not lead to a direct transfer of the applied heating rate to the whole product container which slows down the real heating rate in contrast to FDM. The operator itself affects also FDM results as the analysis is performed visually. [... middle omitted — see footer ...] * . Williams, Dean (1991). Vial breakage by frozen mannitol solutions: correlation with thermal characteristics and effect of stereoisomerism, additives, and vial configuration. _J. Parenter. Sci. Technol._ 45, 94–100. 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