Advances in Biopharmaceutical Analysis
Biopharmaceuticals are highly vulnerable to degradation and contamination, so careful analysis is required to ensure their safety.
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Biopharmaceuticals – also known as biological therapies or biologics – are usually derived from some form of living system, be it cell culture, animal products or human cell or tissue samples. They are complex and expensive to produce and highly vulnerable to degradation and contamination, requiring careful analysis during research, development and manufacturing stages to ensure quality and integrity.
In this article, we highlight some of the analytical techniques employed in biopharmaceutical analysis, and how the field is evolving to meet new challenges.
Biopharmaceutical characterization and quality
The field of biopharmaceuticals not only includes antibody-based drugs, but also the expanding fields of cell and gene therapies. Across the spectrum of biologics, the most common analytical technologies for characterizing biopharmaceuticals are mass spectrometry (MS) and liquid chromatography (LC), either alone or in tandem.
“Some of the most important properties you’re testing for with biologics are post-translational modifications (PTMs) and protein aggregation, said Jared Auclair, director of bioinnovation at the Biopharmaceutical Analysis and Training Laboratory (BATL), Northeastern University, USA. “For PTMs, you might use reverse-phase LC in tandem with MS, whereas size exclusion chromatography might be used to detect aggregation in the development of antibody-based drugs.”
However, although there exists an entire toolbox of methods required to characterize protein and antibody-based products, method development for cell and gene therapy is lagging, says Auclair. “I liken it to different stages of construction of a house: for antibodies, the house is built, albeit not 100% complete, and you can live in it. For gene therapies, we have a basic structure, but for cell therapies, we’ve only just got the building blocks. Cell-based therapies are about 5–10 years behind where antibody-based biologics are in terms of analytics.”
What’s universal to all biopharmaceuticals, though, is that whichever characterization method is used, it needs to be carried through the entire development process from research to commercialization. “This doesn’t only include collecting the same data, if you’re going to monitor PTMs, for example, you should be using as close to the same instrumentation as possible,” said Auclair. “And if the quality of the product is paramount to its efficacy, then continued monitoring is key and we need to be looking at opportunities to monitor in real-time, so we can kill products that are below quality before they advance too far towards commercialization.”
Process innovation for biopharmaceuticals
While developing and improving methods to monitor impurities is a major focus of the research at BATL, it sits alongside a focus on process analytics. Here, method development goes hand-in-hand with engineering to determine the pain points of biopharmaceutical production processes and find solutions, such as new in-line sensors that can monitor different aspects of the process. But there are challenges.
“One of the major challenges is integration, especially where different equipment manufacturers and software providers are involved. The technologies are there, but it’s just a matter of implementation and automation,” said Auclair. “In our lab, we have instrumentation that pulls samples from the bioreactor so we can test different quality checks and try to make that process better and more robust.”
Another headache is data analysis. “The hardware is always ahead of the data analysis tools, in terms of capability, speed and ease of use,” says Auclair. “In the research space, it’s OK to have a really complex process, but as you move down the commercialization space, you need technologies and processes that don’t require 30 years’ experience in informatics to run.”
One area that is gaining momentum is the multi-attribute method (MAM), an MS-based protocol that monitors several properties, such as PTMs, sequence coverage and new peak detection, in the same experiment.1 Auclair’s team is looking at how they can optimize MAM as development processes change, how to integrate it in-line with bioreactors and how to apply the approach to gene and cell therapies.
“In drug development, especially academic drug development, many people don’t think about product quality from the beginning. And if you don’t think about that from the beginning then your product doesn’t get to market, it dies in the valley of death,“ says Auclair. “The ultimate goal of our work is to innovate these product development and commercialization processes, so that more good medicines are discovered and don’t fail because the quality of the product wasn’t considered throughout the process.”
Microbial monitoring in biopharmaceutical development
Another major area of biopharmaceutical analysis is monitoring for contamination by microbes and viruses. It is essential to ensure that biopharmaceuticals are not contaminated with pathogenic or environmental bacteria, fungi or viruses, which, if introduced even in small amounts, can potentially multiply to high levels in the bulk material during manufacturing or in the final drug product.
Pathogens can potentially be introduced via cell lines, raw materials, use of contaminated human or animal products during manufacture or from the environment – such as human contact during sampling.2 Many recombinant protein therapeutics, vaccines and plasma products are produced using cell culture, which is susceptible to contamination. Although a rare occurrence, this can reduce the yield of the drug, affect the quality of the end product and can have considerable financial costs for industry and potentially serious safety implications for patients.3
“As the manufacture of biomedicines is a complex, multi-step process, typically involving potentially non-sterile biological ingredients, a sound microbial and viral safety strategy is required,” said Dr Oleg Krut, head of the microbial safety section at the Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Germany. “The use of sensitive methods to detect bacteria or fungi themselves is an integral part of this strategy. All ingredients must be tested using the most sensitive assays to ensure that any microbes and viruses are detected before use and that contaminated material will not be used during manufacture.”
Bacteria and fungi are usually detected by growth-based methods, that is, detection of turbidity in liquid microbial culture or the enumeration of colonies on agar plates. “The main methodological challenges are the time required to obtain a valid result (up to 14 days), and the high sensitivity required to detect a single microorganism,” explains Krut. “A further challenge is that introducing more advanced microbiological techniques that maintain the highest sensitivity with shorter time-to-result is slow, because of the necessary validation requirements.”
Viruses have traditionally been detected by inoculating samples into animals or indicator cell cultures that are monitored for signs of infection.2,3 However, only a limited range of viruses can be detected through animal testing.3
“Replacement of animal testing is being considered through the revision of regulatory guidelines such as ICH Guideline Q5A(R2) on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin, in line with the 3R principles for animal welfare,” said Dr. Johannes Blümel, head of the viral safety section, at the Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Germany. “The use of indicator cells is still a very valuable method for testing cell cultures for viral contamination as it has an excellent sensitivity for many viruses. However, the range of viruses that can be detected is still limited by the ability of the indicator cells to support viral replication and to show signs of infection that are easily visible under a light microscope.” Virus testing using indicator cells is complemented by nucleic acid amplification technologies such as polymerase chain reaction (PCR). However, viral variants or newly emerging viruses may pose a threat.
“Viruses can be specifically detected by very sensitive methods if their genomes or host cells are known,” said Blümel. “However, although PCR has excellent sensitivity, the technology remains limited towards detection of specific viruses. A battery of several different PCR tests therefore needs to be designed according to a risk assessment of the virus species that could contaminate.”
Advances in microbial monitoring methods
There have been two major trends in microbial safety in recent years, says Krut. “The first is the automation of classical cell culture methods, such as automated liquid culture and automated colony counting. These improvements shorten the time to results, reduce hands-on time and operator bias and increase throughput and data integrity. As they are modifications of classical methods, they are easier to validate.”
The second trend is the emergence of rapid microbiological methods (RMMs), such as nucleic acid-based techniques, adenosine triphosphate (ATP)-based contamination detection, solid-state cytometry and MS. “Unlike classical microbial culture, which takes two weeks, RMMs can detect microbial contamination within days or even hours,” says Krut. “Such methods are needed for biomedicines with short shelf-lives where classical methods are simply too slow. However, they require extensive validation, which is often seen as a major barrier to their implementation in biopharmaceutical production.”
Dr Krut’s team is evaluating culture-independent flow or solid-state cytometry for bacterial detection and enumeration, which it is hoped will allow faster time to results, more rapid Official Medicines Control Laboratories (OMCLs) provision of certificates and earlier governmental batch release of biomedicine batches to the market.
In viral safety, using next-generation sequencing (NGS) or high-throughput sequencing (HTS) in a metagenomic approach (i.e. detecting all viral sequences in a sample) has the potential to overcome the limitations of current PCR-based assays.4 Research at the Paul-Ehrlich-Institut is focused on optimizing HTS technology for the sensitive detection of human viruses in blood samples.
“Our multidisciplinary team of medical virologists, molecular biologists and bioinformaticians is particularly interested in reducing host cell background and optimizing library construction in order to increase the sensitivity of the method, as well as in applying artificial intelligence and machine learning methods to advance the bioinformatics pipeline further,” says Blümel. “Our method allows us to address any type of emerging agent, using technology that meets regulatory requirements, and will ensure we keep pace with emerging viruses and are prepared.”
1. Millán-Martín S, Jakes C, Carillo S, et al. Multi-attribute method (MAM): An emerging analytical workflow for biopharmaceutical characterization, batch release and cGMP purity testing at the peptide and intact protein level. Crit Rev Anal Chem. 2023;1-18. doi: 10.1080/10408347.2023.2238058
2. Valiant WG, Cai K, Vallone PM. A history of adventitious agent contamination and the current methods to detect and remove them from pharmaceutical products. Biologicals. 2022;80:6-17. doi: 10.1016/j.biologicals.2022.10.002
3. Barone PW, Wiebe ME, Leung JC, et al. Viral contamination in biologic manufacture and implications for emerging therapies. Nat Biotechnol. 2020;38(5):563-572. doi: 10.1038/s41587-020-0507-2
4. Khan AS, Mallet L, Blümel J, et al. Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals [published online ahead of print, 2023 Jul 19]. Biologicals. 2023;83:101696. doi: 10.1016/j.biologicals.2023.101696