Applications of the Methylotrophic Yeast Komagataella phaffii in the Context of Modern Biotechnology.

Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.

Highly selective chemical modification of poly-His tagged peptides and proteins.

Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His6 (Gly-His tag) or the sequence Hism-Lys-Hisn (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal Nα-amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys Nε-amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody.

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells.

Cell engineering strategies typically rely on energy-consuming overexpression of genes or radical gene-knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine-tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters.

Expediting online liquid chromatography for real-time monitoring of product attributes to advance process analytical technology in downstream processing of biopharmaceuticals.

The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

Microfluidic Cartridge for Bead-Based Affinity Assays.

Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.

Repurposing of biologics and biopharmaceuticals.

The field of drug repurposing is gaining attention as a way to introduce pharmaceutical agents with established safety profiles to new patient populations. This approach involves finding new applications for existing drugs through observations or deliberate efforts to understand their mechanisms of action. Recent advancements in bioinformatics and pharmacology, along with the availability of extensive data repositories and analytical techniques, have fueled the demand for novel methodologies in pharmaceutical research and development. To facilitate systematic drug repurposing, various computational methodologies have emerged, combining experimental techniques and in silico approaches. These methods have revolutionized the field of drug discovery by enabling the efficient repurposing of screens. However, establishing an ideal drug repurposing pipeline requires the integration of molecular data accessibility, analytical proficiency, experimental design expertise, and a comprehensive understanding of clinical development processes. This chapter explores the key methodologies used in systematic drug repurposing and discusses the stakeholders involved in this field. It emphasizes the importance of strategic alliances to enhance the success of repurposing existing compounds for new indications. Additionally, the chapter highlights the current benefits, considerations, and challenges faced in the repurposing process, which is pursued by both biotechnology and pharmaceutical companies. Overall, drug repurposing holds great promise in expanding the use of existing drugs and bringing them to new patient populations. With the advancements in computational methodologies and the collaboration of various stakeholders, this approach has the potential to accelerate drug development and improve patient outcomes.

Is Lyophilization Key for Transportation and Storage of Biopharmaceutical Drug Products in Space?

Long-lasting space missions as well as space tourism are technically possible today and economically in reach. It is a matter of time until the use of biopharmaceutical drug products in space will be common practice. Until drug product manufacturing in space is possible, the products need to be brought to space with rockets, which means that stable and light-weight products are preferred. Lyophilization is a promising approach to reduce weight during transportation and achieve storage stability at room temperature without cold-chain demands. This implies that recycled water in space needs to be used for reconstitution which poses a microbiological challenge and should be considered during formulation development. Furthermore, administration of the injectable drugs in space has an impact on the chosen packaging material which needs to be considered during drug product development.

The use of proteins and peptides-based therapy in managing and preventing pathogenic viruses.

Therapeutic proteins have been employed for centuries and reached approximately 50 % of all drugs investigated. By 2023, they represented one of the top 10 largest-selling pharma products ($387.03 billion) and are anticipated to reach around $653.35 billion by 2030. Growth hormones, insulin, and interferon (IFN α, γ, and ß) are among the leading applied therapeutic proteins with a higher market share. Protein-based therapies have opened new opportunities to control various diseases, including metabolic disorders, tumors, and viral outbreaks. Advanced recombinant DNA biotechnology has offered the production of therapeutic proteins and peptides for vaccination, drugs, and diagnostic tools. Prokaryotic and eukaryotic expression host systems, including bacterial, fungal, animal, mammalian, and plant cells usually applied for recombinant therapeutic proteins large-scale production. However, several limitations face therapeutic protein production and applications at the commercial level, including immunogenicity, integrity concerns, protein stability, and protein degradation under different circumstances. In this regard, protein-engineering strategies such as PEGylation, glycol-engineering, Fc-fusion, albumin conjugation, and fusion, assist in increasing targeting, product purity, production yield, functionality, and the half-life of therapeutic protein circulation. Therefore, a comprehensive insight into therapeutic protein research and findings pave the way for their successful implementation, which will be discussed in the current review.

Novel EDTA mediated ethanol protein precipitation method and the application for polysorbate quantification in high protein concentration biopharmaceuticals.

Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10-250 mg/mL).

Development of a high throughput oxidation profiling strategy for monoclonal antibody products.

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

Bioanalytical technologies using temperature-responsive polymers.

In recent decades, various bioanalytical technologies have been investigated for appropriate medical treatment and effective therapy. Temperature-responsive chromatography is a promising bioanalytical technology owing to its functional properties. Temperature-responsive chromatography uses a poly(N-isopropylacrylamide)(PNIPAAm) modified stationary phase as the column packing material. The hydrophobic interactions between PNIPAAm and the analyte could be modulated by changing the column temperature because of the temperature-responsive hydrophobicity of PNIPAAm. Thus, the chromatography system does not require organic solvents in the mobile phase, making it suitable for therapeutic drug monitoring in medical settings such as hospitals. This review summarizes recent developments in temperature-responsive chromatography systems for therapeutic drug monitoring applications. In addition, separation methods for antibody drugs using PNIPAAm are also summarized because these methods apply to the therapeutic drug monitoring of biopharmaceutics. The temperature-responsive chromatography systems can also be utilized for clinical diagnosis, as they can assess multiple medicines simultaneously. This highlights the significant potential of temperature-responsive chromatography in medicine and healthcare.

Impact of Glycosylation on Protein-Protein Self-Interactions of Monoclonal Antibodies.

Protein self-interactions measured via second osmotic virial coefficients (B22) and dynamic light scattering interaction parameter values (kD) are often used as metrics for assessing the favorability of protein candidates and different formulations during monoclonal antibody (MAb) product development. Model predictions of B22 or kD typically do not account for glycans, though glycosylation can potentially impact experimental MAb self-interactions. To the best of our knowledge, the impact of MAb glycosylation on the experimentally measured B22 and kD values has not yet been reported. B22 and kD values of two fully deglycosylated MAbs and their native (i.e., fully glycosylated) counterparts were measured by light scattering over a range of pH and ionic strength conditions. Significant differences between B22 and kD of the native and deglycosylated forms were observed at a range of low to high ionic strengths used to modulate the effect of electrostatic contributions. Differences were most pronounced at low ionic strength, indicating that electrostatic interactions are a contributing factor. Though B22 and kD values were statistically equivalent at high ionic strengths where electrostatics were fully screened, we observed protein-dependent qualitative differences, which indicate that steric interactions may also play a role in the observed B22 and kD differences. A domain-level coarse-grained molecular model accounting for charge differences was considered to potentially provide additional insight but was not fully predictive of the behavior across all of the solution conditions investigated. This highlights that both the level of modeling and lack of inclusion of glycans may limit existing models in making quantitatively accurate predictions of self-interactions.

Computer-assisted multifactorial method development for the streamlined separation and analysis of multicomponent mixtures in (Bio)pharmaceutical settings.

The (bio)pharmaceutical industry is rapidly moving towards complex drug modalities that require a commensurate level of analytical enabling technologies that can be deployed at a fast pace. Unsystematic method development and unnecessary manual intervention remain a major barrier towards a more efficient deployment of meaningful analytical assay across emerging modalities. Digitalization and automation are key to streamline method development and enable rapid assay deployment. This review discusses the use of computer-assisted multifactorial chromatographic method development strategies for fast-paced downstream characterization and purification of biopharmaceuticals. Various chromatographic techniques such as reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and supercritical fluid chromatography (SFC) are addressed and critically reviewed. The most significant parameters for retention mechanism modelling, as well as mapping the separation landscape for optimal chromatographic selectivity and resolution are also discussed. Furthermore, several computer-assisted approaches for optimization and development of chromatographic methods of therapeutics, including linear, nonlinear, and multifactorial modelling are outlined. Finally, the potential of the chromatographic modelling and computer-assisted optimization strategies are also illustrated, highlighting substantial productivity improvements, and cost savings while accelerating method development, deployment and transfer processes for therapeutic analysis in industrial settings.

Engineering mammalian cell growth dynamics for biomanufacturing.

Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a "gas pedal", based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a "brake pedal". A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the "gas and brake pedals", enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications.

Ionic liquids and deep eutectic solvents for the stabilization of biopharmaceuticals: A review.

Biopharmaceuticals have allowed the control of previously untreatable diseases. However, their low solubility and stability still hinder their application, transport, and storage. Hence, researchers have applied different compounds to preserve and enhance the delivery of biopharmaceuticals, such as ionic liquids (ILs) and deep eutectic solvents (DESs). Although the biopharmaceutical industry can employ various substances for enhancing formulations, their effect will change depending on the properties of the target biomolecule and environmental conditions. Hence, this review organized the current state-of-the-art on the application of ILs and DESs to stabilize biopharmaceuticals, considering the properties of the biomolecules, ILs, and DESs classes, concentration range, types of stability, and effect. We also provided a critical discussion regarding the potential utilization of ILs and DESs in pharmaceutical formulations, considering the restrictions in this field, as well as the advantages and drawbacks of these substances for medical applications. Overall, the most applied IL and DES classes for stabilizing biopharmaceuticals were cholinium-, imidazolium-, and ammonium-based, with cholinium ILs also employed to improve their delivery. Interestingly, dilute and concentrated ILs and DESs solutions presented similar results regarding the stabilization of biopharmaceuticals. With additional investigation, ILs and DESs have the potential to overcome current challenges in biopharmaceutical formulation.

Molecular Dynamics Simulations for Rationalizing Polymer Bioconjugation Strategies: Challenges, Recent Developments, and Future Opportunities.

The covalent modification of proteins with polymers is a well-established method for improving the pharmacokinetic properties of therapeutically valuable biologics. The conjugated polymer chains of the resulting hybrid represent highly flexible macromolecular structures. As the dynamics of such systems remain rather elusive for established experimental techniques from the field of protein structure elucidation, molecular dynamics simulations have proven as a valuable tool for studying such conjugates at an atomistic level, thereby complementing experimental studies. With a focus on new developments, this review aims to provide researchers from the polymer bioconjugation field with a concise and up to date overview of such approaches. After introducing basic principles of molecular dynamics simulations, as well as methods for and potential pitfalls in modeling bioconjugates, the review illustrates how these computational techniques have contributed to the understanding of bioconjugates and bioconjugation strategies in the recent past and how they may lead to a more rational design of novel bioconjugates in the future.

Capillary electrophoresis in the analysis of therapeutic peptides-A review.

Therapeutic peptides are a growing class of innovative drugs with high efficiency and a low risk of adverse effects. These biomolecules fall within the molecular mass range between that of small molecules and proteins. However, their inherent instability and potential for degradation underscore the importance of reliable and effective analytical methods for pharmaceutical quality control, therapeutic drug monitoring, and compliance testing. Liquid chromatography-mass spectrometry (LC-MS) has long time been the "gold standard" conventional method for peptide analysis, but capillary electrophoresis (CE) is increasingly being recognized as a complementary and, in some cases, superior, highly efficient, green, and cost-effective alternative technique. CE can separate peptides composed of different amino acids owing to differences in their net charge and size, determining their migration behavior in an electric field. This review provides a comprehensive overview of therapeutic peptides that have been used in the clinical environment for the last 25 years. It describes the properties, classification, current trends in development, and clinical use of therapeutic peptides. From the analytical point of view, it discusses the challenges associated with the analysis of therapeutic peptides in pharmaceutical and biological matrices, as well as the evaluation of CE as a whole and the comparison with LC methods. The article also highlights the use of microchip electrophoresis, nonaqueous CE, and nonconventional hydrodynamically closed CE systems and their applications. Overall, the article emphasizes the importance of developing new CE-based analytical methods to ensure the high quality, safety, and efficacy of therapeutic peptides in clinical practice.

Prediction of CHO cell line stability using expression of DNA repair genes.

Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long-standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a, Lig4, Palb2, Pari, and Xrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO-DG44 cell lines. Notably, two genes, Lig4 and Xrcc6, showed higher expression in unstable CHO-DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long-term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

Automated method for quantification of 20 amino acids in cell culture media during biopharmaceutical development.

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present in cell culture media is described. The protocol facilitates detection of both primary and secondary amino acids through a two-step precolumn derivatization strategy using ortho-phthalaldehyde and 9-fluorenylmethyl chloroformate as derivatizing agents. The separation of derivatized amino acids with varying hydrophobicity is achieved through reverse-phase chromatography. The amino acids are simultaneously detected in a single workflow through the use of Variable Wavelength Detector at 338 and 262 nm. The protocol is applicable for both mammalian and bacterial cell culture matrices with an option for automation of precolumn derivatization.