Biological pharmaceuticals such as proteins, peptides, and oligonucleotides are typically supplied as aqueous solutions or lyophilized solids. Such products generally contain other components like buffers, surfactants, cryoprotectants, and lyoprotectants. Even when a drug product is a solution, the active pharmaceutical ingredient (API) has often been produced as a solid prior to formulation.
In order for biological products to perform consistently, solid materials which lead to or comprise those products must be produced with consistent properties. In general, these properties include the following:
- molecular structure (primary, secondary, tertiary, and quaternary) and conformation
- solid form (crystalline, amorphous, cocrystal)
- solid-state stability (chemical and physical)
- thermal behavior (melting point, glass transition temperature)
- solubility and dissolution rate
- integrity on reconstitution
- surface activity (propensity to aggregate or denature)
- pharmacokinetics
Strategies need to be developed for measuring these properties and developing manufacturing processes that ensure lot-to-lot consistency. Standard solid evaluation techniques that are commonly used in small molecule development can often be applied to good effect to biological APIs and products.
Development activities related to both small molecule and biological products are covered by ICH guidelines. Of particular importance are the guidelines relating to quality (CTD4), specifications (Q6B), stability (Q1), and methods validation (Q2).
The quality guidance directs inclusion of analytical methods for control of API as well as adventitious agents. It also specifies that the physicochemical and biological properties of biomolecules be defined, especially as they relate to manufacturing. Acceptance criteria are also recommended. The discussion of pre-IND and end of phase 2 meetings suggests that CMC issues related to unique physicochemical or biological properties should be addressed.
The guidance on specifications indicates that an identity test be developed that is highly specific for the material of interest. In some cases, such specificity may best be obtained using a solid-state technique such as x-ray powder diffraction or infrared spectroscopy. Since most peptides are lyophilized, the FDA requests that exact lyophilization conditions be part of regulatory filings. In addition, a description of the nature of the solid form is recommended.
The FDA requires extensive characterization of purified, unmodified monoclonal antibodies. The characterization steps include determination of structural integrity using methods such as mass spectrometry. Reference standards are needed to provide assurance on consistency among lots. The FDA also recommends stability testing programs and lot-to-lot quality control.
Stability tests are needed, especially for smaller molecule biologics. Peptides containing methionine, tryptophan, or cysteine are sensitive to oxygen, asparagine is sensitive to cyclization, and dipeptide units conatining glycine, alanine, or proline can cyclize to give diketopiperazines. The guidance on stability of peptides recommends tests of both biological activity and chemical stability, and states that "Preservation of biological activity is not a guarantee of chemical integrity."
Modern analytical chemistry provides a range of techniques for evaluation of solid properties. Most techniques can be applied to both API and drug product. With proper method development it is possible to quantitatively analyze the state of an API in a product, often at very low levels. X-ray powder diffraction is useful for determining crystallinity, degree of anisotropism, and packing motifs. Infrared spectroscopy and Raman spectroscopy are powerful techniques for determination of both molecular and aggregate structures. For example, infrared spectroscopy has been used to determine the conformation of nucleotides and the percentages of beta-pleated sheets, alpha-helices, and random coils in proteins. Circular dichroism is a complementary technique to infrared spectroscopy in the secondary structural information it provides. Solid-state nuclear magnetic resonance spectroscopy is a powerful tool not only for structure elucidation but for understanding system dynamics. Thermal methods, such as differential scanning calorimetry and thermogravimetry, also can be used to probe molecular mobility in solids, as well as the extent of crystallinity and the presence of volatile components. Water sorption/desorption behavior, as measured by an automated sorption analyzer, reveals information about the affinity of a solid to water and the ability of water to cause solid form changes. Sequencing of proteins or oligonucleotides in conjunction with mass spectrometry can provide proof of chemical integrity through measurement of primary structure and molecular mass. Particle size analysis is critical to understanding equivalence and expected behaviors of lyophilized products.
Typically, a combination of the above techniques, and sometimes other techniques, are necessary to fully characterize an API or drug product. Starting with a new substance, a wide variety of techniques should be utilized to determine which ones provide critical information.
Comparability protocols for biologics are defined in a 1996 guidance from the FDA. These protocols are based on the realization that changes in a manufacturing process, equipment, or facilities could result in changes in a biological product that would affect the product's safety and efficacy. Comparability protocols are aimed at reducing the risk of such changes by outlining strategies for comparing the properties of pre- and post-change materials.
The 1996 guidance suggests that comparison of pre- and post-change materials should be based on extensive chemical, physical, and biological assays. Tests should include those routinely used, as well as those aimed at fully evaluating the effect of the change on the final product. Analytical tests should be selected to provide the maximum amount of information. If a manufacturer can demonstrate comparability, additional clinical safety or efficacy trials may not be needed.
During drug development and immediately prior to product launch, significant changes are often made as the process is scaled up. In some cases, the FDA has required additional clinical data each time a process is changed. Comparability protocols define a strategy for establishing equivalence after a process or scale change. Comparability protocols can avoid additional clinical trials and the associated delays in product development and can facilitate post-launch changes.
Strategies need to be developed for comparing properties and ensuring equivalence. These strategies involve determining which techniques provide the most information and are the most sensitive to critical properties. The selected techniques can then be used to determine a baseline for pre-change lots. Once this baseline is established, post-change lots are examined using the same techniques and equivalence is evaluated.
How can chemical stability and solid-state transformations be analyzed and predicted? To begin to address this question, basic mechanisms of degradation need to be understood. Is the degradation physical or chemical? Is water involved in the degradation, and does it accelerate the degradation process? What role does the formulation play?
The mechanisms of instability can often be determined by combining the results of chemical analyses and physical analyses. Especially important is an understanding of the role of water in the mechanism of physical (crystallization, aggregation, or denaturation) or chemical instabilities.
A comparability protocol can be used to guide stability studies. API and drug product are subject to ambient and accelerated stability conditions and analyzed using techniques like those discussed above. Additionally, techniques that provide information on chemical degradation routes, such as high performance liquid chromatography or mass spectrometry, should be incorporated.
The methods described above can be used to assist in the design of stabilized formulations. For example, measurement of the glass transition temperature and molecular mobility can provide information on the possible role that cryoprotectants might play in enhancing stability. Another possible approach to achieve stability is to use crystalline proteins. In many cases, crystalline materials are more stable than amorphous compounds.
According to the FDA's draft guidance on PAT, the desired state of pharmaceutical manufacturing is that:
- Product quality and performance are ensured through the design of effective and efficient manufacturing processes.
- Product and process specifications are based on a mechanistic understanding of how formulation and process factors affect product performance.
- Quality assurance is continuous and real time.
- Relevant regulatory policies and procedures are tailored to accommodate the most current level of scientific knowledge.
- Risk-based regulatory approaches recognize both the level of scientific understanding and the capability of process control related to product quality and performance.
The PAT initiative is consistent with the current FDA belief that quality cannot be tested into products, but should be built-in or by design.
Biomolecules are included in the FDA's new initiative, "Pharmaceutical cGMPs for the 21st Century: A Risk-Based Approach." This initiative touches every aspect of pharmaceutical research and manufacturing. It demands knowledge of the properties and characteristics of solid drug substances and ingredients, expert insight into the mechanistic aspects of the manufacturing process, identification of critical variables, and integrated quality control assessment throughout the manufacturing process. Firms with the knowledge and ability to achieve quality by design will have strategic advantages over firms that do not, including the possibility of fewer inspections and the ability to cope with process upsets more quickly and effectively.
Early applications of PAT have included the use of sensors on fermentation equipment. These include pH sensors, temperature sensors, oxygen sensors, etc. As PAT evolves, sensors for solids will become more prevalent. For example, sensors for water content during lyophilization and precipitation will become widely used. Furthermore, it is possible to envision that on-line sensors for crystallinity will be used to ensure the desired nature of drug substances and drug products. Ultimately, sensors will be used in a cycle with process controllers to monitor and adjust critical process parameters real-time.
The rational approach to quality by design requires a thorough understanding of the dependence of product properties on manufacturing parameters. Use of fundamental preformulation studies and modern analytical methods is necessary to achieve the appropriate level of understanding. Application of these modern methods promises to lead to new and improved methods for manufacturing biological pharmaceutical products.
SSCI Inc. is a cGMP contract research laboratory providing a wide range of research and analytical services focused on pharmaceutical and industrial chemical solids. We are experts in crystallization, characterization, and the chemistry of solid materials. SSCI operates at the forefront of technology in these areas:
- Polymorphism Studies and Salt Selection
- Production Control
- Development of Analytical Methods and Strategies (traditional and on-line)
- Amorphous Form Generation and Stabilization
- Solid-State Analytical Services
- Characterization of Small Molecule and Biological Solids
- Analysis of Drug Substance and Dosage Form
- Verification of Sameness for Clinical Trials
- Expert Services and Litigation Assistance
We invite your queries on these important developments in Process Analytical Technology and Quality by Design. We believe our extensive experience in cGMP solid-state research and analysis will help you meet the PAT challenges today and for the future.
For detailed information on SSCI's research, analytical, and consulting capabilities, and for upcoming conference presentations and short courses, visit the SSCI Web site: www.ssci-inc.com
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