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Protein characterization is a critical part of drug development, but as there are still limitations with available techniques, industry needs to look at technological advances to meet the specific requirements of complex molecule characterization.
Protein therapeutics are a promising class of drugs that are increasingly coming to the fore in development pipelines as a result of their utility in difficult-to-treat diseases. Yet, these complex, large-molecule drugs pose significant challenges for developers and regulatory bodies alike.
An important issue for protein-based drug developers is that of structural characterization, which can help in understanding whether or not a drug product will be stable and of sufficient quality to be launched at all. “It is critically important that the complete and in-depth characterization of therapeutic proteins is performed throughout all stages of the drug discovery and development process,” says Simon Cubbon, senior global marketing manager, chromatography and mass spectrometry, Thermo Fisher Scientific. “Ultimately, this facilitates the transfer of appropriate knowledge throughout the pipeline, ensuring product consistency, safety, and efficacy.”
In agreement, Jeff Zonderman, chief commercial officer of RedShiftBio, highlights the concerns around how stress conditions, manufacturing processes, and storage may affect protein structure and adds that drug developers are limited by current analytical tools that do not offer sufficient insight. “This is especially the case with stability and aggregation, where earlier detection and monitoring in the development and manufacturing processes result in better, more effective drugs,” he notes. “Promising biological drug candidates, those that exhibit therapeutic activity and inherent stability, as assessed via simple screening techniques, become subject to increasing levels of structural elucidation as they progress through the pharmaceutical pipeline.”
Protein characterization, along with demonstrating a product’s consistency and reproducibility, are imperative for developers aiming for a quality product, states Brian R. Berquist, director of process development and technology transfer at iBio. “Obtaining information on your protein early on is a way to examine the quality and consistency throughout the drug development cycle,” he asserts. “Protein characterization is critical even in the earliest stages of process and drug development. The process development phase is when one can get a first glimpse at the potential product molecule and how the purification processes applied affect it.”
During early development stages, it is possible to make changes easily to address any potential purity issues. “In some instances, the product may be susceptible to proteolytic activity, either during expression or during purification, so being able to identify small changes in product mass and the amino acid location of that proteolytic activity allow one to address the issue in a logical manner early on instead of having a process where each step must be re-examined due to changes made,” Berquist says. “Additionally, as purification steps are developed, one is selecting based upon properties of the protein molecule itself. Sometimes a purification step or steps can enrich a certain proteoform, multimer, or glycoform that is not highly desirable. Again, it is easier early on to obtain this knowledge and change the purification accordingly.”
Going beyond the early stages of development, and as processes start to become more fixed in nature, Berquist explains that protein characterization can be used to examine the vigor of the purification process on the whole, as well as for each unit operation. “Protein characterization provides critical information regarding the reproducibility and robustness of each step in the process and the purification in its entirety,” he says. “When the process is locked, and manufacturing begins, it is essential to characterize each lot produced to ensure that a quality product is being generated routinely and consistently.”
“In today’s challenging pharmaceutical environment, research scientists are pushed to screen drug candidates as quickly as possible to increase chances of reaching clinical trials,” says Bill Barrett, product specialist at W.L. Gore’s PharmBIO Products business. “Using monoclonal antibodies as an example, speeding the purification process leads to more candidates and higher productivity.”
Adding to the requirements of reduced time and cost, and improved research productivity, Scott Peterman, senior global marketing manager, chromatography and mass spectrometry, Thermo Fisher Scientific, emphasizes that scientists are also now developing advanced protein drugs that are more extensively engineered and, as such, more complex. “As complexity increases, there are opportunities for greater levels of post-translational modifications and molecular heterogeneity,” he says. “Understanding the complexity of a protein-based therapeutic, and being able to control these modifications and variations, is becoming increasingly more difficult but remains critically important.”
Therefore, bio/pharma scientists are required to employ a cornucopia of various analytical technologies and strategies to adequately characterize protein-based therapeutics, continues Peterman. “Each analytical technology or strategy presents its own knowledge requirements and challenges, which scientists and vendors alike must address,” he notes.
Looking at analytical techniques in particular, Richard Moseley, chief technologist at Microsaic Systems, highlights that most can be categorized into two facets-either on-line with low specificity or off-line with high specificity-potentially challenging the ability to obtain answers fast. “In addition, the protein products are in complex chemical cell media making exact identification difficult. Therefore, some critical quality attributes are difficult to characterize and require complex workflows,” he says.
Furthermore, Moseley stresses that established analytical techniques suited to smallâmolecule drugs have been found to be unsuitable for bioprocessing. “So, biopharma scientists need to look to new techniques, specifically developed for their challenges and workflows to meet the complex needs of bioprocessing,” he asserts.
For Zonderman, challenges lie in the structure of drug studies, irrespective of dosage requirements. “Defining an optimal formulation and manufacturing route relies on assessing the impact of variables such as processing conditions including temperature and applied shear stress, and storage,” he says. “Stress-induced structural changes may have significant consequences including a loss of efficacy, and in the worst case present a safety risk, so demonstrating comparability (that successive stages of formulation, manufacture, and storage do not materially impact the structure of the drug) and stability up to the point of administration is essential.”
Aggregation-where proteins bind together and form undesirable impurities-can be detrimental to protein-based therapeutic development. These clusters of molecules can result in an incorrect drug dosage, or unwanted and even fatal immune responses to the drug, stresses Peterman.
“Consequently, monitoring protein aggregates is important for safety and quality assurance,” he adds. “Complete characterization and in-depth structural insights allow scientists to better understand what factors can lead to aggregation and undesirable outcomes, aiding clone selection and the delivery of a robust biotherapeutic that will not aggregate undesirably.”
This common indicator of protein instability, aggregation, can occur both upstream and downstream and can result in a product being deemed unfit for launch, continues Zonderman. “Characterization can help by giving insight into the onset of aggregation under certain conditions and help developers better formulate drugs to minimize aggregation or eliminate bad drug candidates earlier in the process,” he notes. “Being able to predict aggregation and resolve when you have true aggregation or self-association is critical.”
“Protein aggregation typically has been observed to be detrimental for both product activity and stability, as well as leading to the formation of higher levels of anti-product neutralizing antibodies in-vivo,” summarizes Berquist. “By applying rigorous size-exclusion chromatography (SEC) methodologies, we monitor even low levels of protein aggregation and use these data to optimize drug formulation to prevent aggregate formation.”
It is well-known that many branded protein-based biopharmaceutical products are facing patent expiration in the coming years, and so, the growth of the biosimilars market is an inevitability. As specified by regulatory bodies around the world, in some form or another, biosimilar products must be proven to be highly similar to its reference product with no clinically meaningful differences in terms of safety, purity, and potency.
“Determining similarity for biologics is much more challenging than with small molecules due to their larger size and greater structural complexity,” explains Zonderman. “Along with functional comparisons, measurement and analysis of the structural similarity between proteins are an effective method of demonstrating bioequivalence. I believe for biosimilar work it is important that testing is done at the same concentration and formulation conditions. Today, for complete structural analysis including secondary structure, current techniques are not sensitive to achieve this challenge.”
The primary goal of protein characterization for biosimilars is to provide sufficient evidence of similarity to the originator product, adds Berquist. “However, the problems with this are multifaceted,” he continues. “First, there are difficulties in obtaining sufficient supplies of the innovative drug product for comparison. Second, there is technological gap between original drug product characterization results and the instrumentation available for characterization of the biosimilar. The challenge is to prove that any observed differences are not significant and do not have clinical relevance.”
Concurring with Berquist, Cubbon further explains that biopharmaceutical companies exhaustively characterize therapeutics and associated manufacturing processes to improve the specificity of the patents, and these specifics may not be accessible to the biosimilar developers. “Consequently, the biosimilar developers are required to perform characterization to the same exhaustive levels,” he continues, “for example, to determine protein drug glycosylation, aggregation, and charge variant profile.”
As a result of this level of specificity that may be required when approaching biosimilar development, costs may not be reduced as significantly as is possible when approaching a small-molecule generic drug, for example. “The use of biosimilars, with such closely comparable performance to an original drug that can be used interchangeably, has the potential to drive down healthcare costs but is highly dependent on the rigorous demonstration of similarity in a wide range of attributes including protein content, activity, and stability,” asserts Zonderman.
Currently, there are many methods available that look at different attributes of the protein molecule, however, these methods are not without limitations. “Many of the techniques and tools available are inherited from pharmaceutical’s roots in smallâmolecule drugs, which are normally unsuited to the complex bioprocessing workflows and cell media used,” says Moseley.
Traditional assays, such as capillary electrophoresis (CE) or liquid chromatography coupled with ultraviolet (LC–UV), are robust and reliable techniques; however, they can only provide limited information on the critical quality attributes (CQAs) that need to be measured, stresses Cubbon. “This means that numerous assays are required to confidently cover these CQAs and ensure the correct information is obtained,” he adds.
According to Zonderman, there is a dependency on what technology is used and how it is applied, but in general, key limitations of current techniques include dynamic range, sensitivity, and automation (both analysis and data processing). “Many of the currently available technologies may meet some of the needs for research, but are a challenge for biopharma to move into more downstream, QA/QC [quality assurance/quality control], and process monitoring applications,” he notes.
The detailed definition of the structure of a drug molecule can provide a basis for scientists to identify structure-function relationships that enable an understanding of how a drug is efficacious, explains Zonderman, but beyond this the structural characterization of proteins plays a critical role through the drug development lifecycle. “In particular, investigating structural changes is the key to understanding and controlling the factors and mechanisms associated with stability and aggregation,” he says.
“Characterizing proteins during drug development is essential in reducing drug development times and manufacturing costs, and is critical for safety reasons,” agrees Moseley. “Furthermore, through protein characterization, industry is now capable of creating personalized medicine for patients.”
Yet, challenges and limitations remain in the ability to characterize these complex molecules sufficiently, and industry needs to be fully capable of managing the complexity of the information that is obtained through characterization, says Berquist. “As technology advances, industry will gain an ability to address increasingly intricate questions about protein products,” he summarizes. “Today, we have the capabilities to analyze intact macromolecules to the detail of detecting microheterogeneities leading to difficulties associated with data interpretation and refinement.”
Vol. 32, No. 8
When referring to this article, please cite it as F. Thomas, “Accepting the Challenge of Protein Characterization,” BioPharm International 32 (8) 2019.