High-concentration Formulation Research Articles

AF4 and PEG Precipitation as Predictive Assays for Antibody Self-Association.

Monoclonal antibodies (mAbs) are often formulated as high-protein-concentration solutions, which in some cases can exhibit physical stability issues such as high viscosity and opalescence. To ensure that mAb-based drugs can meet their manufacturing, stability, and delivery requirements, it is advantageous to screen for and select mAbs during discovery that are not prone to such behaviors. It has been recently shown that both these macroscopic properties can be predicted to a certain extent from the diffusion interaction parameter (kD), which is a measure of self-association under dilute conditions.1 However, kD can be challenging to measure at the early stage of discovery, where a relatively large amount of a high-purity material, which is required by traditional methods, is often not available. In this study, we demonstrate asymmetric field-flow fractionation (AF4) as a tool to measure self-association and therefore identify antibodies with problematic issues at high concentrations. The principle lies on the ability to concentrate the sample close to the membrane during the injection mode, which can reach formulation-relevant concentrations (>100 mg/mL).2 By analyzing a well-characterized library of commercial antibodies, we show that the measured retention time of the antibodies allows us to pinpoint molecules that exhibit issues at high concentrations. Remarkably, our AF4 assay requires very little (30 μg) sample under dilute conditions and does not need extensive sample purification. Furthermore, we show that a polyethylene glycol (PEG) precipitation assay provides results consistent with AF4 and moreover can further differentiate molecules with issues of opalescence or high viscosity. Overall, our results delineate a two-step strategy for the identification of problematic variants at high concentrations, with AF4 for early developability screening, followed by a PEG assay to validate the problematic molecules and further discriminate between opalescence or high-viscosity issues. This two-step antibody selection strategy enables us to select antibodies early in the discovery process, which are compatible with high-concentration formulations.

An Intercompany Perspective on Practical Experiences of Predicting, Optimizing and Analyzing High Concentration Biologic Therapeutic Formulations

Developing high-dose biologic drugs for subcutaneous injection often requires high-concentration formulations and optimizing viscosity, solubility, and stability while overcoming analytical, manufacturing, and administration challenges. To understand industry approaches for developing high-concentration formulations, the Formulation Workstream of the BioPhorum Development Group, an industry-wide consortium, conducted an inter-company collaborative exercise which included several surveys. This collaboration provided an industry perspective, experience, and insight into the practicalities for developing high-concentration biologics. To understand solubility and viscosity, companies desire predictive tools, but experience indicates that these are not reliable and experimental strategies are best. Similarly, most companies prefer accelerated and stress stability studies to in-silico or biophysical-based prediction methods to assess aggregation. In addition, optimization of primary container-closure and devices are pursued to mitigate challenges associated with high viscosity of the formulation. Formulation strategies including excipient selection and application of studies at low concentration to high-concentration formulations are reported. Finally, analytical approaches to high concentration formulations are presented. The survey suggests that although prediction of viscosity, solubility, and long-term stability is desirable, the outcome can be inconsistent and molecule dependent. Significant experimental studies are required to confirm robust product definition as modeling at low protein concentrations will not necessarily extrapolate to high concentration formulations.

Stable High-Concentration Monoclonal Antibody Formulations Enabled by an Amphiphilic Copolymer Excipient.

Monoclonal antibodies are a staple in modern pharmacotherapy. Unfortunately, these biopharmaceuticals are limited by their tendency to aggregate in formulation, resulting in poor stability and often requiring low concentration drug formulations. Moreover, existing excipients designed to stabilize these formulations are often limited by their toxicity and tendency to form particles such as micelles. Here, we demonstrate the ability of a simple "drop-in", amphiphilic copolymer excipient to enhance the stability of high concentration formulations of clinically-relevant monoclonal antibodies without altering their pharmacokinetics or injectability. Through interfacial rheology and surface tension measurements, we demonstrate that the copolymer excipient competitively adsorbs to formulation interfaces. Further, through determination of monomeric composition and retained bioactivity through stressed aging, we show that this excipient confers a significant stability benefit to high concentration antibody formulations. Finally, we demonstrate that the excipient behaves as an inactive ingredient, having no significant impact on the pharmacokinetic profile of a clinically relevant antibody in mice. This amphiphilic copolymer excipient demonstrates promise as a simple formulation additive to create stable, high concentration antibody formulations, thereby enabling improved treatment options such as a route-of-administration switch from low concentration intravenous (IV) to high concentration subcutaneous (SC) delivery while reducing dependence on the cold chain.

S1057 A Randomized Phase I Study Comparing Pharmacokinetics, Safety and Immunogenicity of High Concentration Formulation (100 mg/mL) With GP2017 Formulation (50 mg/mL), an Adalimumab Biosimilar, in Healthy Male Subjects

Introduction: GP2017 (adalimumab-adaz) is an approved biosimilar of adalimumab (ADL)1, with a concentration of 50 mg/mL. A high-concentration formulation (HCF) (100 mg/mL) can reduce the injection volume, and consequently decrease the number of injections required for patients who need to administer a higher dose. A phase I study (randomized, double-blind, parallel, 2-arm study with healthy male subjects) was conducted to demonstrate pharmacokinetics (PK) comparability between a newly developed GP2017 citrate-free -HCF and the currently approved GP2017. Methods: Healthy male subjects were randomized to receive a single 40 mg s.c. injection of either GP2017 or GP2017-HCF. PK, safety, and immunogenicity were assessed over 72 days post-injection. Primary endpoints were maximum serum concentration (Cmax), area under the concentration–time curve from time zero to infinity (AUC0–inf) and area under the concentration–time curve from time zero to 360 hours (AUC0-360); area under the concentration–time curve from time zero to the last quantifiable concentration (AUC0–last) was assessed as a secondary endpoint. PK comparability was concluded when the 90% confidence intervals (CIs) of the ratios of geometric means were within the predefined comparability margin of 0.80 to 1.25. Results: 331 subjects were randomized, of which 330 received study treatment (162 GP2017-HCF; 168 GP2017). The 90% CI of the ratios (GP2017-HCF / GP2017) of geometric means for the PK endpoints (Cmax, AUCinf, AUC0-360 and AUC0–last) were all contained within the pre-defined comparability limits of 0.80 to 1.25 (Figure). Safety was similar across groups (Table). The numbers and proportions of subjects with positive anti-drug antibodies (ADA) and with neutralizing antibodies (NAb) were comparable overall (up to day 72: GP2017-HCF 69.1% / 63%; GP2017 64.9% / 61.3%, respectively) and at all individual visits. Conclusion: PK comparability between GP2017-HCF and GP2017 was demonstrated, supporting the development of the new formulation of GP2017. Safety and immunogenicity profiles were comparable between treatment groups and consistent with previously reported adalimumab data.Figure 1.: Forest plot to compare primary and secondary PK endpoints between the treatment groups Table 1. - TEAEs by primary system organ class (at least 2% of subjects in any treatment group) Primary system organ classPreferred term GP2017-HCFN=162n (%) GP2017N=168n (%) Number of subjects with at least 1 event 80 (49.4) 95 (56.5) General disorders and administration site conditions 55 (34.0) 69 (41.1) Investigations 16 (9.9) 24 (14.3) Musculoskeletal and connective tissue disorders 8 (4.9) 14 (8.3) Nervous system disorders 8 (4.9) 6 (3.6) Skin and subcutaneous tissue disorders 10 (6.2) 4 (2.4) Gastrointestinal disorders 8 (4.9) 3 (1.8) Respiratory, thoracic and mediastinal disorders 2 (1.2) 6 (3.6) Eye disorders 1 (0.6) 4 (2.4) Injury, poisoning and procedural complications 4 (2.5) 1 (0.6)

Antibody apparent solubility prediction from sequence by transfer learning

SummaryDeveloping therapeutic monoclonal antibodies (mAbs) for the subcutaneous administration requires identifying mAbs with superior solubility that are amenable for high-concentration formulation. However, experimental screening is often material and labor intensive. Here, we present a strategy (named solPredict) that employs the embeddings from pretrained protein language modeling to predict the apparent solubility of mAbs in histidine (pH 6.0) buffer. A dataset of 220 diverse, in-house mAbs were used for model training and hyperparameter tuning through 5-fold cross validation. solPredict achieves high correlation with experimental solubility on an independent test set of 40 mAbs. Importantly, solPredict performs well for both IgG1 and IgG4 subclasses despite the distinct solubility behaviors. This approach eliminates the need of 3D structure modeling of mAbs, descriptor computation, and expert-crafted input features. The minimal computational expense of solPredict enables rapid, large-scale, and high-throughput screening of mAbs using sequence information alone during early antibody discovery.

Pharmacokinetics, blood and urine profile effects, and injection site histopathology following three daily injections of subcutaneous high concentration buprenorphine in New Zealand white rabbits (Oryctolagus cuniculus)

Buprenorphine is a common analgesic administered to rabbits and high concentration formulations can reduce handling stress without sacrificing pain relief. The objective of this study was to evaluate the pharmacokinetics of high concentration buprenorphine and its metabolites following multiple subcutaneous doses in the rabbit. Laboratory variables (complete blood cell count , biochemistry profile, urinalysis) were compared for drug effects and injection sites were evaluated via histopathology. High concentration buprenorphine (HCB) was administered subcutaneously (0.24 mg/kg) to six, 17-week-old, intact female, New Zealand white rabbits ( Oryctolagus cuniculus ) for three doses, 24 hours apart. Two control animals received an equal volume of saline. Blood samples were collected at -72, 0, 0.33, 0.66, 1.5, 3, 6, 12, 24, 48, 72, 96, 120, and 144 hours. Buprenorphine and its metabolites were measured via liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). Blood and urine profiles were collected prior to and following HCB administration and compared within and between groups. Injection sites and major organ systems were evaluated grossly and microscopically for drug effects. High concentration buprenorphine administered once every 24 hours for three doses had a variable accumulation index of 1.68 (1.24–2.29), reflecting variability in terminal half-life. Subsequently, the metabolites buprenorphine-3-glucuronide and norbuprenorphine-3-glucuronide had accumulation indices of 1.79 (1.25–2.77) and 1.84 (1.43–2.88), respectively. No significant laboratory changes were attributed to multiple dose drug administration. Local subcutaneous vasculitis and panniculitis were reported in rabbits receiving HCB. Ante-mortem examination did not find clinical disease, however 6 of 8 rabbits, including controls, had multifocal inflammatory changes in the liver and lungs on histopathology. Subcutaneous high concentration buprenorphine in rabbits has substantial pharmacokinetic variability and accumulation of both the parent drug and metabolites occurs with multiple dosing regimens. Subclinical illness may have impacted these findings and is a notable study limitation. Local inflammation in subcutaneous fat at injection sites was noted in rabbits administered HCB. High concentration buprenorphine has a quick onset of action when administered subcutaneously in the rabbit, but individual variability is notable. Subclinical inflammatory illness may prolong buprenorphine metabolism and clearance and should be considered in determining dosing regimens in rabbits, especially if disease is present or suspected. Inflammation at HCB injection sites is a drug sequela; monitoring injection sites is recommended.

GP2017-HCF, a high concentration formulation, demonstrates similar pharmacokinetics, immunogenicity and safety to GP2017, an approved adalimumab biosimilar

ABSTRACT Background GP2017 is an adalimumab biosimilar. The objective of this study is to compare the pharmacokinetics (PK) of GP2017 in its approved formulation and GP2017-high concentration formulation (HCF) in a randomized, double-blind, two-arm PK bridging study. Research design and methods Healthy male subjects received a single 40 mg subcutaneous injection of either GP2017-HCF (n = 162) or GP2017 (n = 168). PK, safety, and immunogenicity were assessed over 72 days post-injection. Results The 90% confidence intervals [CIs] of geometric mean ratios between GP2017-HCF and GP2017 for Cmax, AUC0-inf, AUC0-360 and AUC0-last were within the pre-defined margin of 0.80 to 1.25; thus, PK comparability between GP2017-HCF and GP2017 was demonstrated. Subgroup analysis of PK comparability by anti-drug antibody (ADA) subpopulation showed that the 90% CIs of geometric mean ratios between GP2017-HCF and GP2017 for Cmax, AUC0-inf, AUC0-360 and AUC0-last were within the margin of 0.80 to 1.25 in ADA-positive and ADA-negative subjects. The proportions of subjects with positive ADA responses and with neutralizing antibodies were comparable between the GP2017-HCF and GP2017 groups. GP2017-HCF and GP2017 were well tolerated, and there were no reports of deaths or other serious adverse events. Conclusion Results show PK comparability between GP2017-HCF and GP2017 and comparable safety and tolerability.

Abstract CT198: Subcutaneous delivery of amivantamab in patients with advanced solid malignancies: Initial safety and pharmacokinetic results from the PALOMA study

Abstract Background: Amivantamab, an epidermal growth factor receptor (EGFR)-MET bispecific antibody, is approved for patients with advanced EGFR exon 20 insertion non-small cell lung cancer after progression on platinum-based chemotherapy. First-dose intravenous (IV) delivery leads to infusion-related reactions (IRR) among 66% of patients, resulting in dose interruptions and slower infusion restart rates (infusion duration ranges 2-4 hours) and necessitates splitting of the dose over 2 days (Park Ann Oncol 32[suppl_5]:S981). Subcutaneous (SC) administration of amivantamab, which could simplify and accelerate administration, is being investigated in an ongoing phase 1 study (PALOMA; NCT04606381). Preliminary safety (including IRR) and pharmacokinetics (PK) of SC formulations of amivantamab ± recombinant human hyaluronidase (rHuPH20) for enhanced absorption were evaluated. Methods: PALOMA is an ongoing phase 1 dose escalation study of amivantamab SC in patients with advanced solid tumors who may derive benefit from EGFR or MET-directed therapy. Eligible patients must have progressed after standard-of-care therapy for metastatic disease, be ineligible for, or have declined current standard therapies. The study objectives were to evaluate the feasibility of administration, safety, and PK of a low concentration formulation, 50 mg/mL of amivantamab ± rHuPH20 (Part 1) and a high concentration formulation, 160 mg/mL of amivantamab ± rHuPH20 (Part 2). Patients in Part 1 and Part 2 received the currently approved dosage of amivantamab, 1050 mg (1400 mg for bodyweight ≥80 kg) SC (weekly for the first 4 weeks and every other week thereafter). This study also evaluated administering the full dose of amivantamab on the first day. Results: The full safety, PK, bioavailability, and receptor occupancy data of patients enrolled in Part 1 (n=16) and Part 2 (n=17) will be presented. Compared to IV administration, initial SC experience demonstrates the co-formulation of high concentration amivantamab with rHuPH20 shortened the needed infusion time to less than 5 minutes, with initial bioavailability of approximately 65% of IV administration. Saturation of soluble free EGFR and MET was achieved after the first SC dose. The incidence of IRRs was 18.2%, with all events of grade 1-2 severity. The full amivantamab SC dose was safely given at first administration to 14 patients, potentially obviating the need for split dosing. Conclusions: Initial SC amivantamab ± rHuPH20 was well tolerated with improvements in time and ease of administration and associated with a meaningful reduction in IRRs, eliminating the need for split dosing compared with IV administration. Higher SC dose levels and alternative dosing schedules are being explored. Citation Format: Matthew G. Krebs, Melissa L. Johnson, Byoung Chul Cho, Se-Hoon Lee, Rachel Kudgus-Lokken, Donna Zemlickis, Anna Mitselos, Eileen Berkay, Joshua M. Bauml, Roland E. Knoblauch, Peter Hellemans, Anna Minchom. Subcutaneous delivery of amivantamab in patients with advanced solid malignancies: Initial safety and pharmacokinetic results from the PALOMA study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT198.

Assessment of inhalation toxicity of cigarette smoke and aerosols from flavor mixtures: 5-week study in A/J mice.

Most flavors used in e‐liquids are generally recognized as safe for oral consumption, but their potential effects when inhaled are not well characterized. In vivo inhalation studies of flavor ingredients in e‐liquids are scarce. A structure‐based grouping approach was used to select 38 flavor group representatives (FGR) on the basis of known and in silico‐predicted toxicological data. These FGRs were combined to create prototype e‐liquid formulations and tested against cigarette smoke (CS) in a 5‐week inhalation study.Female A/J mice were whole‐body exposed for 6 h/day, 5 days/week, for 5 weeks to air, mainstream CS, or aerosols from (1) test formulations containing propylene glycol (PG), vegetable glycerol (VG), nicotine (N; 2% w/w), and flavor (F) mixtures at low (4.6% w/w), medium (9.3% w/w), or high (18.6% w/w) concentration or (2) base formulation (PG/VG/N). Male A/J mice were exposed to air, PG/VG/N, or PG/VG/N/F‐high under the same exposure regimen. There were no significant mortality or in‐life clinical findings in the treatment groups, with only transient weight loss during the early exposure adaptation period. While exposure to flavor aerosols did not cause notable lung inflammation, it caused only minimal adaptive changes in the larynx and nasal epithelia. In contrast, exposure to CS resulted in lung inflammation and moderate‐to‐severe changes in the epithelia of the nose, larynx, and trachea. In summary, the study evaluates an approach for assessing the inhalation toxicity potential of flavor mixtures, thereby informing the selection of flavor exposure concentrations (up to 18.6%) for a future chronic inhalation study.

Viscosity Reduction and Stability Enhancement of Monoclonal Antibody Formulations Using Derivatives of Amino Acids

Monoclonal antibodies are complex molecules that often require high concentration formulations to meet clinical requirement and bulk drug substance storage. Stability is a common concern in both high and low concentration formulations, however, high concentration formulations are further complicated by an increase in viscosity, leading to manufacturing and administration challenges. To manage stability and viscosity, drug developers typically use salt, amino acids, sugars, polyols, and surfactant. It is possible that excipients control some of the product quality attributes (CQAs), at the cost of several others. In this paper, we have evaluated several amino acid derivatives and identified bis acetyl lysine and propionyl serine to be efficient and superior to commonly used parenteral excipients for minimizing antibody solution viscosity, as well as mitigating physical and chemical degradation by controlling protein-protein interactions and deamidation.

Stepping Forward to the Next Level: Totality of Evidence for the First High-Concentration Adalimumab Biosimilar, CT-P17.

Biosimilar regulatory evaluation considers the totality of evidence gathered through analytical, non-clinical and clinical studies. CT-P17 is the first high-concentration (100 mg/mL), citrate-free adalimumab biosimilar to receive regulatory approval in Europe for all indications held by reference adalimumab, following comprehensive non-clinical and clinical development programmes. State-of-the art physicochemical and biological methods demonstrated quality, analytical and functional comparability between CT-P17 and reference adalimumab; non-clinical in vivo studies supported biosimilarity. Three phase I and two phase III studies were conducted, with pharmacokinetic equivalence of CT-P17 and reference adalimumab shown in healthy volunteers, and equivalent efficacy demonstrated in patients with rheumatoid arthritis. Safety and immunogenicity profiles were comparable between CT-P17 and reference adalimumab across studies. CT-P17 is available for administration by autoinjector/prefilled pen (AI/PFP), prefilled syringe (PFS) and PFS with needle guard, providing diverse self-injection options for patients. Equivalent pharmacokinetics and comparable overall safety and usability were demonstrated between AI/PFP and PFS devices during the clinical development programme. All CT-P17 devices include fine, 29-gauge needles; combined with the citrate-free, high-concentration formulation, these characteristics reflect the newer reference adalimumab formulation (100 mg/mL) and are associated with reduced injection-site pain. The high-concentration formulation also facilitates treatment delivery via fewer injections. Compared with reference adalimumab, CT-P17 remains stable for longer at room temperature, enhancing ease of storage for patients and healthcare providers. In summary, the totality of evidence supports the biosimilarity of CT-P17 to high-concentration reference adalimumab, and several distinctive features differentiate it from existing adalimumab biosimilars.

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation.

Nonionic surfactant polysorbates, including PS-80 and PS-20, are commonly used in the formulation of biotherapeutic products for both preventing surface adsorption and acting as stabilizer against protein aggregation. Trace levels of residual host cell proteins (HCPs) with lipase or esterase enzymatic activity have been shown to degrade polysorbates in biologics formulation. The measurement and control of these low abundance, high-risk HCPs for polysorbate degradation are an industry-wide challenge to achieve desired shelf life of biopharmaceuticals in liquid formulation, especially for high-concentration formulation product development. Here, we reviewed the challenges, recent advances, and future opportunities of analytical method development, risk assessment, and control strategies for polysorbate degradation during formulation development with a focus on enzymatic degradation. Continued efforts to advance our understanding of polysorbate degradation in biologics formulation will help develop high-quality medicines for patients.

Molecular Flexibility of Antibodies Preserved Even in the Dense Phase after Macroscopic Phase Separation.

Antibody therapies are typically based on high-concentration formulations that need to be administered subcutaneously. These conditions induce several challenges, inter alia a viscosity suitable for injection, sufficient solution stability, and preservation of molecular function. To obtain systematic insights into the molecular factors, we study the dynamics on the molecular level under strongly varying solution conditions. In particular, we use solutions of antibodies with poly(ethylene glycol), in which simple cooling from room temperature to freezing temperatures induces a transition from a well-dispersed solution into a phase-separated and macroscopically arrested system. Using quasi-elastic neutron scattering during in situ cooling ramps and in prethermalized measurements, we observe a strong decrease in antibody diffusion, while internal flexibility persists to a significant degree, thus ensuring the movement necessary for the preservation of molecular function. These results are relevant for a more dynamic understanding of antibodies in high-concentration formulations, which affects the formation of transient clusters governing the solution viscosity.

US FDA-approved therapeutic antibodies with high-concentration formulation: summaries and perspectives.

Thirty four (34) of the total US FDA approved 103 therapeutic antibody drugs, accounts for one third of the total approved mAbs, are formulated with high protein concentration (100 mg/mL or above) which are the focus of this article. The highest protein concentration of these approved mAbs is 200 mg/mL. The dominant administration route is subcutaneous (76%). Our analysis indicates that it may be rational to implement a platform formulation containing polysorbate, histidine and sucrose to accelerate high concentration formulation development for antibody drugs. Since 2015, the FDA approval numbers are significantly increased which account for 76% of the total approval numbers, i.e., 26 out of 34 highly concentrated antibodies. Thus, we believe that the high concentration formulations of antibody drugs will be the future trend of therapeutic antibody formulation development, regardless of the challenges of highly concentrated protein formulations.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Subcutaneous delivery of amivantamab in patients with advanced solid malignancies: PALOMA, an open-label, multicenter, dose escalation phase 1b study.

TPS3150 Background: Amivantamab, an epidermal growth factor receptor (EGFR)-MET bispecific antibody with immune cell-directing activity, targets activating/resistance EGFR mutations and MET mutations/amplifications. Amivantamab has demonstrated antitumor activity in patients (pts) with EGFR-mutant NSCLC and also in pts with EGFR tyrosine kinase inhibitor-resistant disease. The recommended phase 2 dose (RP2D) is 1050 mg (1400 mg, ≥80 kg) administered as intravenous (IV) infusions weekly (QW) for the first 28-day cycle and every other week (Q2W) thereafter. A subcutaneous (SC) formulation of amivantamab has the potential to reduce pt and physician burden by reducing administration time. The safety and pharmacokinetics (PK) of amivantamab administered SC ± recombinant human hyaluronidase (rHuPH20) will be evaluated. Methods: PALOMA is an ongoing phase 1b dose escalation study of amivantamab SC in pts with advanced solid tumors that may derive benefit from EGFR or MET-directed therapy (NCT04606381). Pts must have progressed on standard of care therapy for metastatic disease, be ineligible for, or have refused current standard therapies. The primary endpoints are trough concentration at the end of QW dosing and safety of SC administration. The objective of part 1 is to evaluate the feasibility, safety, and PK of SC administration of a low concentration (50 mg/mL) formulation of amivantamab alone (Ami-LC) or admixed with rHuPH20 (Ami-LC-MD). Approximately 8 pts will be enrolled to receive either 1050/1400 mg amivantamab SC using Ami-LC-MD (Cohort 1a) or Ami-LC (Cohort 1b) QW in cycle 1 and Q2W thereafter. The objective of part 2 is to evaluate the safety and PK of SC administration of a high concentration (160 mg/mL) formulation of amivantamab alone (Ami-HC) or with rHuPH20 (Ami-HC-CF) and to determine a dose, schedule, and formulation for SC administration that achieves similar exposure as observed at the RP2D of amivantamab IV, with acceptable safety. Pts enrolled in part 2 will initially receive 1050/1400 mg amivantamab SC using Ami-HC-CF in Cohort 2a or Ami-HC in Cohort 2b. ≤10 pts may be enrolled in either cohort. Additional cohorts of ≤10 pts may be enrolled to support dose, schedule, and formulation selection as guided by safety and PK observations in earlier cohorts. To mitigate infusion related reactions (IRR), medication with steroid, paracetamol, and antihistamine will be given pre-infusion and as clinically indicated post-infusion. Safety assessments include monitoring adverse events, laboratory abnormalities, vital signs, IRR, and injection site reactions. Blood samples will be collected to assess PK, pharmacodynamics, and immunogenicity. A Study Evaluation Team composed of investigators and sponsor representatives will review safety and PK data to make decisions about dose escalation and cohort expansion throughout the conduct of the study. Clinical trial information: NCT04606381.

Pharmacokinetics of a high-concentration formulation of buprenorphine (Simbadol) in male dogs

ObjectiveTo describe the pharmacokinetics of buprenorphine in dogs following administration of a high-concentration formulation of buprenorphine. Study designProspective, randomized, crossover study. AnimalsA total of six healthy male intact Beagle dogs, aged 9–13 months and weighing 10.3 ± 1.4 kg (mean ± standard deviation). MethodsDogs were randomized to be administered buprenorphine (0.12 mg kg–1; Simbadol, 1.8 mg mL–1) via the intravenous (lateral saphenous) or subcutaneous (dorsal interscapular) route followed by the alternative route of administration after a 14 day interval. Blood was sampled before administration and at set times up to 72 hours after injection. Plasma buprenorphine concentration was measured using liquid chromatography–tandem mass spectrometry. ResultsA three-compartment model with zero or biphasic rapid and slow first-order input in (intravenous or subcutaneous data, respectively) and first-order elimination from the central compartment best fitted the data. The rapid first-order input accounted for 63% of the dosage absorption. Typical values (% interindividual variability) for the three compartment volumes were 900 (33), 2425 (not estimated) and 6360 (28) mL kg–1. The metabolic and two distribution clearances were 25.7 (21), 107.5 (74) and 5.7 (61) mL minute–1 kg–1. The absorption half-life for the fast absorption phase was 8.9 minutes with a 0.7 (103) minute delay. The absorption half-life for the slow absorption phase was 347 minutes with a 226 (42) minute delay. Median (range) bioavailability calculated from noncompartmental analysis was 143 (80–239)%. Calculated terminal half-life was 963 minutes. Conclusions and clinical relevanceThe high-concentration formulation of buprenorphine administered subcutaneously had a large volume of distribution and a rapid absorption phase followed by slower, delayed absorption. The high estimate of bioavailability should be interpreted with caution as values above 100% are most commonly related to experimental issues.

Effects of monoclonal antibody concentration and type of bulking agent on critical quality attributes of lyophilisates

Biopharmaceuticals represent one of the fastest growing areas in the pharmaceutical industry. Previously, protein formulations for intravenous applications were most widespread, while now the subcutaneous route of administration is gaining importance. Subcutaneous administration requires high concentrations of protein drugs. Lyophilisation represents the method of choice for drying such formulations. In this study the effects of monoclonal antibody (mAb) ‘drug’ concentration on the thermal characteristics and viscosities of pre-lyophilised formulations were determined. Additionally, the effects of mAb concentration on reconstitution time, cake appearance and mAb stability were evaluated. The main aim of the study was to investigate the effects of amino acids as selected bulking agents and their ratios to sucrose on the critical quality attributes of lyophilisate formulations at low and high mAb concentrations, as a comparison with the most commonly used mannitol formulations. Harsher aggressive drying conditions did not have any major impact on the critical quality attributes of lyophilisates or on mAb stability, while primary drying time was shortened by up to 80%. The isoleucine:sucrose ratio of 1:4 provided a lyophilised cake with superior macroscopic appearance for low and high mAb concentrations despite aggressive drying, which greatly shortened lyophilisation time. However, the drawback of isoleucine in high protein concentration formulations is the long reconstitution time. In contrast, for low mAb concentrations, an isoleucine:sucrose ratio of 1:4 is promising for replacement of mannitol, as it provides suitable reconstitution time, cake appearance and mAb stability.

The thermal antinociceptive effects of a high-concentration formulation of buprenorphine alone or followed by hydromorphone in conscious cats

ObjectiveTo evaluate the thermal antinociceptive effects of a high-concentration formulation of buprenorphine alone or followed by hydromorphone in conscious cats. Study designRandomized, blinded, placebo-controlled crossover study design. AnimalsA total of six purpose-bred, adult female ovariohysterectomized Domestic Short Hair cats. MethodsCats were allocated into three treatments each consisting of two injections, subcutaneous then intravenous (IV) administration, 2 hours apart: treatment SS, two injections of 0.9% saline; treatment BS, buprenorphine (0.24 mg kg–1, 1.8 mg mL–1) and saline; and treatment BH, buprenorphine (0.24 mg kg–1) and hydromorphone (0.1 mg kg–1). Skin temperature (ST) and thermal threshold (TT) were recorded before (baseline) and for 24 hours following first injection. TT data were analyzed using mixed linear models and a Benjamini–Hochberg sequential adjustment procedure (p < 0.05). ResultsThere were no significant differences among treatments for baseline ST and TT values, treatment SS over time and between treatments BS and BH. Compared with baseline, TT was significantly increased at all time points in treatments BH and BS except at 2 hours in treatment BS. TT was significantly higher than SS at 3–18 hours and 4–12 hours for treatments BS and BH, respectively. Maximal increases in TT were 47.5 °C at 2 hours, 53.9 °C at 3 hours and 52.4 °C at 6 hours in treatments SS, BS and BH, respectively. Conclusions and clinical relevanceAdministration of IV hydromorphone following high-concentration buprenorphine provided no additional antinociception and decreased the duration of effect when compared with high-concentration buprenorphine alone. Alternative analgesics should be considered if additional analgesia is required after administration of high-concentration buprenorphine.