Biological reagents are especially sensitive to temperature changes, necessitating specific storage conditions to keep these bioactive for longer periods of time. Dehydrating reagents reduces this sensitivity, and this is a leading factor in the increasing use of lyophilized bead technology in the pharmaceutical and diagnostic industries.
Dr. Mattia Cassanelli - Technical Business Manager at Biopharma Group, UK - recently delivered a webinar on the utilization of Lyobead technology in the pharmaceutical and diagnostic sectors, outlining the use of these beads across a range of different applications. This article provides a summary of that webinar.
What are lyobeads?
Lyophilized beads (Lyobeads) are spheres of customizable lyophilized material. These spheres contain a defined volume of material per unit.
One of the most frequent and pertinent uses of these beads in the diagnostic and pharmaceutical industries is found in the encapsulation of the reagents in PCR assays, allowing these to be stored for longer periods at room temperature. Lyobeads are also found in drug delivery, microfluidics, and encapsulation of bacteria; particularly when aiming to meet increased market-led demands.
Why use lyobeads?
Lyobeads’ primary benefit stems from the capacity to develop a single formulation that can be modified for use in different applications at various temperatures or in different containers - reducing R&D investment. It is also possible to increase production, with the Lyobeads’ stability making them suitable for high throughput loading into the lyophilizer and bulk storage prior to packaging.
The spherical nature of the Lyobeads maximizes their surface area, meaning that reconstitution time following lyophilization can be reduced.
How are lyobeads made?
Lyobeads can be created by using liquid nitrogen to snap freeze the liquid formulation of reagents, then dehydrating this using a freeze dryer or via polymeric gelation.
Polymeric gelation sees a gelling polymer anionic alginate inserted into a solution that contains calcium ions. As the alginate encounters divalent cations, this results in the creation of a stable polymer that forms a membrane, protecting any reagents in the solution.
Use of lyobeads in R&D
Choosing an appropriate liquid formulation of excipients for lyophilization into Lyobeads involves three key testing stages: compatibility, shortlisting and screening.
The compatibility step sees a range of up to thirty excipients recommended based on prior experience and an array of initial thermal analysis results. Each of these excipients is tested to verify its compatibility in the liquid state, ensuring that there was no test interference.
A shortlist of promising candidates in the liquid state is chosen from these initial tests. These candidates will be lyophilized during the screening process, with each formulation candidate then processed using an identical freeze-drying cycle. A true comparison is possible, because all samples are dried under the same process conditions. Next, the most favorable dried candidates will be characterized, and results combined to determine the lead formulations.
In some circumstances, a statistical analysis or Design of Experiment (DoE) approach can be used to enlarge the formulation candidate list, identifying the ideal formulation based on key parameters; for example, the mechanical properties of the lyophilized beads, the glass transition temperature (Tg) of the active pharmaceutical ingredient (API), and any specific post-lyophilization product activity.
This approach also facilitates the definition of a design space that identifies optimal lyophilization conditions for each batch while ensuring appropriate regulatory compliance.
When the Lyobead formulation has been successfully developed, this can then be scaled up for manufacturing.
Diagnostics - PCR assays
A study was conducted with a view to extending the shelf life of a liquid formulation of an existing COVID-19 detection PCR assay. It was necessary to store the reagents at room temperature in high throughput (96-well) format with no impact on reaction quality. There was also the possibility that manufacturing would be ramped up to millions of reactions a month in a short timeframe.
The liquid formulation’s thermal behavior was analyzed using a freeze-drying microscope, employing a steady increase in temperature to visually identify the point of collapse. Further analysis was conducted via a Lyotherm (Biopharma Group), using a combination of electrical impedance and differential thermal analysis (DTA), to ascertain the formulation’s glass transition temperature.
The results from these investigations prompted a reformulation of the product into a new Lyobead mastermix with the capacity to include all formulation components (buffer, enzymes, probes, and primers).
As part of the compatibility, shortlisting and screening processes, Biopharma performed a number of tests to find the most promising candidates.
Visual assessment was used to evaluate the bead’s morphology and elegance, and this was supplemented with modified differential scanning calorimetry (DSC) - further characterizing the product’s thermal properties.
Figure 1. R&D - Visual assessment of PCR assay lyobeads. Image Credit: SP Scientific Products
To better comprehend how the product behaves during storage, an assessment was conducted into its water absorption and mechanical stability. Dynamic vapor sorption (DVS) is used to measure how water is absorbed by a sample at a specific temperature, providing an indication of how long a product can be stored before this absorbs an irreparable quantity of water vapor.
Lyophilized products may be subjected to mechanical stresses during transport and storage. To assess this, a MicroPress (Biopharma Group) actuator was employed in the application-controlled pressure on the freeze-dried cake. This allowed researchers to measure the percentage of strain that the Lyobeads could withstand without regaining shape. This is illustrated by breaks in the curve, implying a friable and brittle product.
Figure 2. R&D - Mechanical properties of lyobeads. Image Credit: SP Scientific Products
Assessment was then conducted into the reconstitution of the most robust candidates. The freeze-drying process leaves microscopic pores, meaning that products can be reconstituted far more rapidly and easily. Electron microscopy can be used to investigate the distribution, size, and interconnection of these pores, allowing the optimization of candidates able to reconstitute in a short time.
A number of candidates for the COVID-19 PCR assay were ultimately tested, with comparisons conducted in line with the studies outlined here. Once lead candidates had been identified and cycle conditions had been optimized, validation was performed with several batches. This verified the most optimal Lyobead products, suitable for manufacturing and commercial use.
Figure 3. R&D Analyses – Microstructure. Image Credit: SP Scientific Products
Biologics - bacterial vaccines
A study was conducted in order to develop an appropriate formulation containing an inactivated gram-negative bacterium. This had to be suitable for use in an oral vaccine offering long-term stability and the potential for rapid reconstitution.
An investigation into various methods and formats showed that Lyobeads offered an ideal solution for this product. This study also supported the technology transfer and set up required for a new production line.
Preliminary studies confirmed that snap freezing the Lyobeads caused no damage to the bacteria, so this method was selected for Lyobead synthesis. Compatibility testing and post-process analysis were completed following the first screening cycle, highlighting a series of applicable cryo- or lyo-protectants that were required to be encapsulated within the Lyobeads.
Biopharma supported the client to transfer this technology to its site, assisting in the development of efficiently run processes and offering advice around the selection of an appropriate freeze dryer.
Pharmaceuticals - oral tablets
Oral tablets must be milled and tableted prior to placement in blister packs. A study was conducted to evaluate the capacity of Lyobeads in a pharmaceutical product developed for oral administration. Lyobeads’ design negated the need for milling and tableting – a factor that is both technically and commercially advantageous. The Lyobeads were inserted into the blister packs.
Ensuring appropriate analysis and optimization of the Lyobeads required the use of a DoE approach designed to maximize parameters. This process leveraged several techniques (outlined earlier) to analyze API activity, dissolution speed, and thermal stability in a frozen state, alongside the mechanical properties of the dried sphere.
The DoE process resulted in the characterization of nineteen candidate formulations, each processed in screening and refinement cycles. The mechanical properties resulting from the combination of several excipients is illustrated via a heat plot which identifies limitations relating to product stability.
Residual moisture content calculations alongside modulated differential scanning calorimetry (MDSC) analysis were used to identify the most appropriate time out of the freeze dryer.
These studies resulted in the efficient production of Lyobeads, which absorbed less moisture while being quicker and more cost-effective to produce versus milled and tableted products.
Nutraceuticals - probiotics
A study was conducted into the use of Lyobeads in orally administered probiotics. The final formulation involves millions of probiotic bacteria remaining viable following processing. This formulation was also required to exhibit extended shelf life and short reconstitution times.
Lyobeads were selected as the optimal format. Rather than being snap-frozen, these 2 mm beads were created as polymers from alginate in a buffer containing calcium ions.
Appropriate cryo- or lyo-protectants, and optimal freezing rates were determined via compatibility studies with a number of freeze-drying cycles. A freeze-drying microscope was used to visualize any physical clumping or loss of shape of the Lyobeads.
By altering freeze-drying temperatures and pressure in the freeze-drying cycles, it was also possible to develop an appropriate design space for this product.
Figure 4. Four additional cycles to work out a suitable design space. Image Credit: SP Scientific Products
Lyobeads offer a simple solution to the creation of stable products suitable for long term storage – a key consideration in the diagnostic and pharmaceutical fields. Following manufacture, the beads can be inserted into any container, allowing for different product changes and bulk storage prior to packaging.
Numerous methods exist for the creation of Lyobeads, and conditions must be optimized via robust compatibility testing, shortlisting, and screening. These initial steps requires a notable time investment.
Lyobeads are suitable for use in a diverse range of areas and application, including those outlined in the case studies here. One of the most common applications of these beads is in PCR, whereby a stable lyophilized mastermix stored at room temperature offers the potential to speed up and increase PCR screening assays’ throughput.
Many companies are employing lyophilized beads in their product lines, due to their range of tangible benefits, including reduced environmental impact and cost savings.
Produced from materials originally authored by Dr. Mattia Cassanelli, Ph.D. from Biopharma Group.
About SP Scientific Products
SP is a synergistic collection of well-known, well-established, and highly regarded scientific equipment brands — SP VirTis, SP FTS, SP Hotpack, SP Hull, SP Genevac, SP PennTech, and most recently SP i-Dositecno — joined to create one of the largest and most experienced companies in freeze-drying/lyophilization, complete aseptic fill-finish production lines, centrifugal evaporation and concentration, temperature control/thermal management, glassware washers and controlled environments.
SP is part of SP Industries, Inc., a leading designer, and manufacturer of state-of-the-art laboratory equipment, pharmaceutical manufacturing solutions, laboratory supplies and instruments, and specialty glassware. SP's products support research and production across diverse end-user markets including pharmaceutical, scientific research, industrial, aeronautic, semiconductor, and healthcare. In December 2015, SP Industries was acquired by Harbour Group, a private investment firm founded in 1976. Harbour Group is a privately owned, operations focused company based in St. Louis, Missouri. Headquartered in Warminster, Pennsylvania, SP has production facilities in the USA and in Spain and the UK in Europe and offers a world-wide sales and service network with full product support including training and technical assistance.
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