Patient Centricity Driving Formulation Innovation: Improvements in Patient Care Facilitated by Novel Therapeutics and Drug Delivery Technologies.

Innovative formulation technologies can play a crucial role in transforming a novel molecule to a medicine that significantly enhances patients' lives. Improved mechanistic understanding of diseases has inspired researchers to expand the druggable space using new therapeutic modalities such as interfering RNA, protein degraders, and novel formats of monoclonal antibodies. Sophisticated formulation strategies are needed to deliver the drugs to their sites of action and to achieve patient centricity, exemplified by messenger RNA vaccines and oral peptides. Moreover, access to medical information via digital platforms has resulted in better-informed patient groups that are requesting consideration of their needs during drug development. This request is consistent with health authority efforts to upgrade their regulations to advance age-appropriate product development for patients. This review describes formulation innovations contributingto improvements in patient care: convenience of administration, preferred route of administration, reducing dosing burden, and achieving targeted delivery of new modalities.

Identification of Hetero-aggregates in Antibody Co-formulations by Multi-dimensional Liquid Chromatography Coupled to Mass Spectrometry.

Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo- versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo- and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.

Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Biophysical Analysis of Lipopolysaccharide Formulations for an Understanding of the Low Endotoxin Recovery (LER) Phenomenon.

Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called "Low Endotoxin Recovery (LER)". LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration's (FDA's) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER.

Inclusion of an RGD Motif Alters Invasin Integrin-Binding Affinity and Specificity.

Invasin is a key adhesin displayed on the outer membrane of Yersinia enterocolitica and Y. pseudotuberculosis that mediates the initial stages of infection. Invasin specifically targets microfold (M) cells in the small intestine by binding ß1 integrins and is sufficient to trigger eukaryotic uptake of invasin-coated particles, including Yersinia, Escherichia coli, and latex beads. As a result, invasin has generated interest to mediate oral delivery of vaccines and other biologics. Integrin binding affinity has been shown to correlate with particle uptake; thus we hypothesized that invasin variants with higher affinity would confer enhanced internalization. We first performed alanine scanning of surface-exposed tyrosine residues to identify those contributing to integrin binding. We identified two residues, which, when substituted with alanine, reduced binding to soluble α5ß1 integrin. Next, we constructed four targeted mutagenesis libraries spanning these and other residues known to contribute to binding, followed by enrichment of variants able to mediate Caco-2 cellular invasion and to bind soluble α5ß1 integrin. We identified three amino acid substitutions that increased α5ß1 integrin binding affinity as measured by flow cytometry and ELISA assays, two of which created a novel RGD motif surrounding the D911 residue critical for binding. This variant confers enhanced internalization into CHO cells but not Caco-2 cells when expressed on the E. coli surface. Further analysis showed that inclusion of an RGD expands invasin-integrin specificity, thereby impacting cellular selectivity. This work provides a molecular explanation for the lack of an RGD motif in invasin that is present in many other adhesins.

Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice.

BACKGROUND: Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data. RESULTS: We isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors-(i) replicate sequencing and (ii) sequencing depth-are crucial for robust CDR3 and VDJ detection and ranking. CONCLUSIONS: In summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.

In vivo whole animal fluorescence imaging of a microparticle-based oral vaccine containing (CuInSe(x)S(2-x))/ZnS core/shell quantum dots.

Zinc sulfide-coated copper indium sulfur selenide (CuInSexS2-x/ZnS core/shell) nanocrystals were synthesized with size-tunable red to near-infrared (NIR) fluorescence with high quantum yield (40%) in water. These nanocrystals were tested as an imaging agent to track a microparticle-based oral vaccine administered to mice. Poly(lactic-co-glycolic acid) (PLGA) microparticle-encapsulated CuInSexSe2-x/ZnS quantum dots were orally administered to mice and were found to provide a distinct visible fluorescent marker in the gastrointestinal tract of living mice.

High concentration formulation developability approaches and considerations.

The growing need for biologics to be administered subcutaneously and ocularly, coupled with certain indications requiring high doses, has resulted in an increase in drug substance (DS) and drug product (DP) protein concentrations. With this increase, more emphasis must be placed on identifying critical physico-chemical liabilities during drug development, including protein aggregation, precipitation, opalescence, particle formation, and high viscosity. Depending on the molecule, liabilities, and administration route, different formulation strategies can be used to overcome these challenges. However, due to the high material requirements, identifying optimal conditions can be slow, costly, and often prevent therapeutics from moving rapidly into the clinic/market. In order to accelerate and derisk development, new experimental and in-silico methods have emerged that can predict high concentration liabilities. Here, we review the challenges in developing high concentration formulations, the advances that have been made in establishing low mass and high-throughput predictive analytics, and advances in in-silico tools and algorithms aimed at identifying risks and understanding high concentration protein behavior.

Self-efficacy of family caregivers of older adults with cognitive impairment: A concept analysis.

BACKGROUND: Research demonstrates that increased self-efficacy can help family caregivers of older adults with Alzheimer's and other types of cognitive impairment experience lower burden and depressive symptom severity. AIMS: The purpose of this concept analysis is to address fundamental gaps in the understanding of self-efficacy in family caregivers of older adults with cognitive impairment, including updating the 26-year-old concept analysis with a contemporary definition. METHODS: This study utilizes Walker and Avant's (2019) concept analysis method, an eight-step iterative process that helps to clarify ambiguous concepts. A literature review was conducted from July 1993 through March 2019 using PubMed/MEDLINE, Scopus, CINAHL, and Embase. Inclusion criteria encompassed peer-reviewed research articles and review articles that included family caregivers of older adults with cognitive impairment. RESULTS: Eight defining attributes of this concept are identified. The revised definition of self-efficacy in this population is a family caregiver's confidence in their ability to: manage behaviors and other caregiving stresses, control upsetting thoughts, acquire medical information, manage medical issues, obtain self-care, access community supports, assist with activities of daily living and other care, and maintain a good relationship with a relative, friend, or neighbor of an older adult with cognitive impairment. CONCLUSION: This paper utilizes over a quarter-century of research to build on the original analysis by Mowat and Spence Laschinger (1994) and update the concept's definition. This analysis should provide researchers with a clearer understanding of this concept and a renewed emphasis on the importance of targeting interventions to improve self-efficacy in this vulnerable caregiving population.

Adsorption of non-ionic surfactant and monoclonal antibody on siliconized surface studied by neutron reflectometry.

The adsorption of monoclonal antibodies (mAbs) on hydrophobic surfaces is known to cause protein aggregation and degradation. Therefore, surfactants, such as Poloxamer 188, are widely used in therapeutic formulations to stabilize mAbs and protect mAbs from interacting with liquid-solid interfaces. Here, the adsorption of Poloxamer 188, one mAb and their competitive adsorption on a model hydrophobic siliconized surface is investigated with neutron scattering coupled with contrast variation to determine the molecular structure of adsorbed layers for each case. Small angle neutron scattering measurements of the affinity of Poloxamer 188 to this mAb indicate that there is negligible binding at these solution conditions. Neutron reflectometry measurements of the mAb show irreversible adsorption on the siliconized surface, which cannot be washed off with neat buffer. Poloxamer 188 can be adsorbed on the surface already occupied by mAb, which enables partial removal of some adsorbed mAb by washing with buffer. The adsorption of the surfactant introduces significant conformational changes for mAb molecules that remain on the surface. In contrast, if the siliconized surface is first saturated with the surfactant, no adsorption of mAb is observed. Competitive adsorption of mAb and Poloxamer 188 from solution leads to a surface dominantly occupied with surfactant molecules, whereas only a minor amount of mAb absorbs. These findings clearly indicate that Poloxamer 188 can protect against mAb adsorption as well as modify the adsorbed conformation of previously adsorbed mAb.

Protein-Polydimethylsiloxane Particles in Liquid Vial Monoclonal Antibody Formulations Containing Poloxamer 188.

Surfactants play an important role in stabilizing proteins in liquid formulations against aggregate/particle formation during processing, handling, storage, and transportation. Only 3 surfactants are currently used in marketed therapeutic protein formulations: polysorbate 20, polysorbate 80, and poloxamer 188. While polysorbates are the most widely used surfactants, their intrinsic oxidative and hydrolytic degradation issues highlights the importance of alternative surfactants such as poloxamer 188. Here, we compare polysorbates and poloxamer 188 with regards to their stabilizing properties under various stress and storage conditions for several monoclonal antibody formulations. Our data shows that poloxamer 188 can provide suitable protection of monoclonal antibodies against interfacial stress in liquid formulations in vials. However, visible protein-polydimethylsiloxane (PDMS; silicone oil) particles were observed in vials after long-term storage at 2-8°C for some protein formulations using poloxamer 188, which were not observed in polysorbate formulations. The occurrence of these protein-PDMS particles in poloxamer 188 formulations is a protein-specific phenomenon that may correlate with protein physico-chemical properties. In this study, the primary source of the PDMS in particles found in vials was considered to be from the primary packaging stoppers used. Our findings highlight benefits, but also risks associated with using poloxamer 188 in liquid biotherapeutic formulations.

Footprint of Reports From Low- and Low- to Middle-Income Countries in the Neurosurgical Data: A Study From 2015 to 2017.

OBJECTIVE: In 2015, the Lancet Commission on Global Surgery highlighted the disparities in surgical care worldwide. The aim of the present study was to investigate the research productivity of low-income countries (LICs) and low- to middle-income countries (LMICs) in selected journals representing the worldwide neurosurgical data and their ability to report and communicate globally the existing differences between high-income countries (HICs) and LMICs. METHODS: We performed a retrospective bibliometric analysis using PubMed and Scopus databases to record all the reports from 2015 to 2017 by investigators affiliated with neurosurgical departments in LICs and LMICs. RESULTS: A total of 8459 reports by investigators self-identified as members of neurosurgery departments worldwide were identified. Of these, 6708 reports were included in accordance with our method in the final analysis. The systematic search resulted in 459 studies reported by LICs and LMICs. Of these, 334 reports were included for the full text evaluation. Of the 6708 reports, 303 (4.52%) had been reported with an LMIC affiliation and only 31 (0.46%) with an LIC. The leading countries were India with 182 (54.5% among LMICs and LICs; 2.71% overall), followed by Egypt at 66 (19.76% among the LMICs and LICs; 0.98% overall), with a large difference compared with other countries such as Uganda at 9 (2.69% among the LMICs and LICs) and Tunisia and Pakistan at 8 each (2.4% among the LMICs and LICs). A few reports studies had been generated by collaboration with HIC neurosurgeons. CONCLUSIONS: Our results have shown that research studies from LMICs are underrepresented. Understanding and discussing the reasons for this underrepresentation are necessary to start addressing the disparities in neurosurgical research and care capacity. Future engagements from international journals, more partnership collaboration from HICs, and tailored funding to support investigators, collaborations, and networks could be of help.

Advanced Methodologies in High-Throughput Sequencing of Immune Repertoires.

In recent years, major efforts have been made to develop sophisticated experimental and bioinformatic workflows for sequencing adaptive immune repertoires. The immunological insight gained has been applied to fields as varied as lymphocyte biology, immunodiagnostics, vaccines, cancer immunotherapy, and antibody engineering. In this review, we provide a detailed overview of these advanced methodologies, focusing specifically on strategies to reduce sequencing errors and bias and to achieve high-throughput pairing of variable regions (e.g., heavy-light or alpha-beta chains). In addition, we highlight recent technologies for single-cell transcriptome sequencing that can be integrated with immune repertoires. Finally, we provide a perspective on advanced immune repertoire sequencing and its ability to impact basic immunology, biopharmaceutical drug discovery and development, and cancer immunotherapy.

Approaches to Mitigate the Unwanted Immunogenicity of Therapeutic Proteins during Drug Development.

All biotherapeutics have the potential to induce an immune response. This immunological response is complex and, in addition to antibody formation, involves T cell activation and innate immune responses that could contribute to adverse effects. Integrated immunogenicity data analysis is crucial to understanding the possible clinical consequences of anti-drug antibody (ADA) responses. Because patient- and product-related factors can influence the immunogenicity of a therapeutic protein, a risk-based approach is recommended and followed by most drug developers to provide insight over the potential harm of unwanted ADA responses. This paper examines mitigation strategies currently implemented and novel under investigation approaches used by drug developers. The review describes immunomodulatory regimens used in the clinic to mitigate deleterious ADA responses to replacement therapies for deficiency syndromes, such as hemophilia A and B, and high risk classical infantile Pompe patients (e.g., cyclophosphamide, methotrexate, rituximab); novel in silico and in vitro prediction tools used to select candidates based on their immunogenicity potential (e.g., anti-CD52 antibody primary sequence and IFN beta-1a formulation); in vitro generation of tolerogenic antigen-presenting cells (APCs) to reduce ADA responses to factor VIII and IX in murine models of hemophilia; and selection of novel delivery systems to reduce in vivo ADA responses to highly immunogenic biotherapeutics (e.g., asparaginase). We conclude that mitigation strategies should be considered early in development for biotherapeutics based on our knowledge of existing clinical data for biotherapeutics and the immune response involved in the generation of these ADAs.

Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting.

High-throughput antibody repertoire sequencing (Ig-seq) provides quantitative molecular information on humoral immunity. However, Ig-seq is compromised by biases and errors introduced during library preparation and sequencing. By using synthetic antibody spike-in genes, we determined that primer bias from multiplex polymerase chain reaction (PCR) library preparation resulted in antibody frequencies with only 42 to 62% accuracy. Additionally, Ig-seq errors resulted in antibody diversity measurements being overestimated by up to 5000-fold. To rectify this, we developed molecular amplification fingerprinting (MAF), which uses unique molecular identifier (UID) tagging before and during multiplex PCR amplification, which enabled tagging of transcripts while accounting for PCR efficiency. Combined with a bioinformatic pipeline, MAF bias correction led to measurements of antibody frequencies with up to 99% accuracy. We also used MAF to correct PCR and sequencing errors, resulting in enhanced accuracy of full-length antibody diversity measurements, achieving 98 to 100% error correction. Using murine MAF-corrected data, we established a quantitative metric of recent clonal expansion-the intraclonal diversity index-which measures the number of unique transcripts associated with an antibody clone. We used this intraclonal diversity index along with antibody frequencies and somatic hypermutation to build a logistic regression model for prediction of the immunological status of clones. The model was able to predict clonal status with high confidence but only when using MAF error and bias corrected Ig-seq data. Improved accuracy by MAF provides the potential to greatly advance Ig-seq and its utility in immunology and biotechnology.

Key interactions of surfactants in therapeutic protein formulations: A review.

Proteins as amphiphilic, surface-active macromolecules, demonstrate substantial interfacial activity, which causes considerable impact on their multifarious applications. A commonly adapted measure to prevent interfacial damage to proteins is the use of nonionic surfactants. Particularly in biotherapeutic formulations, the use of nonionic surfactants is ubiquitous in order to prevent the impact of interfacial stress on drug product stability. The scope of this review is to convey the current understanding of interactions of nonionic surfactants with proteins both at the interface and in solution, with specific focus to their effects on biotherapeutic formulations.

Determination of the Density of Protein Particles Using a Suspended Microchannel Resonator.

One of the analytical tools for characterization of subvisible particles, which gained popularity over the last years because of its unique capabilities, is the resonance mass measurement technique. However, a challenge that this technique presents is the need to know the exact density of the measured particles in order to obtain accurate size calculations. The density of proteinaceous subvisible particles has not been measured experimentally yet and to date researchers have been using estimated density values. In this paper, we report for a first-time experimental measurements of the density of protein particles (0.2-5 µm in size) using particles created by stressing three different proteins using four different types of stress conditions. Interestingly, the particle density values that were measured varied between 1.28 and 1.33 g/cm(3) and were lower than previous estimates. Furthermore, it was found that although the density of proteinaceous particles was affected to a very low degree by the stress conditions used to generate them, there is relatively larger difference between particles originating from different classes of proteins (e.g., monoclonal antibody vs. bovine serum albumin).

Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade.

Targeted drug delivery with antibody-drug conjugates such as the HER2-directed ado-trastuzumab emtansine (T-DM1) has emerged as a powerful strategy for cancer therapy. We show that T-DM1 is particularly effective in eliciting antitumor immunity in patients with early breast cancer (WSG-ADAPT trial) and in a HER2-expressing orthotopic tumor model. In the latter, despite primary resistance to immunotherapy, combined treatment with T-DM1 and anti-CTLA-4/PD-1 (cytotoxic T lymphocyte-associated protein-4/programmed cell death protein-1) was curative because it triggered innate and adaptive immunity. Tumor rejection was accompanied by massive T cell infiltration, TH1 (T helper 1) cell polarization, and, notably, a substantial increase in regulatory T cells. Depletion of regulatory T cells resulted in inflammation and tissue damage, implying their essential role in protecting the host during therapy. This study provides insights into the mechanisms of T-DM1's therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

Immunological principles regulating immunomodulation with biomaterials.

The immune system has evolved to recognize and eliminate pathogens; this recognition relies on the identification of structural molecular patterns within unique tissue microenvironments. Therefore, bioengineers can harness these immunological cues to design materials that modulate innate and adaptive immunity in a controlled manner. This review acts as an immunology primer by focusing on the basic molecular and cellular immunology principles governing immunomodulation with biomaterials.