Lyophilization of Synthetic Gene Carriers.

Lyophilization, also known as freeze drying, is a widely used method for stabilization, improvement of long-term storage stability, and simplification of handling of drugs and/or carrier systems. Lyophilization is time-consuming and energy-consuming, and hence optimized processes are required to avoid time loss and higher costs without compromising product stability. Beginning from the last decade, nonviral, synthetic carriers for gene delivery have been of increasing interest. However, these systems suffer from poor physical stability in aqueous solution or suspension. Hence, to ensure long-term storage stability lyophilization of the gene carrier systems is favored. This chapter gives an overview of the basic steps and troubleshooting for successful lyophilization of synthetic gene carriers. Furthermore, the required excipients and their mechanism of action are summarized.

Formulation development of lyophilized, long-term stable siRNA/oligoaminoamide polyplexes.

Polyplexes based on precise oligoaminoamides exhibited promising results in non-viral siRNA delivery. However, one serious limitation is insufficient stability of polyplexes in liquid, which raises the demand for lyophilized, long-term stable formulations. Two different siRNA/oligoaminoamide polyplexes were prepared. Freeze-thaw experiments were performed, in order to test various formulations containing sucrose, trehalose, lactosucrose, and hydroxypropyl-ß-cyclodextrin for their cryoprotective potential and to investigate the influence of the oligoaminoamide structure on particle stability. Selected formulations were lyophilized and tested for storage stability up to 6 months. Moreover, reconstitution of the lyophilisates in reduced volume as a technique to prepare higher concentration formulations was studied. Samples were analyzed for particle size, gene silencing, cytotoxicity, turbidity, subvisible particles, osmolarity, residual moisture content, glass transition temperature, and morphology. Depending on the oligoaminoamide, siRNA polyplexes maintained particle size and gene silencing efficiency in the absence or presence of low amounts (7%) of stabilizers after freeze-thawing, lyophilization, and reconstitution. Particle stability was highly dependent on the oligoaminoamide used, but independent of the presence of cysteines that form intra-particular disulfide bridges. In contrast to all other excipients, hydroxypropyl-ß-cyclodextrin did not provide sufficient stability. For lyophilized 5%/10% sucrose and 7% lactosucrose formulations, long-term stability was demonstrated at 40 °C with retained particle size, retained gene silencing activity, unchanged turbidity values, low numbers of subvisible particles, low residual moisture level, and sufficiently high glass transition temperature. Hence, this work is a promising approach in order to provide long-term stable siRNA polyplex formulations that are ready to use after a simple reconstitution step.

Recent advances and further challenges in lyophilization.

While entering a new century, lyophilization in the pharmaceutical field has been subjected to ongoing development and steady expansion. This review aims to highlight recent advances but also to discuss further challenges in lyophilization. At first, the expanded range of pharmaceutical applications based on lyophilization is summarized. Moreover, novel formulation aspects and novel container systems are discussed, and the importance of the freezing step is outlined. Furthermore, the dogma of "never lyophilize above the glass transition temperature" is argued, and recent insights into novel stabilization concepts are provided. Process analytical technology (PAT) and quality by design (QbD) are now leading issues, and the design of the lyophilization equipment also might have to be reconsidered in the future.

Lyophilization of synthetic gene carriers.

Lyophilization, also known as freeze-drying, is a widely used method for stabilization, improvement of long-term storage stability, and simplification of the handling of drugs and/or carrier systems. Lyophilization is time- and energy-consuming; hence, optimized processes are required to avoid time loss and higher costs without compromising product stability. Since the last decade nonviral, synthetic carriers for gene delivery are of increasing interest. However, these systems suffer from poor physical stability in aqueous solution or suspension. Hence, to ensure long-term storage stability lyophilization of the gene carrier systems is favored. Though, lyophilized products retrieving original carrier size and transfection efficiency after reconstitution are mandatory. This chapter gives an overview of the basic steps and troubleshooting for successful lyophilization of synthetic gene carriers. Furthermore the required excipients and their mechanism of action are summarized.

Implementation and evaluation of an optical fiber system as novel process monitoring tool during lyophilization.

Lyophilization is an important and well-established pharmaceutical drying process. Product temperature is the most critical process parameter during lyophilization as it impacts both product quality and process efficiency. Traditionally, thermocouples (TCs) or resistance temperature detectors (RTDs) and recently, manometric temperatures measurements (MTMs) have been used to monitor the product temperature. But, all of these techniques have several drawbacks. The objective of this study was the implementation and evaluation of an optical fiber system as novel process monitoring tool during lyophilization. Therefore, temperature profiles of mannitol, sucrose, or trehalose were recorded with various prototypes of the optical fiber sensors (OFSs) and compared to data obtained with conventional TCs or Pirani/capacitance manometry with respect to the endpoint of primary drying. The OFS allowed easy handling and easy center bottom positioning. Data obtained with the OFS in contact with product were in good agreement with data obtained via TCs or Pirani/capacitance manometry. The OFSs showed significantly higher sensitivity, faster response, and better resolution compared to TCs. This allowed for the detection of additional excipient crystallization events. It was found that force effects on unshielded sensors enabled to detect glass transitions. Three-dimensional temperature profiles were obtained with an OFS helix configuration. The possible integration of a glass fiber with several OFSs in series into the shelf surface enables non-invasive, automatic loading compatible monitoring of the drying process. In conclusion, these advantages turn the novel optical fiber systems into a highly attractive process monitoring tool during lyophilization.

Near-infrared spectroscopy for in-line monitoring of protein unfolding and its interactions with lyoprotectants during freeze-drying.

This work presents near-infrared spectroscopy (NIRS) as an in-line process analyzer for monitoring protein unfolding and protein-lyoprotectant hydrogen bond interactions during freeze-drying. By implementing a noncontact NIR probe in the freeze-drying chamber, spectra of formulations containing a model protein immunoglobulin G (IgG) were collected each process minute. When sublimation was completed in the cake region illuminated by the NIR probe, the frequency of the amide A/II band (near 4850 cm(-1)) was monitored as a function of water elimination. These two features were well correlated during protein dehydration in the absence of protein unfolding (desired process course), whereas consistent deviations from this trend to higher amide A/II frequencies were shown to be related to protein unfolding. In formulations with increased sucrose concentrations, the markedly decreased amide A/II frequencies seen immediately after sublimation indicated an increased extent of hydrogen bond interaction between the protein's backbone and surrounding molecules. At the end of drying, there was evidence of nearly complete water substitution for formulations with 1%, 5%, and 10% sucrose. The presented approach shows promising perspectives for early fault detection of protein unfolding and for obtaining mechanistic process information on actions of lyoprotectants.

The freezing step in lyophilization: physico-chemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals.

Lyophilization is a common, but cost-intensive, drying process to achieve protein formulations with long-term stability. In the past, typical process optimization has focused on the drying steps and the freezing step was rather ignored. However, the freezing step is an equally important step in lyophilization, as it impacts both process performance and product quality. While simple in concept, the freezing step is presumably the most complex step in lyophilization. Therefore, in order to get a more comprehensive understanding of the processes that occur during freezing, the physico-chemical fundamentals of freezing are first summarized. The available techniques that can be used to manipulate or directly control the freezing process in lyophilization are also reviewed. In addition, the consequences of the freezing step on quality attributes, such as sample morphology, physical state of the product, residual moisture content, reconstitution time, and performance of the primary and secondary drying phase, are discussed. A special focus is given to the impact of the freezing process on protein stability. This review aims to provide the reader with an awareness of not only the importance but also the complexity of the freezing step in lyophilization and its impact on quality attributes of biopharmaceuticals and process performance. With a deeper understanding of freezing and the possibility to directly control or at least manipulate the freezing behavior, more efficient lyophilization cycles can be developed, and the quality and stability of lyophilized biopharmaceuticals can be improved.

Development of a lyophilized plasmid/LPEI polyplex formulation with long-term stability--A step closer from promising technology to application.

Cationic polymer/DNA complexes are limited by their instability in aqueous suspensions and usually have to be freshly prepared prior to administration. Thus, the development of isotonic lyophilized polyplex formulations with long-term stability is a desirable goal. Polyplexes based on 22kDa linear polyethylenimine were prepared using a micro-mixer method. Freeze-thawing and lyophilization were performed on a pilot scale freeze-drier. Several excipients (trehalose, sucrose, lactosucrose, dextran, hydroxypropylbetadex or povidone and combinations thereof) at varying concentrations were evaluated for their stabilizing potential against freezing and dehydration induced stresses. For stability testing the lyophilized samples were stored for 6 weeks at 2-8°C, 20°C and 40°C, respectively. Polyplex samples were characterized for particle size, zeta potential, their in vitro transfection efficiency and metabolic activity in Neuro2A cells. In addition, liquid samples were investigated for turbidity and number of sub-visible particles and solid samples were analyzed for residual moisture content, glass transition temperature and sample morphology. L-histidine buffer pH 6.0 was selected as effective buffer. In isotonic formulations with 14% lactosucrose, 10% hydroxypropylbetadex/6.5% sucrose or 10% povidone/6.3% sucrose, particle size was <170nm for all formulations and did not change after storage for 6weeks at 40°C. Polyplexes formulated with lactosucrose or hydroxypropylbetadex/sucrose showed high transfection efficiencies and cellular metabolic activities. Absence of large aggregates was indicated by turbidity and subvisible particle number measurements. The current standard limits for particulate contamination for small volume parenterals were met for all formulations. All samples were amorphous with low residual moisture levels (<1.3%) and high glass transition temperatures (>90°C).

The establishment of an up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined plasmid/LPEI polyplexes.

Polyplexes based on linear polyethylenimine (LPEI) and plasmid DNA are known as efficient non-viral gene delivery systems. However, the requirement for freshly prepared complexes prior to administration due to their instability in aqueous suspension poses the risk of batch-to-batch variations. Therefore, the aim of the study was the establishment of a reproducible and up-scalable method for the preparation of well-defined polyplexes. Polyplexes consisting of pCMVLuc plasmid and 22 kDa linear polyethylenimine (LPEI) were prepared by classical pipetting or with a micro-mixer method using different mixing speeds and plasmid DNA concentrations (20-400 µg/mL). The z-average diameter of the polyplexes was measured by dynamic light scattering. Metabolic activity and transfection efficiency was evaluated on murine neuroblastoma cells after transfection with polyplexes. When varying mixing speeds of the micro-mixer, polyplex size (59-197 nm) and polydispersity index (0.05-0.19) could be directly controlled. The z-average diameter (65-170 nm) and polydispersity index (0.05-0.22) of the polyplexes increased with increasing plasmid DNA concentration (20-400 µg/mL). The established up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined, transfection-competent plasmid/LPEI polyplexes with high reproducibility.

Structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress.

Aggregation of monoclonal antibodies can be induced by freeze-thawing and elevated temperature, typical stress factors during development, production and storage. Our aim was to characterize structural properties of aggregates formed after freeze-thawing and thermal stressing of humanized monoclonal IgG(1) antibody (IgG). Formulations with 1.0mg/ml IgG in 100mM phosphate pH 7.2 were subjected to freeze-thawing and heating and characterized by spectroscopic techniques (UV-absorption, CD, ATR-FTIR and fluorescence), light obscuration, dynamic light scattering, SDS-PAGE, AF4 with UV and MALLS detection, and HP-SEC with UV and online fluorescent dye detection. Thermal stress led to an increased formation of dimers and soluble oligomers (HP-SEC, AF4). Aggregates smaller than 30nm were measured (DLS), next to slightly elevated particle levels in the mum range (light obscuration). Aggregates created by heating were in part covalently linked (SDS-PAGE) and made up of conformationally perturbed monomers (CD, ATR-FTIR, extrinsic dye fluorescence). Aggregation after freeze-thawing was manifested primarily in particle formation in the mum range. These aggregates were noncovalently linked (SDS-PAGE) and composed of native-like monomers, as obvious from CD, ATR-FTIR and extrinsic dye fluorescence spectroscopy. In conclusion, the complementary methods used in this study revealed that heating and freeze-thawing induced aggregates differ significantly in their physico-chemical characteristics.