A Review of the Latest Spectroscopic Research in Pharmaceutical and Biopharmaceutical Applications

A close up of a lab technician monitoring biopharmaceutical production © Cheetose - stock.adobe.com

Spectroscopic analytical techniques play an essential role in the pharmaceutical and biopharmaceutical industries, providing tools for detailed classification and quantification of processes and finished products. Among the tools used are inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES) for trace elemental analysis, offering high sensitivity and precision. Raman spectroscopy, including its enhanced variants surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS), which is used for molecular imaging, fingerprinting, and detecting low concentrations of substances. Fourier-transform infrared spectroscopy (FT-IR) is applied for identifying chemical bonds and functional groups within molecules. Powder X-ray diffraction (PXRD), technically a diffraction method, is a technique used to assess the crystalline identity of active drug compounds. Ultraviolet-visible spectroscopy (UV-vis) measures absorbance and concentration of analytes, while fluorescence spectroscopy detects the emission of light by substances, often used for tracking molecular interactions, kinetics, and dynamics. Nuclear magnetic resonance (NMR) spectroscopy provides detailed information about the molecular structure and conformational subtleties through the interaction of nuclear spin properties following the application of an external magnetic field. This brief review highlights the most recent advancements and applications of these techniques in the pharmaceutical and biopharmaceutical fields.

ICP-MS
Transition metals can be introduced in therapeutic protein drugs during manufacturing, potentially affecting efficacy, safety, and stability. Understanding protein-metal interactions is crucial. A new strategy using size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS) differentiates between ultra-trace levels of metals (cobalt, chromium, copper, iron, nickel) interacting with proteins and free metals in solution. In a recent study, monoclonal antibodies were coformulated, stored, and analyzed to measure metal-protein interactions (1 . SEC-ICP-MS offers valuable insights for measuring these interactions during drug development.

The metal content in Chinese hamster ovary (CHO) cell culture media (CCM) significantly impacts process productivity and critical quality attributes (CQAs). Metals in CCM can change forms during the monoclonal antibody (mAb) production cycle, affecting uptake and metabolism. To address this, a methodology using high-performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) was developed to speciate and quantify five target metals (Mn, Fe, Co, Cu, Zn) in CCM (2). This method identifies metal speciation and concentration deviations, aiding in quality control, contaminant identification, and assessment of media stability and cell metal uptake.

ICP-OES
Liquid crystalline nanoparticles (LCNPs) are promising in cancer nanomedicines due to its high surface area, stability, and sustained-release profile. Recent work has resulted in a novel LCNP co-encapsulating Bi2O3 and hydrophilic doxorubicin (DOX), functionalized with folic acid (FA) for targeted CT-scan imaging and chemotherapy of melanoma. In this study LCNPs were prepared using glycerol monooleate-pluronic F-127 (GMO/PF127/water) and exhibited superior stability and encapsulation efficiency. FA-Bi2O3-DOX-NPs showed high cytotoxicity and cellular internalization in folate-receptor overexpressing cells, and effective tumor suppression with increased survival in tumorized mice (3). The targeted theranostic FA-Bi2O3-DOX-NPs demonstrated significant potential for melanoma diagnostics and treatment.

Intravenous iron-carbohydrate complexes are nanomedicines used to treat iron deficiency and anemia. Understanding pharmacokinetics is challenging due to difficulties in measuring intact iron nanoparticles versus endogenous iron, incomplete knowledge of iron metabolism, and significant interpatient variability in parameters like ferritin. Additionally, the absence of traditional receptor/enzyme interactions complicates modeling. A recently published review paper examines the known parameters of bioavailability, distribution, metabolism, and excretion for these nanomedicines and discusses future challenges that hinder the direct application of physiologically-based pharmacokinetic or other computational modeling techniques (4).

Raman (general)
Process analytical technologies are critical for advancing biopharmaceutical manufacturing by addressing clinical, regulatory, and cost challenges. Raman spectroscopy, a key technology for inline product quality monitoring, faces limitations due to laborious calibration and computational chemometric modeling. A paper published in 2023 showcases real-time measurement of product aggregation and fragmentation during clinical bioprocessing using hardware automation and machine learning (5). By integrating existing workflows into a robotic system, calibration and validation efforts were reduced, and increased data throughput allowed for accurate product quality measurements every 38 seconds. Raman in-process analytics enhance process understanding and ensure consistent product quality through controlled bioprocesses (5).

A 2024 published paper applying Raman spectroscopy has aimed at optimizing cell culture processes by employing inline Raman technology for real-time monitoring (6). By introducing a method to identify and eliminate anomalous spectra, the research established Raman-based models for 27 components crucial in cell culture (6). The derived models exhibited high accuracy, with Q2 values (predictive R-squared values) surpassing 0.8 and relative percent differences (between reference values and estimated values) above 2.0, except for glucose. Furthermore, the study demonstrated the effectiveness of control charts in detecting normal and abnormal conditions like bacterial contamination. In instances of unexplained anomalies, the proposed workflow has provided valuable insights for this type of application.

Raman (SERS and TERS)
A 2023 review article has addressed key challenges in bioprocessing, which include cost-effectiveness and process understanding. Online data access aids in comprehending process dynamics and monitoring critical parameters, aligning with quality-by-design principles. Raman spectroscopy offers noninvasive, versatile analysis, facilitating enhanced control strategies. The article highlights Raman's recent applications of its variant techniques, such as SERS, in protein production and its potential in virus, cell therapy, and mRNA processes, underscoring its relevance in advancing bioprocessing techniques (7).

Protein unfolding and aggregation are linked to diseases like Alzheimer's and Parkinson's, posing challenges for understanding and monitoring under physiological conditions. Raman spectroscopy and its plasmon-enhanced variants (SERS and TERS) offer nondestructive, real-time analysis of protein dynamics and aggregation mechanisms (8). These techniques provide insights into molecular events with potential applications in diverse fields, including biopharmaceuticals and point-of-care devices. Raman, SERS, and TERS enable precise chemical analysis of protein aggregation from various sources, promising advancements in disease research and practical applications.

FT-IR
Stability testing of pharmaceuticals, especially protein drugs, is intricate and time-consuming. Utilizing Fourier-transform infrared spectroscopy (FT-IR) has allowed weekly samples of three protein drugs stored under varying conditions to be routinely analyzed. Employing hierarchical cluster analysis (HCA) in the Python programming language, researchers assessed the similarity of secondary protein structures (9). Results showed that stability was maintained across temperature conditions, with closer similarity among samples than anticipated. This suggests FT-IR coupled with HCA could be a valuable tool for future drug stability studies, offering a more nuanced understanding of drug behavior.

PXRD
A 2023 study aimed to enhance the drug norfloxacin's biopharmaceutical properties by synthesizing co-crystals with water-insoluble antibacterial antibiotics. Three co-crystals were formed using nicotinamide, cinnamic acid, and sorbic acid as co-formers via liquid-assisted grinding. Characterization via various analytical techniques confirmed crystalline identity, with crystal structures determined using Material Studio Software. Co-crystals exhibited sustained structures through hydrogen bond networks. Physicochemical properties, pharmacokinetics, and antimicrobial activity were assessed, revealing significant improvements in solubility (8 to 3-fold), dissolution (6 to 2-fold), and peak plasma concentration (2 to 1.5-fold higher) compared to norfloxacin alone (10).

UV-vis
A porous agarose bead matrix compatible with UV–vis imaging was developed for mimicking transport within human tissue, like subcutaneous tissue. Introducing high-molecular-weight dextrans enhanced optical clearing, improving transmittance and resolution. Real-time UV–vis imaging illustrated diffusive and convective transport dynamics. Incorporating ion-exchange resins and revealing their impact on biotherapeutic retention. This imaging technique could assess electrostatic interactions of injected biotherapeutics, with potential applications to predict size-exclusion chromatography (SEC) characterization of injectables (11).

Research from 2023 had a goal to enhance biomanufacturing using Process Analytical Technology (PAT) by optimizing Protein A affinity chromatography for monoclonal antibody (mAb) purification and minimizing host cell proteins (HCPs). Utilizing inline UV-viis monitoring at 280 nm (for mAb) and 410 nm (for HCPs), separation conditions were optimized. Results showed optimal conditions: 12 CV loading, pH 3.5 elution, and starting collection at 0.5 CV, achieving 95.92% mAb recovery and 49.98% HCP removal compared to the whole elution pool. This study underscores the potential of UV-vis-based inline monitoring for real-time control and optimization of Protein A affinity chromatography in mAb purification (12).

Fluorescence
Conventional quality control methods for biopharmaceuticals involve destructive lab-based techniques like gel electrophoresis and chromatography, compromising sterility and product integrity. A recent study explored non-invasive in-vial fluorescence analysis to monitor heat- and surfactant-induced denaturation of bovine serum albumin (BSA), eliminating the need for sample removal. A bespoke setup measures fluorescence polarization to assess denaturation, validated against circular dichroism and size-exclusion chromatography (SEC). In-vial fluorescence proves effective for monitoring protein stability. This method offers a cost-effective, portable solution for assessing biopharmaceutical stability from production to patient administration, promising streamlined quality control processes (13).

One study introduced a rapid screening method for high-producing bacterial strains in microbial protein expression. Mutagen-induced genotype variants were encapsulated in microemulsions and cultured to secrete proteins, detected by a fluorescent immunosensor (Q-body). In this method a cell sorter selects strains based on fluorescence intensity. Corynebacterium glutamicum secretes proteins in emulsions, demonstrating the concept. Screening productive strains of fibroblast growth factor 9 (FGF9) yields a threefold increase in secretion. This simple method, requiring only Q-body addition, accelerates industrial strain development for biopharmaceutical production, promising wide applicability and shorter development periods (14).

NMR
One published review underscores the importance of high-resolution nuclear magnetic resonance (NMR) spectroscopy in biologics formulation development, addressing the need for advanced analytical techniques in the biopharmaceutical industry. Protein conformational changes during formulation can affect stability, necessitating precise characterization of protein-protein and protein-excipient interactions. Solution NMR emerges as a potent tool, with 1D NMR monitoring monoclonal antibody (mAb) structural changes and interactions, while 2D NMR, such as the XL-ALSOFAST-[1H–13C]-heteronuclear multiple-quantum correlation (HMQC) method, detects higher-order structural changes and interactions. However, studying proteins in formulations, where they are at low concentrations amidst high concentrations of excipients, poses challenges in signal detection and experimental time. Innovative NMR approaches, like XL-ALSOFAST, tackle these challenges, enhancing sensitivity and selectivity. The review discusses the utility of XL-ALSOFAST in analyzing investigational protein formulations, advocating for NMR's expanded role in biologics formulation development (13).

Monoclonal antibodies (mAbs) are crucial biotherapeutic drugs, yet robust characterization of their higher-order structure (HOS) remains challenging, particularly in solid preparations. Solution-state nuclear magnetic resonance (NMR) spectroscopy has gained attention for aqueous-based mAb characterization. However, solid-state NMR (ssNMR) fingerprinting offers a novel approach to directly assess HOS in solid mAb formulations. Using lyophilized NISTmAb samples with varied formulations, 1H–13C cross-polarization (hC-CP) buildup spectra at natural isotopic abundance reveal formulation differences. Principal component analysis (PCA) aids in user-independent differentiation of samples, while expert analysis elucidates structural insights. This study establishes ssNMR as a valuable tool for solid-phase mAb HOS characterization, advancing understanding and quality assessment of solid-state biotherapeutics.

Summary
This brief review highlights numerous applications in pharmaceutical and biopharmaceutical research, such as analyzing trace elements in therapeutic proteins and cell culture media to improve drug development and production quality. It discusses advancements in cancer treatment through targeted chemotherapy and imaging, optimizing bioprocessing by monitoring product quality and aggregation, and enhancing drug stability and solubility. Other applications include evaluating crystallinity of drug products, developing new imaging techniques to mimic human tissue transport, optimizing protein purification processes, and improving quality control methods for biopharmaceuticals. Additionally, the review emphasizes the importance of understanding protein dynamics, aggregation mechanisms, and higher-order structures to advance drug formulation and characterization.

References

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