It usually takes around a billion dollars and ten years to deliver a new drug to market. Developing the formation takes between one and a half to two years. With the urgency to manufacture an annual flu vaccine and also to create a SARS-CoV-2 vaccine or drug, there is an urgent requirement for fast throughput screening methods.
The development of a formulation that will be proven effective, safe, and stable for at least two years after manufacturing is key. Lyophilized products are more stable even though liquids are easier and cheaper to produce, so the ideal formulation should be capable of freeze-drying.
For the best formulation development, knowledge of not just the protein itself but how the final product will be packaged and utilized are both required. This will help the development team in screening excipients that are appropriate, including buffers, for the target product.
In order to speed up this process, high-throughput techniques have been developed to assess different conditions quickly and to move through the drug development processes faster.
Dr. Jeff Schwegman, CEO at Labyrinth BioPharma, LLC, USA, recently gave a webinar discussing how to select the correct buffer and excipient for high-throughput formulation development; this article summarizes the webinar.
Building a stable product – formulation design
Building a stable product is a methodical process that begins with the development stage that characterizes the protein, followed by a number of compatibility studies to identify the most stable product that can be lyophilized and, lastly, the manufacturing process.
Some additional formulation components, sufficient amounts of purified protein, and an understanding of storage, dosage, and delivery technique are all needed before this development process can begin.
Formulation components are available in a variety of forms, from buffers to balance the pH to bulking agents, stabilizers, and surfactants, which work together with the active ingredient. In the early development stages, it is typical to ‘partially formulate’ the product with enough stability to get through the trial, even in Phase I Clinical Trials.
This is typically created as a frozen presentation to be thawed and administered at the clinic. This enables the drug to undertake clinical trials while the formulation is still being developed to become more stable, ideally in a lyophilized state.
FDA website (www. fda.gov) offers information about excipients approved for use in drug formulations. Image Credit: SP Scientific Products
Although during the manufacturing of a drug a number of these non-active pharmaceutical agents are crucial, it is worth considering the addition of excipients early in the development process, as these substances can influence the active ingredient’s properties.
Devastating effects can come about as a result of a bad choice of excipient, even fatalities. Detailed information about excipients that are approved for utilization in previous drug formulations can be seen on a number of websites, including the United States Food and Drug Administration (FDA) website, which can supply a useful resource when designing a new drug.
Keeping the concentration of any excipient to the minimum will help to ensure that they have minimal effects on the target product, particularly during lyophilization.
Two or three buffers are initially chosen during compatibility studies, which have a pKa that is near to the stable pH of the target product. Due to a chemical reaction in the formulation, for example, oxidation, degradation, or hydrolysis, alterations in pH can happen in drug products, but this can also occur through the freezing process, and in some instances, the purified protein can have some buffering capacity.
Due to both the ‘freezing stress’ and the ‘drying stress’ of lyophilization, biological molecules are particularly susceptible to loss of activity. A good stabilizer stays amorphous, is preferentially excluded from the protein surface, and is capable of hydrogen bonding with the protein.
Trehalose and sucrose are both well suited to protect biological molecules from these stresses. Knowing how much stabilizer to add is vital; adding too little will not protect the protein and adding too much can significantly reduce drying time, which could collapse the lyophilized cake or lead to destabilization.
Salts decrease the collapse temperature during freeze-drying as they increase the amount of unfrozen water and can prevent or delay the crystallization of other components. The tonicity of a product can be altered using glycine or mannitol if it is necessary to attain a product that is isotonic.
4. Bulking agents
When the total amount of active ingredient and other excipients are too small to give adequate structural support to the cake, then bulking agents are utilized. Mannitol is the most commonly used in lyophilized products, but its crystalline behavior can cause issues and vial breakage can be significant.
Products can stick to stainless steel, glass, filters, process tubing or any product contact surface. Surfactants have the ability to decrease surface tension and are able to block certain surfaces where protein molecules can stick or even stop them from sticking to each other. Polysorbates between 20 and 80 are typically utilized in formulations, particularly 20Tween80.
Dr. Schwegman describes a number of methods that can be used to study formulation variables during the webinar. Differential Scanning Fluorimetry (DSF) and Nano Differential Scanning Calorimetry (DSC) enable the solution to be stressed by temperature in different formulation conditions.
The more stable the initial solution condition is then the higher the temperature; the protein will keep its folded, native state structure. Nano DSC quantifies the heat that is being absorbed as unfolding or degradation happens. It is a sensitive technique, but testing requires a lot of material and is time-consuming.
The product temperature is raised during DSF until denaturation happens, changing the fluorescence because of an extrinsic fluorophore. These techniques can be used with a 96-well format, allowing a variety of conditions to be tested in high throughput at the same time.
Soluble and insoluble aggregates can be quantified during freeze-thawing or lyophilization of product by utilizing colloidal methods like static/dynamic light scattering, Size Exclusion Chromatography (SEC)-HPLC, flow imaging microscopy, as seen in Figure 1.
Figure 1. Size Exclusion Chromatography (Soluble Aggregates) with UV Detection. Image Credit: SP Scientific Products
Adjustments can be made to the excipients after measurements and the process can be repeated until aggregation (soluble and insoluble) is eliminated or minimized.
Another screening technique analyzes protein secondary structure by measuring hydrogen bonding in the different structural elements of a folding protein, including beta-sheets, alpha helices, beta turns, etc.
Infrared analysis of proteins (FTIR) distinguishes Amide I vibrational frequency alterations, which are caused by C=O backbone stretching vibrations when the oxygen in the carbonyl bonds with hydrogen in the different folded structural elements.
Developing a stable formulation for a biologically-based therapeutic product is a time consuming and expensive process. Excipient choice, including buffer choice, has typically been a trial-and-error process involving the preparation of numerous different formulation variations and placing them on accelerated stability.
High-throughput screening methods, including but not limited to nano-scale DSC, FTIR and DSF, are extremely helpful tools for screening excipient variations quickly to establish how they will affect the short/long term stability and on the effectiveness of stabilizing against the freezing and drying stresses of lyophilization.
In addition to keeping good lines of communication open between the analytical and manufacturing groups, high-throughput screening techniques will help to decrease the time and cost of production. These methods also optimize the chances of success for producing a safe, stable and effective product for consumers.
Produced from materials originally authored by Dr. Jeff Schwegman Ph. D from Labyrinth BioPharma
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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