Development Of Therapeutic Proteins Research Articles

Physiologically Based Pharmacokinetic Modeling of Therapeutic Proteins

Biologics or therapeutic proteins are becoming increasingly important as treatments for disease. The most common class of biologics are monoclonal antibodies (mAbs). Recently, there has been an increase in the use of physiologically based pharmacokinetic (PBPK) modeling in the pharmaceutical industry in drug development. We review PBPK models for therapeutic proteins with an emphasis on mAbs. Due to their size and similarity to endogenous antibodies, there are distinct differences between PBPK models for small molecules and mAbs. The high-level organization of a typical mAb PBPK model consists of a whole-body PBPK model with organ compartments interconnected by both blood and lymph flows. The whole-body PBPK model is coupled with tissue-level submodels used to describe key mechanisms governing mAb disposition including tissue efflux via the lymphatic system, elimination by catabolism, protection from catabolism binding to the neonatal Fc (FcRn) receptor, and nonlinear binding to specific pharmacological targets of interest. The use of PBPK modeling in the development of therapeutic proteins is still in its infancy. Further application of PBPK modeling for therapeutic proteins will help to define its developing role in drug discovery and development.

An Improved Method of Predicting Extinction Coefficients for the Determination of Protein Concentration.

Concentration determination is an important method of protein characterization required in the development of protein therapeutics. There are many known methods for determining the concentration of a protein solution, but the easiest to implement in a manufacturing setting is absorption spectroscopy in the ultraviolet region. For typical proteins composed of the standard amino acids, absorption at wavelengths near 280 nm is due to the three amino acid chromophores tryptophan, tyrosine, and phenylalanine in addition to a contribution from disulfide bonds. According to the Beer-Lambert law, absorbance is proportional to concentration and path length, with the proportionality constant being the extinction coefficient. Typically the extinction coefficient of proteins is experimentally determined by measuring a solution absorbance then experimentally determining the concentration, a measurement with some inherent variability depending on the method used. In this study, extinction coefficients were calculated based on the measured absorbance of model compounds of the four amino acid chromophores. These calculated values for an unfolded protein were then compared with an experimental concentration determination based on enzymatic digestion of proteins. The experimentally determined extinction coefficient for the native proteins was consistently found to be 1.05 times the calculated value for the unfolded proteins for a wide range of proteins with good accuracy and precision under well-controlled experimental conditions. The value of 1.05 times the calculated value was termed the predicted extinction coefficient. Statistical analysis shows that the differences between predicted and experimentally determined coefficients are scattered randomly, indicating no systematic bias between the values among the proteins measured. The predicted extinction coefficient was found to be accurate and not subject to the inherent variability of experimental methods. We propose the use of a predicted extinction coefficient for determining the protein concentration of therapeutic proteins starting from early development through the lifecycle of the product.LAY ABSTRACT: Knowing the concentration of a protein in a pharmaceutical solution is important to the drug's development and posology. There are many ways to determine the concentration, but the easiest one to use in a testing lab employs absorption spectroscopy. Absorbance of ultraviolet light by a protein solution is proportional to its concentration and path length; the proportionality constant is the extinction coefficient. The extinction coefficient of a protein therapeutic is usually determined experimentally during early product development and has some inherent method variability. In this study, extinction coefficients of several proteins were calculated based on the measured absorbance of model compounds. These calculated values for an unfolded protein were then compared with experimental concentration determinations based on enzymatic digestion of the proteins. The experimentally determined extinction coefficient for the native protein was 1.05 times the calculated value for the unfolded protein with good accuracy and precision under controlled experimental conditions, so the value of 1.05 times the calculated coefficient was called the predicted extinction coefficient. Comparison of predicted and measured extinction coefficients indicated that the predicted value was very close to the experimentally determined values for the proteins. The predicted extinction coefficient was accurate and removed the variability inherent in experimental methods.

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

ABSTRACTCo-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging. Aiming for molecules of simple and stable configuration, we used TNFSF ligands in a single-chain format (scTNFSF), i.e., three units of the ectodomain connected by polypeptide linkers, folding into an intramolecular trimer. By fusing tumor-directed scFv antibody fragments directed against EpCAM or FAP to co-stimulatory scTNFSF molecules (sc4-1BBL, scOX40L, scGITRL or scLIGHT), a set of monomeric scFv-scTNFSF fusion proteins was generated. In comparison to the scFv-TNFSF format, defined by intermolecular homotrimerization via the TNFSF part, scFv-scTNFSF showed equal or enhanced co-stimulatory activity despite reduced avidity in antibody binding. In addition, enhanced serum stability and improved bioavailability in mice were observed. We show that the scFv-scTNFSF format can be applied to various members of the TNFSF, presenting targeting-dependent co-stimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.

An Opportunity for Industry–Academia Partnership: Training the Next Generation of Industrial Researchers in Characterizing Higher Order Protein Structure

Training researchers for positions in the United States biopharmaceutical industry has long been driven by academia. This commentary explores how the changing landscape of academic training will impact the industrial workforce, particularly with regard to the development of protein therapeutics in the area of biophysical and higher order structural characterization. We discuss how to balance future training and employment opportunities, how academic-industrial partnerships can help young scientists acquire the skills needed by their future employer, and how an appropriately trained workforce can facilitate the translation of new technology from academic to industrial laboratories. We also present suggestions to facilitate the coordinated development of industrial-academic educational partnerships to develop new training programs, and the ability of students to locate these programs, through the development of authoritative public resources.

Stapled peptides: providing the best of both worlds in drug development.

Peptide-based drug discovery has experienced a remarkable resurgence within the past decade due to the emerging class of inhibitors known as stapled peptides. Stapled peptides are therapeutic protein mimetics that have been locked within a specific conformational structure by hydrocarbon stapling. These peptides are highly important in selectively impairing disease-relevant protein-protein interactions and exhibit significant pharmacokinetic advantages over other forms of therapeutics in terms of affinity, specificity, size, synthetic accessibility and resistance to proteolytic degradation. A series of stapled peptides are currently in development, and the potential successes of these peptides, either as single-agent treatments or as combinational treatments with other therapeutic modalities, could potentially change the landscape of protein therapeutic development. Here, we provide examples of successful discovery efforts to illustrate the research strategies of stapled peptides in drug design and development.

Novel Displacement Agents for Aqueous 2-Phase Extraction Can Be Estimated Based on Hybrid Shortcut Calculations

The purification of therapeutic proteins is a challenging task with immediate need for optimization. Besides other techniques, aqueous 2-phase extraction (ATPE) of proteins has been shown to be a promising alternative to cost-intensive state-of-the-art chromatographic protein purification. Most likely, to enable a selective extraction, protein partitioning has to be influenced using a displacement agent to isolate the target protein from the impurities. In this work, a new displacement agent (lithium bromide [LiBr]) allowing for the selective separation of the target protein IgG from human serum albumin (represents the impurity) within a citrate–polyethylene glycol (PEG) ATPS is presented. In order to characterize the displacement suitability of LiBr on IgG, the mutual influence of LiBr and the phase formers on the aqueous 2-phase system (ATPS) and partitioning is investigated. Using osmotic virial coefficients (B22 and B23) accessible by composition gradient multiangle light-scattering measurements, the precipitating effect of LiBr on both proteins and an estimation of both protein partition coefficients is estimated. The stabilizing effect of LiBr on both proteins was estimated based on B22 and experimentally validated within the citrate–PEG ATPS. Our approach contributes to an efficient implementation of ATPE within the downstream processing development of therapeutic proteins.

Integrated process for high conversion and high yield protein PEGylation.

Over the past decades, PEGylation has become a powerful technique to increase the in vivo circulation half-life of therapeutic proteins while maintaining their activity. The development of new therapeutic proteins is likely to require further improvement of the PEGylation methods to reach even better selectivity and yield for reduced costs. The intensification of the PEGylation process was investigated through the integration of a chromatographic step in order to increase yield and conversion for the production of mono-PEGylated protein. Lysozyme was used as a model protein to demonstrate the feasibility of such approach. In the integrated reaction/separation process, chromatography was used as fractionation technique in order to isolate and recycle the unreacted protein from the PEGylated products. This allows operating the reactor with short reaction times so as to minimize the production of multi-PEGylated proteins (i.e., conjugated to more than one polymer). That is, the reaction is stopped before the desired product (i.e., the mono-PEGylated protein) can further react, thus leading to limited conversion but high yield. The recycling of the unreacted protein was then considered to drive the protein overall conversion to completion. This approach has great potential to improve processes whose yield is limited by the further reaction of the product leading to undesirable by-products. Biotechnol. Bioeng. 2016;113: 1711-1718. © 2016 Wiley Periodicals, Inc.

The transgenic animal platform for biopharmaceutical production.

The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.

Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation

Facile labeling of proteins of interest is highly desirable in proteomic research as well as in the development of protein therapeutics. Herein we report a novel method that allows for fast and selective labeling of proteins with an N-terminal cysteine. Although N-terminal cysteines are well known to conjugate with aldehydes to give thiazolidines, the reaction requires acidic conditions and suffers from slow kinetics. We show that benzaldehyde with an ortho-boronic acid substituent readily reacts with N-terminal cysteines at neutral pH, giving rate constants on the order of 103 M-1 s-1. The product features a thiazolidono boronate (TzB) structure and exhibits improved stability due to formation of the B-N dative bond. While stable at neutral pH, the TzB complex dissociates upon mild acidification. These characteristics make the TzB conjugation chemistry potentially useful for the development of drug-protein conjugates that release the small molecule drug in acidic endosomes.

A Tobacco-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice.

Thymosin β4 (Tβ4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tβ4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tβ4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bp Tβ4 gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify 4×Tβ4 expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tβ4 protein concentrations as high as 3 μg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tβ4 was more effective than Tβ4 either obtained commercially or derived from expression in Escherichia coli at promoting splenocyte proliferation in vitro and wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.

Fundamentals of large-molecule protein therapeutic bioanalysis using ligand-binding assays.

This article provides an overview of ligand-binding assays, including the origin and evolution of the primary concepts, in addition to reviewing commonly used assay formats. The birth of ligand-binding assays began with a radioimmunoassay developed to measure insulin in 1960. Fundamental to ligand-binding assay design is the requirement of at least one protein that interacts with the analyte of interest. Enzyme immunoassay has largely supplanted radioimmunoassay as the ligand-binding assay of choice in today's laboratory environment. This article illustrates various assay formats such as competitive, sandwich and bridging, in addition to, describing critical reagents necessary for their design. The utility of ligand-binding assays in therapeutic protein development and comparison to alternative bioanalysis platforms is discussed.

Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein

Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development. Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5′ un-translated regions were generated and optimized, from which the best vector with expression level at ~50mg/L was selected for production at 2L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q, Fast Flow Q, and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC–HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at ~50mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins.

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

Automated small‐scale protein purification and analysis for accelerated development of protein therapeutics

Small‐scale protein purification presents opportunities for accelerated process development of biotherapeutic molecules. Miniaturization of purification conditions reduces time and allows for parallel processing of samples, thus offering increased statistical significance and greater breadth of variables. The ability of the miniaturized platform to be predictive of larger scale purification schemes is of critical importance. The PerkinElmer JANUS BioTx Pro and Pro‐Plus workstations were developed as intuitive, flexible, and automated devices capable of performing parallel small‐scale analytical protein purification. Preprogrammed methods automate a variety of commercially available ion exchange and affinity chromatography solutions, including miniaturized chromatography columns, resin‐packed pipette tips, and resin‐filled microtiter vacuum filtration plates. Here, we present a comparison of microscale chromatography versus standard fast protein LC (FPLC) methods for process optimization. In this study, we evaluated the capabilities of the JANUS BioTx Pro‐Plus robotic platform for miniaturized chromatographic purification of proteins with the GE ӒKTA Express system. We were able to demonstrate predictive analysis similar to that of larger scale purification platforms, while offering advantages in speed and number of samples processed. This approach is predictive of scale‐up conditions, resulting in shorter biotherapeutic development cycles and less consumed material than traditional FPLC methods, thus reducing time‐to‐market from discovery to manufacturing.

High-throughput screening and automation approaches for the development of recombinant therapeutic proteins

Generation of recombinant therapeutic proteins involves the use of Chinese hamster ovary cells as one of the workhorses for complex protein production. This process requires screening large numbers of transfected cells to identify single clones that have high protein production and drug-specific quality attributes. Traditionally, this process was limited by manual operation; however, high-throughput screening methods and automation have made this process more efficient. Implementation of high-throughput screening and automation within bioprocess development have led to increased screening capacity, higher product yields, reductions in manual operation, reduction in human error and shorter development time lines. This review outlines the high-throughput methodologies and technologies currently used for clone screening, selection and evaluation in bioprocess development.

Dual Effect of (LK)nL Peptides on the Onset of Insulin Amyloid Fiber Formation at Hydrophobic Surfaces.

Soluble proteins are constantly in contact with material or cellular surfaces, which can trigger their aggregation and therefore have a serious impact on the development of stable therapeutic proteins. In contact with hydrophobic material surfaces, human insulin aggregates readily into amyloid fibers. The kinetics of this aggregation can be accelerated by small peptides, forming stable beta-sheets on hydrophobic surfaces. Using a series of (LK)nL peptides with varying length, we show that these peptides, at low, substoichiometric concentrations, have a positive, cooperative effect on insulin aggregation. This effect is based on a cooperative adsorption of (LK)nL peptides at hydrophobic surfaces, where they form complexes that help the formation of aggregation nuclei. At higher concentrations, they interfere with the formation of an aggregative nucleus. These effects are strictly dependent on the their adsorption on hydrophobic material surfaces and highlight the importance of the impact of materials on protein stability. (LK)nL peptides prove to be valuable tools to investigate the mechanism of HI aggregation nuclei formation on hydrophobic surfaces.

Laser-induced breakdown detection of temperature-ramp generated aggregates of therapeutic monoclonal antibody

The detection and characterization of protein aggregation is essential during development and quality control of therapeutic proteins, as aggregates are typically inactive and may trigger anti-drug-antibody formation in patients. Especially large multi-domain molecules, such as the important class of therapeutic monoclonal antibodies (mAbs), can form various aggregates that differ in size and morphology. Although particle analysis advanced over the recent years, new techniques and orthogonal methods are highly valued. To our knowledge, the physical principle of laser-induced breakdown detection (LIBD) was not yet applied to sense aggregates in therapeutic protein formulations. We established a LIBD setup to monitor the temperature-induced aggregation of a mAb. The obtained temperature of aggregation was in good agreement with the results from previously published temperature-ramped turbidity and dynamic light scattering measurements. This study demonstrates the promising applicability of LIBD to investigate aggregates from therapeutic proteins. The technique is also adaptive to online detection and size determination, and offers interesting opportunities for morphologic characterization of protein particles and impurities, which will be part of future studies.

Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy

Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation. In this manuscript on perspectives on subvisible particles in protein therapeutic drug products, we focus on the tools available for detection, characterization, and quantification of these species and the strategy around their application.

Effects of Sucrose and Benzyl Alcohol on GCSF Conformational Dynamics Revealed by Hydrogen Deuterium Exchange Mass Spectrometry

Protein stability, one of the major concerns for therapeutic protein development, can be optimized during process development by evaluating multiple formulation conditions. This can be a costly and lengthy procedure where different excipients and storage conditions are tested for their impact on protein stability. A better understanding of the effects of different formulation conditions at the molecular level will provide information on the local interactions within the protein leading to a more rational design of stable and efficacious formulations. In this study, we examined the roles of the excipients, sucrose and benzyl alcohol, on the conformational dynamics of recombinant human granulocyte colony stimulating factor using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS). Under physiological pH and temperature, sucrose globally protects the whole molecule from deuterium uptake, whereas benzyl alcohol induces increased deuterium uptake of the regions within the α-helical bundle, with even larger extent. The HDX experiments described were incorporated a set of internal peptides (Zhang et al., 2012. Anal Chem 84:4942-4949) to monitor the differences in intrinsic exchange rates in different formulations. In addition, we discussed the feasibility of implementing HDX-MS with these peptide probes in protein formulation development.

AGGRESCAN3D (A3D): server for prediction of aggregation properties of protein structures.

Protein aggregation underlies an increasing number of disorders and constitutes a major bottleneck in the development of therapeutic proteins. Our present understanding on the molecular determinants of protein aggregation has crystalized in a series of predictive algorithms to identify aggregation-prone sites. A majority of these methods rely only on sequence. Therefore, they find difficulties to predict the aggregation properties of folded globular proteins, where aggregation-prone sites are often not contiguous in sequence or buried inside the native structure. The AGGRESCAN3D (A3D) server overcomes these limitations by taking into account the protein structure and the experimental aggregation propensity scale from the well-established AGGRESCAN method. Using the A3D server, the identified aggregation-prone residues can be virtually mutated to design variants with increased solubility, or to test the impact of pathogenic mutations. Additionally, A3D server enables to take into account the dynamic fluctuations of protein structure in solution, which may influence aggregation propensity. This is possible in A3D Dynamic Mode that exploits the CABS-flex approach for the fast simulations of flexibility of globular proteins. The A3D server can be accessed at http://biocomp.chem.uw.edu.pl/A3D/.