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Fully liquid phase oligonucleotide synthesis
No full textRepresentative oligonucleotides (oligos; a 21-mer siRNA strand and a 16-mer ASOlike
gapmer) were synthesized by Nanostar Sieving, a membrane-assisted liquid phase oligo
synthesis (LPOS) method, at 10 mmol scale and 20 mM concentration in a single solvent. This
process is the first total LPOS, i.e., the reagents and intermediates remain in the liquid phase from
the very first reaction to isolation. At all times during the synthesis of the full-length product (FLP),
the concentration of oligo within the Nanostar-10 reactor remained roughly constant. Both
sequences were synthesized four times, twice using membranes sheets clamped flat in circular cells, and twice using scalable spiral wound modules (SWMs). Cleavage and deprotection
continued to maintain the oligo in the liquid phase until the final lyophilization, when yields were
calculated by mass, adjusting for chromatographic and mass spectral purity. Process mass
intensities equal to or better than solid phase oligo synthesis (SPOS) were achieved
A Conceptual Framework for the Crystallizability of Organic Compounds
No full textOver the last few decades, various organic crystal structures long established in industrial-scale crystallization suddenly and unexpectedly switched to a different, more thermodynamically stable crystal arrangement. This drastic shift altered the physical characteristics of the product significantly. We spent two decades perfecting the computation of free energies of organic crystal structures until we were able to reliably predict the thermodynamic determinant of these catastrophic crystallization events, but those predictions alone cannot explain why an unstable crystal packing was isolated for many years. In this contribution we demonstrate how “molecular strain energy” can be calculated and used to conceptually gauge the ease of a molecule to crystallize from solution into the thermodynamically most stable crystal packing arrangement. By calculating the dominant factor affecting the crystallization kinetics of flexible organic molecules in addition to thermodynamic stability, we predict if a thermodynamically more stable crystal packing can be expected in the future. With this discovery, we can rationalize the unexpected crystallization of more stable, novel solid forms and provide the pharmaceutical industry with an instrument to direct appropriate resources to the crystallization of these sought-after, frequently elusive, crystal structures
STP Town Hall Discussion on Virtual Control Groups
No full textThe 2025 Town Hall meeting of the Society of Toxicologic Pathology (STP) discussed virtual control groups (VCG) in nonclinical toxicity testing, which are being pursued by multiple organizations to reduce animal use in product development. The current VCG literature infrequently reflects the toxicologic pathology perspective. Audience members noted that concurrent control groups (CCG) are the gold standard for toxicity studies, essential for replenishing the historical control data (HCD) from which VCG are generated. The utility of VCG is context-dependent: acceptable when test article (TA)-related effects are easily separated from background findings but likely unsuitable where subtle findings must be distinguished. Global regulatory acceptance is of paramount concern in trying to shift from CCG to partly or wholly relying on VCG. Alternative approaches to reduce animal use include eliminating “box-checking” studies, obtaining early regulatory agreement that single-species testing is sufficient, and altering study designs to reduce group numbers, sharing CCG, or blending CCG and VCG. The most effective way for toxicologic pathologists to influence this debate is to dispassionately consider opportunities and challenges of VCG related to pathology diagnoses and interpretations and communicate this perspective clearly to the broader (non-pathologist) scientific community
2025 White Paper on Recent Issues in Bioanalysis SECTION 1 – Gene, Cell, and Vaccine Therapies Immunogenicity & Technologies SECTION 2 – Biotherapeutics & Biosimilars Immunogenicity Assessment & Clinical Relevance
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Predicting and Confirming Bioequivalence of Alpelisib Oral Granules and Tablets for Patients With PIK3CA-Related Disorders.
No full textAlpelisib, an oral α-specific phosphoinositide 3-kinase (PI3K) inhibitor, has been shown to be safe and effective for some patients with gain-of-function mutation in the PIK3CA oncogene. Alpelisib has received US FDA accelerated approval as Vijoice® film-coated tablets to treat severe PIK3CA-Related Overgrowth Spectrum (PROS). PROS typically displays clinical manifestations in the first year of patient life. Therefore, oral granules were developed as an age-appropriate pediatric dosage form. Bioequivalence between alpelisib granules and tablet and the effect of food on granules pharmacokinetics were assessed in a single-center, randomized, three-treatment, six-sequence, three-period, crossover study among 60 healthy adults. Participants were randomly assigned to receive a single 50-mg alpelisib dose as: (i) tablet following a meal, (ii) granules following a meal, and (iii) granules while fasting. Statistical analysis of non-compartmental pharmacokinetic parameters demonstrated bioequivalence between the 50-mg alpelisib granules and tablet forms when administered with food: estimated geometric mean ratios (90% confidence interval) for granules-versus-tablet area under the curve (AUC) from time zero to infinity (AUCinf), to the last measurable concentration (AUClast) and maximum observed concentration (Cmax) were 0.984 (0.952, 1.02), 0.980 (0.946, 1.02), and 0.947 (0.891, 1.01), respectively. No clinically relevant food effect on 50-mg alpelisib granules pharmacokinetics was observed. These results were accurately predicted using physiologically based biopharmaceutical modeling. Alpelisib granules provide a bioequivalent alternative to tablets for patients prescribed a 50-mg dose and have difficulty swallowing tablets, an important consideration for convenience and compliance of this standard-of-care chronic therapy for patients with PROS. This study was registered in ClinicalTrials.gov on January 4, 2022 (NCT05195892)
Noncomplex stability predictions for complex biotherapeutics: advanced kinetic modeling simplified
No full textIn this article, the focus will primarily be on the critical long-term predictions of aggregates for different protein modalities. It will be demonstrated that this concentration-dependent quality attribute can be effectively modelled using a first-order kinetic model. This model characterizes the stability profiles of quality attributes through exponential functions, providing robustness and high precision in stability predictions. The use of a first-order kinetic model emphasizes the vital role of temperature selection in stability studies. By carefully choosing the appropriate temperature conditions, it becomes possible to identify the dominant degradation process and accurately describe it using a simple first-order kinetic model. This approach helps to prevent the activation of additional degradation mechanisms that are not relevant for storage conditions, allowing for the design of a study focused on a single mechanism. The simplicity of the kinetic model obtained from this approach reduces the number of parameters that need to be fitted and minimizes the number of samples that need to be measured. This enhances the robustness and reliability of predictions. Furthermore, by utilizing proper temperature conditions, the majority of quality attributes in biologics, including aggregation, can be successfully modelled using a first-order kinetic framework. This highlights the crucial importance of temperature selection in studying the stability and degradation of biologics
The future of parenteral dosage forms manufacturing
No full textThe article provides a brief historical overview of parenteral pharmaceutical dosage forms production and discusses the fundamental requirements for their manufacturing. Further it focuses on expected future trends in production, such as the introduction of gloveless robotic systems in both industrial manufacturing and hospital pharmacies. Their implementation eliminates human intervention in aseptic environments, which represents the most critical aspect of parenteral pharmaceutical production. At the same time, adjustments in microbiological sampling within aseptic conditions can be expected. These adjustments reflect the need for the introduction of alternative rapid tests for microorganisms detection and a greater emphasis on monitoring critical process parameters that play a crucial role in ensuring the sterility of the entire batch of the pharmaceutical product. Among future directions, it is also important to highlight the continued expansion of the use of pre-sterilized primary packaging, prefilled syringes, and automatic dispensers
Cooperative Free Energy: Induced Protein-Protein Interactions and Cooperative Solvation in Ternary Complexes.
No full textProtein-protein interactions (PPIs) play an essential role in biological processes. Molecules that stabilize or induce PPIs in ternary complexes have received growing attention for their therapeutic potential in engaging "undruggable" targets and their high selectivity. Here, we investigate the thermodynamics of the cooperative phenomenon in ternary complexes. The thermodynamics of cooperativity are characterized by the cooperative free energy, which comprises induced PPIs, cooperative solvation free energy, ligand-associated geometric free-energy costs, and gas-phase correlation. Importantly, the induced PPIs only account for the binding affinity between stabilized conformations of the protein partners, i.e., the free-energy change associated with the conformational transition during protein-ligand binding is not accounted for. By introducing an approximated expression for the cooperative free energy, we developed a rapid computational method, which allowed us to crudely predict cooperativity in eight ternary complexes (Kendall τ = 0.79). We highlight that the term cooperativity used in protein-protein stabilization does not represent the cooperativity phenomenon in three-body systems. We also critically discuss the counterintuitive interpretation of cooperative free energy due to its asymmetric nature. Our study shows how cooperativity stabilizes ternary complexes and provides a thermodynamic basis of cooperativity in protein-ligand-protein complexes
Strategies for the evaluation and characterization of higher-order structures, supramolecular higher-order structures, and aggregates in oligonucleotide therapeutics.
No full textSynthetic oligonucleotides have emerged as a promising therapeutic class, holding significant potential for the treatment of a wide range of indications, including previously undruggable targets. Due to specific primary structure motifs, oligonucleotide therapeutics may form higher-order structures (HOS), supramolecular higher-order structures (sHOS) and/or aggregates, which are influenced by factors such as dissolution medium and oligonucleotide concentration. Although rarely observed, unintended (s)HOS and/or aggregates might influence various stages of drug substance (DS) or drug product (DP) manufacturing. There is no published guidance on how to best approach the characterization of oligonucleotide therapeutics (s)HOS and aggregation in a scientific and systematic manner. In this article, we provide an overview of oligonucleotide therapeutics (s)HOS formation and aggregation behavior. Based on industry experience and available literature, we also propose strategies for their evaluation and characterization under relevant conditions. The recommended approach involves conducting appropriate scientific assessments during product development with the support of designed workflows, which can help anticipate and/or mitigate the formation of unintended species
Clinical significance of drug–drug interaction studies during therapeutic peptide drug development: Follow-up investigation of therapeutic peptides approved between 2021 and 2024
No full textThe risk of clinically relevant drug–drug interactions (DDIs) for therapeutic peptides remains unclear, mandating a comprehensive analysis for this modality. In our prior study, we analyzed DDIs for 31 peptide drugs approved between January 2008 and August 2021. Here, we analyze DDI data for an additional nine peptide drugs (trofinetide, nirmatrelvir, danicopan, odevixibat, rezafungin, motixafortide, zilucoplan, vosoritide, and tirzepatide) approved from September 2021 to September 2024, focusing on in vitro and clinical DDI data for metabolism-and transporter-based
interactions. All nine peptides investigated CYP inhibition in human liver microsomes (HLMs), with low risk identified for larger peptides (> 2 kDa). Likewise, all nine peptides assessed CYP induction in human hepatocytes, with one peptide showing a risk in vitro (danicopan). Phenotyping investigations varied from standard studies (e.g., HLMs with selective CYP inhibitors) to submission packages without classical phenotyping studies. All nine peptides included information related to in vitro transporter properties, but the level of detail varied between submitted packages. Clinical studies investigating metabolism-or transporter-mediated DDIs were performed for four peptides (all < 2 kDa). Area under the curve changes attributed to the peptide drug were < 2.3 fold. Our expanded dataset now includes 40 therapeutic peptides approved since 2008, providing a unique resource for drug developers. The findings reinforce our previous conclusions regarding the low likelihood of DDIs for larger peptides and a higher risk for smaller peptides with xenobiotic structural properties. This collective data will be invaluable in developing clear and meaningful DDI guidelines for therapeutic peptides