Development Of Therapeutic Proteins Research Articles

Half-life extension via ABD-fusion leads to higher tumor uptake of an affibody-drug conjugate compared to PAS- and XTENylation.

A critical parameter during the development of protein therapeutics is to endow them with suitable pharmacokinetic and pharmacodynamic properties. Small protein drugs are quickly eliminated by kidney filtration, and in vivo half-life extension is therefore often desired. Here, different half-life extension technologies were studied where PAS polypeptides (PAS300, PAS600), XTEN polypeptides (XTEN288, XTEN576), and an albumin binding domain (ABD) were compared for half-life extension of an anti-human epidermal growth factor receptor 2 (HER2) affibody-drug conjugate. The results showed that extension with the PAS or XTEN polypeptides or the addition of the ABD lowered the affinity for HER2 to some extent but did not negatively affect the cytotoxic potential. The half-lives in mice ranged from 7.3 h for the construct including PAS300 to 11.6 h for the construct including PAS600. The highest absolute tumor uptake was found for the construct including the ABD, which was 60 to 160% higher than the PASylated or XTENylated constructs, even though it did not have the longest half-life (9.0 h). A comparison of the tumor-to-normal-organ ratios showed the best overall performance of the ABD-fused construct. In conclusion, PASylation, XTENylation, and the addition of an ABD are viable strategies for half-life extension of affibody-drug conjugates, with the best performance observed for the construct including the ABD.

Identification and characterization of rabbit scFv antibodies suitable for immuno-affinity separation of recombinant human kynureninase from Escherichia coli cell lysate

In this study we successfully developed an on-demand affinity chromatographic resin for manufacturing non-Fc-based biopharmaceuticals. Affinity chromatography columns with immobilized rabbit single-chain variable fragments (scFvs) were used for directly purifying the recombinant human kynureninase (KYNase) as a model target therapeutic protein from Escherichia coli cell lysates. Among the 38 different anti-KYNase scFv clones identified, four unique clones were selected as candidates for further characterization owing to their relatively low KYNase binding affinity at pH 4.0, thereby facilitating enzyme elution. Subsequently, all four clones were successfully produced and purified, followed by covalent coupling to NHS-activated HiTrap HP columns. While KYNase was specifically adsorbed to all four scFv-immobilized columns and was eluted at pH 4.0, the respective levels of static binding capacity (SBC) and recovery among the four scFv clones were different at this elution pH. That is, the scFv-immobilized columns captured KYNase with SBC ranging from 1.15 to 2.68 mg/cm3-bed with clone R2-47 exhibiting the highest level of SBC, with a ligand utilization of 39.4%. Moreover, using the scFv column of R2-47, 90.7% of the captured human KYNase was recovered in the first elution step at pH 4.0, and approximately 67% of enzymatic activity was retained. In summary, high-purity human KYNase was obtained from the E.coli cell lysate by one-step affinity purification, and 89.7% of KYNase was recovered in the first elution step. The methodology demonstrated in the current study could be applied for the purification and development of various therapeutic proteins.

Unraveling the Microscopic Mechanism of Molecular Ion Interaction with Monoclonal Antibodies: Impact on Protein Aggregation.

Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.

Limitations of Protein Structure Prediction Algorithms in Therapeutic Protein Development

The three-dimensional protein structure is pivotal in comprehending biological phenomena. It directly governs protein function and hence aids in drug discovery. The development of protein prediction algorithms, such as AlphaFold2, ESMFold, and trRosetta, has given much hope in expediting protein-based therapeutic discovery. Though no study has reported a conclusive application of these algorithms, the efforts continue with much optimism. We intended to test the application of these algorithms in rank-ordering therapeutic proteins for their instability during the pre-translational modification stages, as may be predicted according to the confidence of the structure predicted by these algorithms. The selected molecules were based on a harmonized category of licensed therapeutic proteins; out of the 204 licensed products, 188 that were not conjugated were chosen for analysis, resulting in a lack of correlation between the confidence scores and structural or protein properties. It is crucial to note here that the predictive accuracy of these algorithms is contingent upon the presence of the known structure of the protein in the accessible database. Consequently, our conclusion emphasizes that these algorithms primarily replicate information derived from existing structures. While our findings caution against relying on these algorithms for drug discovery purposes, we acknowledge the need for a nuanced interpretation. Considering their limitations and recognizing that their utility may be constrained to scenarios where known structures are available is important. Hence, caution is advised when applying these algorithms to characterize various attributes of therapeutic proteins without the support of adequate structural information. It is worth noting that the two main algorithms, AlfphaFold2 and ESMFold, also showed a 72% correlation in their scores, pointing to similar limitations. While much progress has been made in computational sciences, the Levinthal paradox remains unsolved.

Experimental procedures to investigate fibrillation of proteins

The unwanted phenomenon of protein fibrillation is observed in vivo and during therapeutic protein development in the industry. Protein aggregation is associated with various degenerative disorders and might induce immune-related challenges post-administration of biopharmaceutics. A pipeline for early detection, identification, and removal of pre-formed fibrils is needed to improve the quality, efficacy, and effectiveness of the formulation. Protein fibril formation is accompanied by unfolding, secondary structural changes and the formation of larger aggregates. However, most detection processes come with extensive sample preparation steps and inefficient repeatability, incurring a financial burden on research. The current article summarizes and critically discusses six simple yet powerful methods to detect aggregation phenomena in the line of detecting fibrillar aggregates in heat-induced bovine serum albumin protein. Comparing the native and heat-induced protein samples would provide insights about aggregates. Easy, inexpensive and optimized protocols for detecting the fibrillation of proteins are explained. The procedures mentioned here detected the appearance of β-sheet-rich fibrils in the heat-induced protein sample. The aggregation is characterized by enhanced thioflavin-T fluorescence, alteration in the intrinsic fluorescence, decrease in helicity and subsequent increase in β-sheet and appearance of particles with larger hydrodynamic diameters.•This article summarizes various analytical techniques to easily characterize the fibrillation of proteins.•Various techniques to detect the formation of β-sheet rich structures, changes in the secondary structures and size of aggregates have been discussed.•The stated methodologies are validated on a model protein, albumin.

Improving Protein Therapeutic Development through Cloud-based Data Integration.

Pharma 4.0 is a digital evolution of the pharmaceutical industry that automates scientists' traditional workflows with the implementation of modern technologies like cloud pipelines, artificial intelligence, robotic platforms, and augmented reality. Lab data capture (LDC) is an essential strategy for initiating Pharma 4.0 that aggregates and harmonizes siloed lab data from analytical instruments, reporting systems, and operational platforms. This publication describes the execution of LDC within a quantitative PCR (qPCR) workflow using the Tetra Data Platform (TDP). We selected this workflow because the qPCR instrument, the ViiA7, generates discrete file-based data that documents execution of individual assays for quantifying residual DNA throughout biologics process development and product profiling. TDP executes LDC through the deployment of file scanning software agents, scanning and ingestion processes, and a cloud-based parsing pipeline that harmonizes source data. Web applications were developed to query, visualize, and interpret harmonized qPCR data for automated experiment data processing and process control charting from the TDP platform. Our implementation of LDC enables analytical researchers to harness FAIR (Findable, Accessible, Interoperable, Reproducible) data practices across the organization and establishes a "compliance-by-code" culture in development labs.

A graph-based machine learning framework identifies critical properties of FVIII that lead to hemophilia A.

Introduction: Blood coagulation is an essential process to cease bleeding in humans and other species. This mechanism is characterized by a molecular cascade of more than a dozen components activated after an injury to a blood vessel. In this process, the coagulation factor VIII (FVIII) is a master regulator, enhancing the activity of other components by thousands of times. In this sense, it is unsurprising that even single amino acid substitutions result in hemophilia A (HA)-a disease marked by uncontrolled bleeding and that leaves patients at permanent risk of hemorrhagic complications. Methods: Despite recent advances in the diagnosis and treatment of HA, the precise role of each residue of the FVIII protein remains unclear. In this study, we developed a graph-based machine learning framework that explores in detail the network formed by the residues of the FVIII protein, where each residue is a node, and two nodes are connected if they are in close proximity on the FVIII 3D structure. Results: Using this system, we identified the properties that lead to severe and mild forms of the disease. Finally, in an effort to advance the development of novel recombinant therapeutic FVIII proteins, we adapted our framework to predict the activity and expression of more than 300 in vitro alanine mutations, once more observing a close agreement between the in silico and the in vitro results. Discussion: Together, the results derived from this study demonstrate how graph-based classifiers can leverage the diagnostic and treatment of a rare disease.

Hydrodynamic considerations for spring-driven autoinjector design

In recent years, significant progress has been made in the studies of the spring-driven autoinjector, leading to an improved understanding of this device and its interactions with tissue and therapeutic proteins. The development of simulation tools that have been validated against experiments has also enhanced the prediction of the performance of spring-driven autoinjectors. This paper aims to address critical hydrodynamic considerations that impact the design of spring-driven autoinjectors, with a specific emphasis on sloshing and cavitation. Additionally, we present a framework that integrates simulation tools to predict the performance of spring-driven autoinjectors and optimize their design. This work is valuable to the pharmaceutic industry, as it provides crucial insights into the development of spring-driven autoinjectors and therapeutic proteins. This work can also enhance the efficacy and safety of the delivery of therapeutic proteins, ultimately improving patient outcomes.

A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation.

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction. Herein, we report a method to make PAL-mediated intermolecular ligation irreversible by coupling it to glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation. In this method, the acyl donor substrate of PALs is designed to have glutamine at the P1' position of the Asn-P1'-P2' tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group in which the exposed N-terminal glutamine is cyclized by QC, quenching the Gln Nα-amine in a lactam. Using this method, PAL-mediated ligation can achieve near-quantitative yields even at an equal molar ratio between the two ligation partners. We have demonstrated this method for a wide range of applications, including protein-to-protein ligations. We anticipate that this cascade enzymatic reaction scheme will make PAL enzymes well suited for numerous new uses in biotechnology.

Using New Peak Detection to Solve Sequence Variants Analysis Challenges in Bioprocess Development.

Recombinant therapeutic proteins have become the major class of drugs to treat various human diseases in recent years. Low levels of protein sequence variants (SVs) have been reported to be present in recombinant therapeutic proteins. The consequences of potential unwanted immune response from SVs of recombinant therapeutic proteins have increasingly drawn attention from regulatory authorities and the biopharmaceutical industry. It is highly desirable to detect low-level SVs during clone selection and early process development as part of the control strategy. Peptide mapping with LC-MS/MS analysis has been applied as a powerful tool to characterize post-translation modifications of therapeutic proteins. Despite the recent advancements in mass spectrometry hardware and software, it is still quite challenging and time-consuming to detect and identify low-level SVs. In this study, we present an optimized approach using new peak detection to detect and identify low level SVs with high confidence and high speed. The new approach makes sequence variants analysis by LC-MS/MS broadly applicable and practical in bioprocess development of therapeutic proteins.

Accelerated CMC workflows to enable speed to clinic in the COVID-19 era: A multi-company view from the biopharmaceutical industry.

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation. To optimize lessons learned from the pandemic and maximize multi-stakeholder (i.e., patients, clinicians, companies, regulatory agencies) benefit, we conducted an industry wide benchmarking survey in September/October 2021. The aims of this survey were to: (i) benchmark current technical practices of key process and product development activities related to manufacturing of therapeutic proteins, (ii) understand the impact of changes implemented in COVID-19 accelerated Ab programs, and whether any such changes can be retained as part of sustainable long-term business practices and (iii) understand whether any accelerative action(s) taken have (negatively) impacted the wider development process. This article provides an in-depth analysis of this data, ultimately highlighting an industry perspective of how biopharmaceutical companies can sustainably adopt new approaches to therapeutic protein development and production.

Identification and Quantification of a Problematic Host Cell Protein to Support Therapeutic Protein Development

Monitoring of residual host cell proteins (HCPs) in therapeutic protein is essential to ensure product quality, safety and efficacy. Despite the development of advanced mass spectrometry techniques and optimized workflows, identifying and quantifying all problematic HCPs present at low levels remain challenging. Here, we developed a practical, effective strategy for the identification and quantification of low abundance HCPs, which facilitates the improvement of downstream purification process to eliminate potentially problematic HCPs. A case study of using this strategy to investigate a problematic HCP is presented. Initially, a commonly used native digestion approach coupled with UPLC-MS/MS was applied for HCP profiling, wherein several lipases and proteases were identified in a monoclonal antibody named mAb1 in early stages of purification process development. A highly active lipase, liver carboxylesterase (CES), was found to be responsible for polysorbate 80 degradation. To facilitate process improvement, after the identification of CES, we developed a highly sensitive LC-MS/MS-MRM assay with a lower limit of quantification of 0.05 ppm for routine monitoring of the CES in mAb1 produced through the different processes. This workflow was applied in low-level lipase identification and absolute quantification, which facilitated the investigation of polysorbate degradation and downstream purification improvement to further remove the problematic HCP. The current MRM method increased the sensitivity of HCP quantification by over 10-fold that in previously published studies, thus meeting the needs for quantification of problematic HCPs at sub-ppm to ppb levels during drug development. This workflow could be readily adapted to the detection and quantification of other problematic HCPs present at extremely low levels in therapeutic protein drug candidates.

Mass spectrometry-based multi-attribute method for mutation analysis in the early development of therapeutic proteins

The early intervention is essential, and later development cannot compensate for this initial generation of an antibody drug. Especially for sequence variants (SVs), should cause concern during the early bioprocess development. The advancement of bioprocess development is paralleled by development of state-of-the-art analytical methods that will provide further information. In the present study, a mass spectrometry (MS)-based multi-attribute method (MAM) was used to simultaneously monitor the SVs and other quality attributes in the early bioprocess development of ofatumumab, and a sequence variant (SV) was detected by a subunit-based MAM. Subsequently, the variant was further identified by MS/MS and confirmed by adding a synthetic peptide. Furthermore, the content of the SV was detected via DNA sequencing. The levels of the variant (T175A mutant) in the light chain were demonstrate to be nearly consistent at the DNA and protein levels, suggesting that the mutation may have negligible effect on both the transcriptional and translational levels. Collectively, these results indicate that broad-spectrum, rapid, and accurate platform such as MS-based MAM should be implemented to quality control for the early development of therapeutic proteins, it will also be important to establish an effective and integrated MAM to control SVs during therapeutic proteins development.

Structure-Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin.

Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart’s molecular characteristics.

Quantification of Structural Integrity and Stability Using Nanograms of Protein by Flow-Induced Dispersion Analysis.

In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and integrity influence drug efficacy, and ultimately patient safety. Existing analytical methodologies solely rely on relative changes in optical properties such as fluorescence or scattering upon thermal or chemical perturbation. Here, we present an absolute analytical method for assessing protein stability, structure, and unfolding utilizing Taylor dispersion analysis (TDA) and LED-UV fluorescence detection. The developed TDA method measures the change in size (hydrodynamic radius) and intrinsic fluorescence of a protein during in-line denaturation with guanidinium hydrochloride (GuHCl). The conformational stability of the therapeutic antibody adalimumab and human serum albumin were characterized as a function of pH. The simple workflow and low sample consumption (40 ng protein per data point) of the methodology make it ideal for assessing protein characteristics related to stability in early drug development or when having a scarce amount of sample available.

Identification, Efficacy, and Stability Evaluation of Succinimide Modification With a High Abundance in the Framework Region of Golimumab.

Succinimide (Asu) is the intermediate for asparagine deamidation in therapeutic proteins, and it can be readily hydrolyzed to form aspartate and iso-aspartate residues. Moreover, Asu plays an important role in the protein degradation pathways, asparagine deamidation, and aspartic acid isomerization. Here, Asu modification with a high abundance in the framework region (FR) of golimumab was first reported, the effect of denaturing buffer pH on the Asu modification homeostasis was studied, and the results revealed that it was relatively stable over a pH range of 6.0–7.0 whereas a rapid decrease at pH 8.0. Then, the peptide-based multi-attribute method (MAM) analyses showed that the Asu formation was at Asn 43 in the FR of the heavy chain. Meanwhile, the efficacy [affinity, binding and bioactivity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity] and stability of the Asu modification of golimumab were evaluated, and the current results demonstrated comparable efficacy and stability between the Asu low- and high-abundance groups. Our findings provide valuable insights into Asu modification and its effect on efficacy and stability, and this study also demonstrates that there is a need to develop a broad-spectrum, rapid, and accurate platform to identify and characterize new peaks in the development of therapeutic proteins, particularly for antibody drugs.

New Aspects in the Integration of MS Technologies in the Biopharmaceutical Industry

In the past decade, advances in both separations and mass spectrometry (MS) technologies have enabled new, streamlined, and data-rich approaches to monitor product quality attributes and their relationship with process parameters throughout the lifecycle of therapeutic proteins. As we enter a new decade of technology and method development, MS-based approaches utilized in the biopharmaceutical industry are evolving further. In this mini-review, we explore key developments that could inspire and improve the future of therapeutic protein development.

Engineered protein nanodrug as an emerging therapeutic tool.

Functional proteins are the most versatile macromolecules. They can be obtained by extraction from natural sources or by genetic engineering technologies. The outstanding selectivity, specificity, binding activity, and biocompatibility endow engineered proteins with outstanding performance for disease therapy. Nevertheless, their stability is dramatically impaired in blood circulation, hindering clinical translations. Thus, many strategies have been developed to improve the stability, efficacy, bioavailability, and productivity of therapeutic proteins for clinical applications. In this review, we summarize the recent progress in the fabrication and application of therapeutic proteins. We first introduce various strategies for improving therapeutic efficacy via bioengineering and nanoassembly. Furthermore, we highlight their diverse applications as growth factors, nanovaccines, antibody-based drugs, bioimaging molecules, and cytokine receptor antagonists. Finally, a summary and perspective for the future development of therapeutic proteins are presented.

Microphysiological endothelial models to characterize subcutaneous drug absorption.

The high variability in subcutaneous bioavailability of protein therapeutics is poorly understood, contributing to critical delays in patient access to new therapies. Preclinical animal and in vitro models fail to provide a physiologically relevant testbed to parse potential contributors to human bioavailability, therefore new strategies are necessary. Here, we present a microphysiological model of the human hypodermal vasculature at the injection site to study the interactions of administered protein therapeutics within the microenvironment that influence subcutaneous bioavailability. Our model combines human dermal endothelial cells, fibroblasts, and adipocytes, self-assembled into three-dimensional, perfusable microvessels that express relevant extracellular matrix. We demonstrate the utility of the model for measurement of biophysical parameters within the hypodermal microenvironment that putatively impact protein kinetics and distribution at the injection site. We propose that microphysiological models of the subcutaneous space have applications in preclinical development of protein therapeutics intended for subcutaneous administration with optimal bioavailability.

Opalescence Measurements: Improvements in Fundamental Knowledge, Identifying Sources of Analytical Biases, and Advanced Applications for the Development of Therapeutic Proteins.

Opalescence of biopharmaceutical solutions can indicate suboptimal colloidal stability and is therefore a generally undesirable attribute that requires investigation and potentially remediation. While there are numerous instrumentation options available for measuring opalescence, cross-instrument comparisons and detailed knowledge of analytical biases have been limited. Here, we highlight key findings from a multi-instrument investigation where differences in reported opalescence values are explained with particular emphasis on how the optical configuration and detector properties of each instrument affect the response of the sample and the primary formazin standards required for instrument calibration. In doing so, the particle size distribution, angular-dependent light scattering properties and refractive index of the primary formazin standard material are characterized and presented. Finally, the advanced application of a 90° angle light scattering instrument is presented as a suitable approach for making low volume, temperature controlled, nephelometric measurements of opalescence. Moreover, we demonstrate how this approach enables the simultaneous evaluation of key physical properties, such as hydrodynamic size, that are pertinent to investigations of opalescent biopharmaceuticals but have historically required the use of separate instrumentation. The findings reported here address key knowledge gaps and provide opportunities for improving the efficiency and inter-laboratory comparability of opalescence measurements for biopharmaceuticals.