Biopharma Analysis

Biotherapeutics, also known as biologics or biopharmaceuticals, are derived from biological sources, such as plants, microorganisms and genetically modified cells and organisms. They include high-molecular mass drugs comprising polymers of nucleotides, i.e., RNA or DNA, or amino acids (peptides and proteins).

Biotherapeutics based on nucleic acids, such as small interfering RNA (siRNA) and DNA, are gaining increasing attention due to their long-lasting and potential curative effects, yet few nucleic acid-based drugs are currently approved for therapeutic use. Peptides and proteins, on the other hand, represent a major class of biotherapeutics, owing to their versatile physiological functions. 

“Analytical techniques, for example chromatography and mass spectrometry, along with various tools such as cloning, CRISPR/Cas, single-use equipment, machine learning [and] use of advanced sensors and automation have helped in the advancement of biopharma research,” said Dr. Johannes Buyel, head of the department of Bioprocess Engineering at the Fraunhofer IME and associate professor at the RWTH Aachen University. Buyel’s work focuses on recombinant protein expression, overall bioprocess integration, the modeling of manufacturing processes and their digitalization.

Various biopharmaceutical analysis techniques are used to gain vital insights into the composition, quality, stability and safety of complex biotherapeutics throughout the development pipeline. In this article, we take a closer look at some of the strategies employed by scientists in the field.

Biotherapeutic discovery

Genome mining

Genome mining involves the identification of genes that are associated with novel biosynthetic pathways in microorganisms.  A common bottleneck in identifying such pathways is the re-discovery of compounds that have already been discovered previously. Gene cluster analysis, along with spectroscopic techniques, can accelerate the identification of unknown natural products and also define the stereochemistry of metabolites.

Examples of the platforms that aid in genome mining from a single genome to entire genera are BiG-SCAPE and CORASON. BiG-SCAPE provides sequence similarity analysis of biosynthetic gene clusters, whereas CORASON helps elucidate evolutionary relationships between gene clusters via the phylogenomic approach. CRISPR–Cas9 is an extremely important technique that can effectively activate biosynthetic gene clusters in multiple Streptomyces species. This enables the production of unique metabolites, e.g., novel polyketide in Streptomyces viridochromogenes. This technology leads to the production of rare and unknown variants of antibiotics, such as amicetin, thiolactomycin, phenanthroviridin and 5-chloro-3-formylindole.

METLIN is an important metabolite identification platform that includes a high-resolution MS/MS database with a fragment similarity search function. It has immensely helped in the rapid identification of unknown compounds. Other in silico tools used in biopharma research are Compound Structure Identification (CSI): FingerID and Input-Output Kernel Regression (IOKR). Scientists are developing advanced molecular network-based databases with taxonomic information from the bioactive compounds to improve the confidence of annotation.

Metabolomics-based methods

Recent advancements in analytical tools such as chromatography and spectrometry, coupled with computational approaches, have enabled the application of metabolomics in natural product-based drug discovery.

Metabolomics-based methods allow for the simultaneous analysis of many metabolites in biological samples, providing accurate information on the metabolite composition in crude extracts. This enables the rapid interpretation and identification of unknown compounds. Metabolomics-based methods can also detect differences between metabolite compositions across various physiological states of cells and organisms, generating detailed metabolite profiles to provide a phenotypic characterization at the molecular level. These profiles are important to understand the molecular mechanisms of action of bioactive compounds.

Analytical tools used in the biopharma pipeline

A wide range of analytical techniques are required during the development and manufacturing of biotherapeutics. These approaches are necessary for the continuous assessment of biotherapeutic products, helping to identify and characterize post-translational modifications (e.g., glycosylation), structural heterogeneity, stability, conformation, etc. Through such assessments, developers are able to evaluate and adjust the upstream and downstream bioprocesses involved in biomanufacturing and implement changes as required to ensure efficiency of the process and purity of the final product.

Bioactive compounds are analyzed using various methods, such as NMR spectroscopy and high-resolution mass spectrometry (HRMS). NMR analysis provides quantitative data and structural characterization of a product. However, it possesses a relatively low sensitivity and is, therefore, typically used to profile major constituents. Several chromatographic tools such as ion exchange, affinity and gel filtration chromatography are used to purify recombinant protein-based biopharmaceutical products.

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Mass spectrometry imaging in biopharma     

Mass spectrometry imaging (MSI) is used to determine the spatial distribution of bioactive compounds within living organisms. Different types of MSI, such as matrix-assisted laser desorption/ionization (MALDI), desorption electrospray ionization (DESI), nanoparticle laser desorption/ionization (nano-PALDI) and secondary ion mass spectrometry (SIMS) are used in drug discovery. These tools differ in terms of spatial resolution and molecular information. Studies related to compound distribution are particularly important for preclinical safety assessment, clinical studies and mechanistic understanding.

Theodore Alexandrov, team leader at the Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, is involved in the development of experimental and computational tools that are used in novel fields such as spatial and single-cell metabolomics. He said, “Over the past decade, MSI became the tool of choice for localizing drugs and drug metabolites in tissue sections.” According to Alexadrov, this is because MSI is faster and more sensitive than the original FDA-approved method for localizing drugs – whole body autoradiography (WBAR).

“MSI can detect drug metabolites that may be missed by WBAR, yet are bioactive," noted Alexandrov. He further explained that this analytical tool helps to speed up the preclinical and clinical stages of biotherapeutic development.

Liquid chromatography in biopharma

Liquid chromatography (LC) is a separation technique that is based on a liquid mobile phase, where molecules and ions present in the samples are dissolved. The liquid phase containing the dissolved sample is passed through a column packed with absorbent particles, known as the stationary phase. Separation is based on each component's affinity for the mobile phase – affinity impacts the speed at which the components migrate through the column. LC is coupled with various detectors such as fluorescence, ultraviolet-visible (UV-Vis) and light-scattering detectors for the characterization of biotherapeutics during development. It can be used to identify specific proteins and helps to determine their structure. Ion-exchange chromatography coupled with a UV detector can be used to determine protein–protein interactions in native state. For separation and qualitative and quantitative analysis of abundantly present isomers in crude extracts, scientists use combinational methods involving LC and HRMS.

Over the past decade, the combination of high performance liquid chromatography (HPLC) and mass spectrometry (MS) has proved to be an invaluable analytical tool for the discovery of biologics. These techniques are used to identify active compounds as well as to determine the purity of a drug substance. Also, liquid chromatography coupled with mass spectrometry (LC-MS), has exhibited a broad range of applicability to determine the purity of a drug. This technique is commonly used in the characterization of protein biopharmaceuticals and is applied for peptide mapping analysis.

LC-MS helps improve the confidence of the analysis and minimize the ambiguity related to data interpretation. Recently, scientists have been successful in improving the quality of data from such experiments by standardizing experimental procedures, optimizing instrument parameters and upgrading mass spectrometers. In the case of protein biotherapeutics, LC-MS is performed and is paired with ultraviolet (UV) detection tools to provide UV fingerprints, which could be used for quality control (QC) purposes and to study drug release.

Strategies to enhance the production of biopharmaceuticals

Traditionally, biotherapeutic research begins with the biological screening of "crude" extracts and this ultimately leads to the isolation of promising compounds with bioactivity. As this process is time-consuming and laborious, various strategies have now been employed by researchers to overcome these drawbacks. One approach has been the creation of libraries that are attuned with high-throughput screening. This process accelerates the process of finding a specific drug for a biological target (e.g., viral protein, enzyme, etc.,).

Optimization of production processes, i.e., from separation of a bioactive metabolite from crude extract to purification, is a complex task. To overcome various associated complexities, scientists miniaturized the process (e.g., miniaturized purification technology, miniaturized bioreactor, etc.) such that they can take a closer look at the entire process and make necessary modifications to develop optimized production methods.

Automated liquid handling technology aids effective and precise handling of liquid samples for biopharma companies. To accelerate the drug development process, crude extracts are pre-fractioned into sub-fractions such that they are suitable for automated liquid handling technology. This technique increases the possibility of obtaining a larger amount of targeted bioactive compound, while minimizing the time required.

Current challenges and future research in biopharma analysis

Generally, drug development is an extremely complex and expensive process. According to the Tufts Center for the Study of Drug Development, the development of a drug requires almost 12–15 years of extensive research and the cost of production may exceed $2 billion. One of the objectives for the future is to develop methods that reduce this time-frame and associated costs.

However, challenges in biopharmaceutical analysis remain. Recombinant proteins are developed via genetically engineered cells and maintaining the optimal growing conditions for these cells is important for obtaining high-quality products. Also, industrial-scale production of recombinant proteins is expensive. Another challenge associated with biopharmaceuticals is denaturation and aggregation when the biomolecules are exposed to multiple stresses during production, storage and transportation. Degraded or aggregated protein components may lose their efficacy and may also become toxic. These changes in the protein structure could be extremely small and the available analytical tools might not be sensitive enough to detect them. Therefore, there is a need for the continuous development of high-precision analytical tools that ensure superior quality and reduce the cost of production.