Although biopharmaceuticals represent a rapidly growing segment for ethical products, the ability to progress these products from research to development and ultimately manufactured products can be hindered by the complex structures and multiple degradation pathways that are associated with biopolymers.1,2,3 Traditional pharmaceutical development has often relied on real-time stability studies and standard analytics to develop product formulations that confer the two year shelf life typically required for drug products. Although these approaches have proven useful, there is a need to develop and apply techniques that report on the structural stability of biopharmaceuticals and reduce the need to rely on real-time stability studies so extensively. Differential scanning calorimetry, which monitors the apparent excess heat capacity of a protein solution as a function of temperature, is one of several tools that have been proven to be useful in this setting.4,5 Even when used in a comparative or rank-ordered fashion DSC can provide data on protein stability that correlates well with real-time stability studies.
A key element in the ability to apply DSC or any other technique that may report on protein structure or stability is an organized approach that allows for the rigorous statistical evaluation of preformulation data. Recent regulatory guidance6 has highlighted the need both for well-characterized biopharmaceutical products as well as a systematic development approach that allows for the understanding of both critical parameters that affect product stability as well as any interactions between those factors that may exist. Here we describe a systematic approach to preformulation development that leverages biophysical characterization and standard analytics in the context of statistical design with the goal of shortening the pharmaceutical development cycle while improving the quality of the development studies.
Tools for pharmaceutical development
A variety of techniques that are capable of monitoring either protein secondary or tertiary structure have been extensively used in support of protein preformulation development; although this is not an exhaustive list, these include differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), and fluorescence spectroscopy.7,8,9 While each of these techniques has its advantages and disadvantages, we have found DSC to be one of the more generally applicable biophysical techniques for preformulation development for the following reasons: most proteins exhibit measureable thermal transitions during unfolding, the technique is relatively insensitive to a variety of potential buffer components and there is no requirement to know the protein structure or relative proportion of alpha-helical or beta-sheet segments in order to generate useful preformulation data. In addition to DSC or other biophysical techniques, however, standard analytical techniques are also employed to more fully understand product quality in the context of accelerated stability studies. The suite of analytical tools typically includes size exclusion chromatography (SEC) for the detection of aggregates, ion exchange (IEX) for monitoring charge variants and deamidation, peptide mapping with mass spectrometry for chemical degradants and either SDS-PAGE or capillary gel electrophoresis (CGE) for monitoring covalent aggregates and degradants. The system that our organization employs extensively to provide an organized framework for experimental design and data interpretation is design of experiments (DOE),10 because it maximizes the quantitative nature of DSC and many of the standard analytical techniques used during preformulation. DOE allows for the identification and statistical assessment of critical factors and their interactions and an example of a DOE interaction plot is shown in Figure 1. The data in Figure 1 illustrate the effect of buffer type and pH on aggregation as assessed by size exclusion chromatography. Had either buffer system alone been used for the pH study, one would have incorrectly concluded that pH either did or did not have an effect on aggregation; however, by conducting a DOE it was possible to demonstrate that an interaction exists between pH and buffer type which was helpful in ruling out one buffer system for subsequent studies.
Figure 1: Interaction plot showing the effect of buffer type on aggregation. For each sample, the percent high molecular weigh (HMW) species were summed and then reported in the statistical design as a function of pH and buffer type. For the first buffer system (shown in red), the percent HMW species varies as a function of pH and is generally higher than is observed for the second buffer system. The black data points show the same data for the second buffer system, but in this case the percent HMW species is generally lower and also does not appear to vary as a function of pH, suggesting that buffer two is a more suitable formulation both from a stability standpoint and from a manufacturing standpoint, since in this buffer system the pH would not have to be as tightly controlled to produce a product of acceptable quality.
The type of design selected usually reflects the level of understanding and/or place in the product development lifecycle for a particular therapeutic: for pre-clinical products or products where there is limited information, factorial or fractional factorial designs are used to identify critical factors/interactions from a larger set of potential factors. For later-stage products or those products where some initial studies to identify critical factors have been performed, response surface designs such as central composite or Box-Behnken designs are more suitable because those designs offer greater granularity within the design space and can be used to model complex or quadratic surfaces.
Platform preformulation
Workflow for Preformulation Development
In addition to the desire to more rapidly develop products, biopharmaceutical manufacturers also have a strong desire to leverage core competencies and create economy of scale across multiple products and processes. For preformulation activities in a contract development setting, this has evolved into a defined workflow designed to clarify scope, timelines and deliverables at each point in the development process. A high-level example of a typical preformulation project is shown in Figure 2 and will be discussed in more detail in the following sections:
Figure 2: Example Workflow for Preformulation Projects
Baseline Biophysical Studies
During the initial stages of preformulation development, several candidate biophysical techniques are employed as part of a “baseline biophysical” study (Figures 3 and 4) to assess their utility and those techniques found to be suitable for a particular molecule are then used as part of a screening design. Because of its general applicability to a wide variety of proteins and tolerance for a large number of buffers and excipients, DSC is almost always found to be suitable for both the initial and subsequent preformulation studies. The goals of this initial task are twofold: first it allows identification of the most suitable techniques for a particular molecule while giving the formulation scientist a chance to optimize and standardize acquisition parameters and more importantly, it allows the scientist to eliminate categoric factors that do not appear to be suitable for a particular molecule so subsequent designs are centered around buffer, pH and ionic strength regimes where the molecule appears to be most stable.
Figure 3: Differential Scanning Calorimetry for several different buffer types. The selected monoclonal antibody was exhaustively dialyzed into four different buffer types prior to analysis. Sample concentrations were approximately 2 mg/mL with a scan rate of 60°C/hr. Data presented here are after buffer subtraction and concentration normalization
Figure 4: Example circular dichroism scans for baseline biophysical screening. Although CD is not as generally applicable as DSC because of potential buffer incompatibilities, certain proteins lend themselves well to CD analysis. Data above show far-UV CD scans of a protein that contains an appreciable amount of alpha-helix, a secondary structural element for which CD is well-suited. Solubility Increasingly, sponsors are requesting solubility studies to generate high concentration (>100 mg/mL) therapeutics because more concentrated drug products allow in-home administration as opposed to restricting administration solely to a clinical setting. While appealing from a marketing point of view, the need for high concentrations where the same dose is delivered in a small volume places extreme demands on the stability of the protein therapeutic. These studies often employ a variety of pH and buffer candidates, typically with a variety of potential excipients. It is important to note, however, that conditions where the molecule is most soluble do not necessarily reflect conditions where the therapeutic is most stable. >> Download the full Application Note as PDF
Malvern Instruments provides the materials and biophysical characterization technology and expertise that enable scientists and engineers to understand and control the properties of dispersed systems. These systems range from proteins and polymers in solution, particle and nanoparticle suspensions and emulsions, through to sprays and aerosols, industrial bulk powders and high concentration slurries. Used at all stages of research, development and manufacturing, Malvern’s materials characterization instruments provide critical information that helps accelerate research and product development, enhance and maintain product quality and optimize process efficiency. Our products reflect Malvern’s drive to exploit the latest technological innovations and our commitment to maximizing the potential of established techniques. They are used by both industry and academia, in sectors ranging from pharmaceuticals and biopharmaceuticals to bulk chemicals, cement, plastics and polymers, energy and the environment. Malvern systems are used to measure particle size, particle shape, zeta potential, protein charge, molecular weight, mass, size and conformation, rheological properties and for chemical identification, advancing the understanding of dispersed systems across many different industries and applications. Headquartered in Malvern, UK, Malvern Instruments has subsidiary organizations in all major European markets, North America, Mexico, China, Japan and Korea, a joint venture in India, a global distributor network and applications laboratories around the world. www.malvern.com severine.michel@malvern.com
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