Gene therapy represents an innovative new treatment strategy that is transforming our ability to treat a wide range of diseases. Manufacturing gene therapy products that are safe and effective for patients often requires reagent optimization, which can involve a range of laboratory tests.
One important measurement that is used extensively in pharmaceutical manufacturing is osmolality, a measure of solute concentration. In this article, we introduce the concept of osmolality, detail the uses and benefits of osmolality testing, and also explore a recent example of how we used osmolality measurements to rapidly optimize a buffer for a gene therapy manufacturer.
Osmolality, a measure of solute concentration, is defined as the total number of moles of solute (the osmoles, or Osm) divided by the weight of the solvent (kg) (Figure 1A). However, to get an idea of what makes osmolality such a useful measurement during manufacture of gene therapy products, it is first important to understand the difference between osmolality and the closely related concept of osmolarity.
While osmolality uses weight in the denominator (Osm/kg), osmolarity uses the volume of solvent (Osm/L), which means that the latter is automatically dependent on both temperature and pressure (Figure 1B). This makes obtaining consistent measurements of osmolarity challenging during manufacturing, as engineers would need to ensure these external parameters are kept constant during the different stages of production.
Figure 1. Comparison of osmolality (A) and osmolarity (B).
Unlike volume, the weight of a solvent is constant at different temperatures and pressures, meaning that osmolality provides a simpler way of measuring solute concentration and provides more consistent measurements for a wide range of applications.
For example, osmolality tests on urine and blood are used clinically to diagnose diseases such as diabetes insipidus , and blood osmolality tests are used to diagnose sodium imbalance . Osmolality measurements are also crucial for gene therapy manufacturing at multiple process stages.
Versatile Osmolality Testing in Gene Therapy Production
Measurement of osmolality in gene therapy production is achieved using freezing point depression osmometers, which work on the principle that increasing the number of particles in a solution decreases its freezing point. By comparing the measured freezing points to standards for a particular solution, scientists can obtain reliable and consistent osmolality measurements at multiple stages of gene therapy production.
Osmolality is an important measurement to consider when it comes to cell culture media. For example, as the osmolality of cell culture media can affect the growth rate of cells used for vector production, measurements of this parameter are crucial for optimal yield during gene therapy production. Osmolality of cell culture media can also be used as an indicator of cell viability, making osmolality testing an important part of cell culture monitoring during the upstream processes of gene therapy production.
For viral vectors themselves, evidence suggests that the osmolality of the host cell culture medium can affect the composition of the viral membranes produced, which in turn, affects the stability of the vectors in the final gene therapy products . As the osmolality of the buffer solution used with viral vectors can also directly affect viral stability , manufacturers are also choosing to measure this parameter during downstream processes of gene therapy production.
Another important use of osmolality tests during manufacture of cell and gene therapies is for cryopreservation—in which cells are frozen for storage of drug intermediates or later use. This process requires specific cryopreservatives, which must be tested with the osmometer to ensure the correct composition at multiple production stages, including cell banking, packaging, and shipping.
The dependence of multiple biological processes on osmolality, coupled with the usefulness of the measurement in vector quality control (QC), makes osmolality testing a powerful method for manufacturers to ensure a high yield of safe and effective gene therapy products.
Case Study: Optimization of a Gene Therapy Buffer
As well as providing a powerful method of QC during gene therapy manufacturing, osmolality testing is also important for optimization of gene therapy reagents—which can include buffers, cell culture media, or other product-specific solutions. By offering manufacturers the ability to quickly adapt their formulations based on obtained osmolality data, reagent suppliers such as Teknova can speed up the process of gene therapy development.
In one recent example, a customer asked us to optimize the osmolality of a PBS buffer for a viral vector to make it suitable for injection during animal trials. For vector stability and to prevent pain and tissue damage upon injection, drugs destined for the intravenous route must be isotonic with blood, meaning that the limit on osmolality for the buffer was placed at around 300 mOsm/kg.
After receiving the request, our formulation chemist referenced USP <785> and worked to optimize the buffer with the aim of reaching both target osmolality and a physiological pH range of 7.4 ± 0.04. The first stage of optimization involved conducting experiments that assessed osmolality at different reagent concentrations, with sucrose varied from 5–7% and NaCl varied from 60–120 mM. With this data, regression analysis was then performed to determine whether osmolality increased linearly with NaCl at these concentrations (Figure 2). (Note: see sidebar below to learn more about PBS and common additives used in different applications.)
Similar experiments were also performed in which disodium phosphate (Na2HPO4) and NaCl were varied with the results used to determine the relationship with osmolality by regression analysis (Figure 3). Conductivity measurements were also taken to give an indirect measure of ionic concentration in the different solutions (data not shown).
During the optimization process, some initial experiments gave unexpected, non-linear changes in osmolality when the concentration of sucrose was varied. However, we quickly identified that these problems were a result of the sucrose solution and soon resolved the issue by switching to a higher purity grade chemical. To learn more about the different reagent purity grades and FDA and EMA regulations on their use in advanced medicinal products, read our article, The Importance of Reagent Purity Grades.
After the optimization experiments were complete, our formulation chemist provided the customer with a calculator that could determine the osmolality of the buffer at different concentrations of NaCl and disodium phosphate. With access to this data, the customer was able to make an informed decision on which buffers would be most suited for further testing of their gene therapy product.
Figure 2. The relationship between osmolality and NaCl concentration in solutions with different sucrose concentrations.
Figure 3. The relationship between osmolality and disodium phosphate (Na2HPO4) concentration in solutions with different NaCl concentrations.
Completed in only a couple of weeks, this rapid optimization process highlights the importance of osmolality measurements in gene therapy development and showcases our ability to quickly adapt to the changing needs of customers.
With multiple gene therapies already approved by regulatory authorities, such as the FDA and the EMA, these innovative treatments may begin to revolutionize the way we treat debilitating diseases.
Using suppliers with the capability for rapid and efficient reagent optimization with such methods as osmolality testing can not only speed up the process of gene therapy development but can also ensure that finished products are safe and effective for patients.
- The Royal Children’s Hospital Melbourne. (2021) Diabetes insipidus. [Accessed 20 July 2021.]
- University of Rochester Medical Center. (2021) Osmolality (Blood). [Accessed 20 July 2021.]
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