Anti-KIT monoclonal antibody CDX-0159 induces profound and durable mast cell suppression in a healthy volunteer study.

BACKGROUND: Mast cells (MC) are powerful inflammatory immune sentinel cells that drive numerous allergic, inflammatory, and pruritic disorders when activated. MC-targeted therapies are approved in several disorders, yet many patients have limited benefit suggesting the need for approaches that more broadly inhibit MC activity. MCs require the KIT receptor and its ligand stem cell factor (SCF) for differentiation, maturation, and survival. Here we describe CDX-0159, an anti-KIT monoclonal antibody that potently suppresses MCs in human healthy volunteers. METHODS: CDX-0159-mediated KIT inhibition was tested in vitro using KIT-expressing immortalized cells and primary human mast cells. CDX-0159 safety and pharmacokinetics were evaluated in a 13-week good laboratory practice (GLP)-compliant cynomolgus macaque study. A single ascending dose (0.3, 1, 3, and 9 mg/kg), double-blinded placebo-controlled phase 1a human healthy volunteer study (n = 32) was conducted to evaluate the safety, pharmacokinetics, and pharmacodynamics of CDX-0159. RESULTS: CDX-0159 inhibits SCF-dependent KIT activation in vitro. Fc modifications in CDX-0159 led to elimination of effector function and reduced serum clearance. In cynomolgus macaques, multiple high doses were safely administered without a significant impact on hematology, a potential concern for KIT inhibitors. A single dose of CDX-0159 in healthy human subjects was generally well tolerated and demonstrated long antibody exposure. Importantly, CDX-0159 led to dose-dependent, profound suppression of plasma tryptase, a MC-specific protease associated with tissue MC burden, indicative of systemic MC suppression or ablation. CONCLUSION: CDX-0159 administration leads to systemic mast cell ablation and may represent a safe and novel approach to treat mast cell-driven disorders.

[COGERIA: coordination of care for the frail elderly]. / COGERIA : Coordination des soins de la personne âgée fragile.

COGERIA, a cantonal program is the fruit of a close collaboration between the Geneva General Directorate of Health and the major health and social partners in the canton. The program aims to improve inter-professional care for the frail elderly and to adapt their care pathways in close collaboration with their primary care physicians and home healthcare providers. Launched in May 2019, the program includes more than 283 beneficiaries and 152 primary care physicians in collaboration with the home healthcare providers in the Servette and Meyrin areas. Preliminary results show a possible trend towards a decrease in hospitalizations, as well as major satisfaction from beneficiaries and the COGERIA partners.

La Coordination des soins de la personne âgée fragile est un dispositif cantonal né d'une étroite collaboration entre la Direction générale de la santé et les grands partenaires de la santé et du social à Genève. Ce dispositif œuvre à améliorer la prise en charge interprofessionnelle autour des personnes âgées fragiles et à adapter leurs parcours de soins, en étroite collaboration avec les médecins traitants et les prestataires de soins à domicile. Lancé en mai 2019, le dispositif compte plus de 283 personnes incluses, avec 152 médecins traitants du réseau primaire en collaboration avec l'Institution genevoise de maintien à domicile, ainsi qu'une ouverture récente à tous les prestataires de soins dans les zones de la Servette et de Meyrin. Des résultats préliminaires mettent en évidence une possible tendance à une baisse d'hospitalisations ainsi qu'une grande satisfaction de la part des bénéficiaires et des partenaires.

A Study of the Radiation Tolerance of CVD Diamond to 70 MeV Protons, Fast Neutrons and 200 MeV Pions.

We measured the radiation tolerance of commercially available diamonds grown by the Chemical Vapor Deposition process by measuring the charge created by a 120 GeV hadron beam in a 50 µm pitch strip detector fabricated on each diamond sample before and after irradiation. We irradiated one group of samples with 70 MeV protons, a second group of samples with fast reactor neutrons (defined as energy greater than 0.1 MeV), and a third group of samples with 200 MeV pions, in steps, to (8.8±0.9) × 1015 protons/cm2, (1.43±0.14) × 1016 neutrons/cm2, and (6.5±1.4) × 1014 pions/cm2, respectively. By observing the charge induced due to the separation of electron-hole pairs created by the passage of the hadron beam through each sample, on an event-by-event basis, as a function of irradiation fluence, we conclude all datasets can be described by a first-order damage equation and independently calculate the damage constant for 70 MeV protons, fast reactor neutrons, and 200 MeV pions. We find the damage constant for diamond irradiated with 70 MeV protons to be 1.62±0.07(stat)±0.16(syst)× 10-18 cm2/(p µm), the damage constant for diamond irradiated with fast reactor neutrons to be 2.65±0.13(stat)±0.18(syst)× 10-18 cm2/(n µm), and the damage constant for diamond irradiated with 200 MeV pions to be 2.0±0.2(stat)±0.5(syst)× 10-18 cm2/(π µm). The damage constants from this measurement were analyzed together with our previously published 24 GeV proton irradiation and 800 MeV proton irradiation damage constant data to derive the first comprehensive set of relative damage constants for Chemical Vapor Deposition diamond. We find 70 MeV protons are 2.60 ± 0.29 times more damaging than 24 GeV protons, fast reactor neutrons are 4.3 ± 0.4 times more damaging than 24 GeV protons, and 200 MeV pions are 3.2 ± 0.8 more damaging than 24 GeV protons. We also observe the measured data can be described by a universal damage curve for all proton, neutron, and pion irradiations we performed of Chemical Vapor Deposition diamond. Finally, we confirm the spatial uniformity of the collected charge increases with fluence for polycrystalline Chemical Vapor Deposition diamond, and this effect can also be described by a universal curve.

Development of CDX-527: a bispecific antibody combining PD-1 blockade and CD27 costimulation for cancer immunotherapy.

CD27 is a costimulatory molecule that provides a complementary target to the PD-1/PD-L1 checkpoint axis on T cells. Combining a CD27 agonist antibody with PD-1/PD-L1 blockade has shown synergistic antitumor activity in preclinical models, which led to clinical studies of the combination in cancer patients. We theorized that coupling CD27 costimulation with PD-1/PD-L1 blockade in a bispecific antibody (BsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced CD27 activation by cross-linking through PD-L1 and Fc receptors. To test this approach, we developed CDX-527, a tetravalent PD-L1xCD27 IgG1-scFv BsAb. CDX-527 potently inhibits PD-1 signaling and induces CD27-mediated T cell costimulation through PD-L1 cross-linking. In mixed lymphocyte reaction assays, CDX-527 is more potent than the combination of the parental antibodies, suggesting that cross-linking through both Fc receptors and PD-L1 results in enhanced CD27 agonist activity. CDX-527 was shown to mediate effector function against tumor cells overexpressing either CD27 or PD-L1. In human CD27 transgenic mice, we observed that antigen-specific T cell responses to a vaccine are greatly enhanced with a surrogate PD-L1xCD27 BsAb. Furthermore, the BsAb exhibits greater antitumor activity than the combination of the parental antibodies in a syngeneic lymphoma model. A pilot study of CDX-527 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-527 effectively combines PD-1 blockade and CD27 costimulation into one molecule that is more potent than combination of the parental antibodies providing the rationale to advance this BsAb toward clinical studies in cancer patients.

Development of CDX-1140, an agonist CD40 antibody for cancer immunotherapy.

Limitations of immunotherapy include poorly functioning events early in the immune response cycle, such as efficient antigen presentation and T cell priming. CD40 signaling in dendritic cells leads to upregulation of cell surface costimulatory and MHC molecules and the generation of cytokines, which promotes effective priming of CD8+ effector T cells while minimizing T cell anergy and the generation of regulatory T cells. This naturally occurs through interaction with CD40 ligand (CD40L) expressed on CD4+ T-helper cells. CD40 signaling can also be achieved using specific antibodies, leading to several agonist CD40 antibodies entering clinical development. Our approach to select a CD40 agonist antibody was to define a balanced profile between sufficiently strong immune stimulation and the untoward effects of systemic immune activation. CDX-1140 is a human IgG2 antibody that activates DCs and B cells and drives NFkB stimulation in a CD40-expressing reporter cell line. These activities are Fc-independent and are maintained using an F(ab')2 fragment of the antibody. CDX-1140 binds outside of the CD40L binding site, and addition of recombinant CD40L greatly enhances DC and B activation by CDX-1140, suggesting that CDX-1140 may act synergistically with naturally expressed CD40L. CDX-1140 also has both direct and immune-mediated anti-tumor activity in xenograft models. CDX-1140 does not promote cytokine production in whole blood assays and has good pharmacodynamic and safety profiles in cynomolgus macaques. These data support the potential of CDX-1140 as part of a cancer therapy regimen, and a phase 1 trial has recently commenced.

An Evaluation of a Multidisciplinary Pediatric Behavioral Health Medication Initiative Workgroup's Interventions on Medication Prescribing in a Population of Medicaid Patients.

BACKGROUND: In response to concerns surrounding pediatric behavioral health medication prescribing, the Massachusetts Medicaid Pharmacy Program implemented a Pediatric Behavioral Health Medication Initiative (PBHMI), proactively requiring prior authorization for specific behavioral health medications and combination regimens. A multidisciplinary therapeutic class management (TCM) workgroup retrospectively reviews complex cases and conducts prescriber outreach to encourage evidence-based practices in Massachusetts. OBJECTIVE: To evaluate recommendation outcomes of telephonic peer-to-peer consultations conducted by the PBHMI TCM workgroup by assessing the percentage of accepted, modified accepted, or rejected recommendations, as well as prescriber satisfaction with consultation. METHODS: This retrospective evaluation reviewed PBHMI TCM workgroup cases with completed peer-to-peer consultations from September 1, 2015, to August 28, 2016. The proportion of medication interventions (e.g., medication changes, dose reductions, and elimination of polypharmacy within or across behavioral health medication classes) accepted, modified accepted, or rejected were assessed based on pharmacy claims data and prior authorization resubmission, following a peer-to-peer consultation. The medication class and prescriber type were categorized in relation to the acceptance, modified acceptance, or rejection outcomes. Satisfaction with the TCM workgroup process was evaluated with an anonymous survey offered to prescribers who participated in prescriber outreach. RESULTS: A total of 70 cases requiring a peer-to-peer consultation by a TCM workgroup child/adolescent psychiatrist had a completed outreach attempt during the evaluation period. Peer-to-peer consultations resulted in a recommendation acceptance rate of 31.4% (22/70), modified acceptance rate of 44.3% (31/70), and a rejection rate of 24.3% (17/70). Recommendations made during a peer-to-peer consultation were rejected by 30% (12/40) of child/adolescent psychiatrists compared with 16.7% (5/30) of nonchild/adolescent psychiatrists with completed peer-to-peer consultations (P = 0.43). Antipsychotics were most frequently recommended for regimen changes. All recommendations pertaining to a benzodiazepine were accepted by the prescriber. Results of an anonymous prescriber survey assessing satisfaction with the peer-to-peer consultation process exhibited variable responses among individual prescribers. CONCLUSIONS: The small sample size in this observational evaluation and lack of a defined control group prevented direct associations between the endpoints and outcomes. Further research is required to determine if prescriber specialty and medication class may be influencing factors on recommendation acceptance. DISCLOSURES: No outside funding supported this study. The authors have nothing to disclose. A poster of this project was presented at the AMCP Managed Care & Specialty Pharmacy Annual Meeting 2017; March 27-30, 2017; in Denver, CO.

Effect of Surfactants on Mechanical, Thermal, and Photostability of a Monoclonal Antibody.

The purpose of this work was to evaluate the effect of commonly used surfactants (at 0.01% w/v concentration) on mechanical, thermal, and photostability of a monoclonal antibody (MAb1) of IgG1 sub-class and to evaluate the minimum concentration of surfactant (Polysorbate 80) required in protecting MAb1 from mechanical stress. Surfactants evaluated were non-ionic surfactants, Polysorbate 80, Polysorbate 20, Pluronic F-68 (polyoxyethylene-polyoxypropylene block polymer), Brij 35 (polyoxyethylene lauryl ether), Triton X-100, and an anionic surfactant, Caprylic acid (1-Heptanecarboxylic acid). After evaluating effect of surfactants and determining stabilizing effect of Polysorbate 80 against mechanical stress without compromising thermal and photostability of MAb1, the minimum concentration of Polysorbate 80 required for mechanical stability was further examined. Polysorbate 80 concentration was varied from 0 to 0.02%. Mechanical stability was evaluated by agitation of MAb1 at 300 rotations per minute at room temperature for 72 h. Samples were analyzed for purity by SEC-HPLC, turbidity by absorbance at 350 nm, visible particles by visual inspection, and sub-visible particles by light obscuration technique on a particle analyzer. All non-ionic surfactants tested showed a similar effect in protecting against mechanical stress and did not exhibit any significant negative effect on thermal and photostability. However, Caprylic acid had a slightly negative effect on mechanical and photostability when compared to the non-ionic surfactants or sample without surfactant. This work demonstrated that polysorbate 80 is better than other surfactants tested and that a concentration of at least 0.005% (w/v) Polysorbate 80 is needed to protect MAb1 against mechanical stress.

Correlating charge heterogeneity data generated by agarose gel isoelectric focusing and ion exchange chromatography methods.

An isoelectric focusing method (IEF) has been used to assess the charge heterogeneity profile of a monoclonal antibody during the early stages of product development. A more precise and sensitive ion exchange chromatography (IEC/CEX) method was developed and implemented as development progressed and was used concurrently with IEF for lot release and to monitor charge heterogeneity. Charge variants resolved by both methods (IEC and IEF) were purified and characterized. Tryptic peptide mapping and N- linked oligosaccharide profile analyses of the IEC and IEF fractions indicated a structural correlation between the charge variants separated by these two methods. The major sources of molecular heterogeneity were due to the variation in the sialyated carbohydrate structure and heavy chain C-terminal lysine truncation. By monitoring the rates of change in the charge heterogeneity profiles of the monoclonal antibody stored at elevated temperatures by the IEC and IEF methods, a positive correlation between the two methods was established. This approach enabled replacement of the IEF method with the more precise IEC method.

CD27-Mediated Regulatory T Cell Depletion and Effector T Cell Costimulation Both Contribute to Antitumor Efficacy.

CD27, a member of the TNFR superfamily, is constitutively expressed in most T cells and plays crucial roles in T cell effector functions. The costimulation and antitumor activity of CD27 agonistic Abs have been well documented in mouse models. Clinical testing of a human IgG1 anti-CD27 Ab, varlilumab (clone 1F5), is ongoing in cancer patients. In this study, we set out to further understand CD27 as an immunomodulatory target and to address the mechanism of antitumor efficacy using different IgG isotypes of 1F5 in human CD27-transgenic mice. 1F5mIgG1, the only isotype engaging inhibitory FcγRIIB expressed in B cells, elicited the most potent and broad immune response, but terminal differentiation, exhaustion, and apoptosis in the activated effector T cells were inevitable. Accordingly, this isotype was the most effective in eradicating BCL1 lymphoma but had limited efficacy in s.c. tumors. Conversely, 1F5mIgG2a, which interacts with cells expressing activating FcγRs, led to moderate immune activation, as well as to prominent reduction in the number and suppressive activity of regulatory T cells. These combined mechanisms imparted potent antitumor activity to 1F5mIgG2a, particularly against the s.c. tumors. 1F5hIgG1, varlilumab, showed balanced agonistic activity that was prominent at lower doses and depleting activity that was greater at higher doses. 1F5hIgG1 had good antitumor activity in all tumor models tested. Thus, both agonist and depleting properties contribute to the antitumor efficacy of CD27-targeted immunotherapy, and modulation of these activities in patients may be achieved by varying the dose and regimen.

A planned care approach and patient registry to improve adherence to clinical guidelines for the diagnosis and management of attention-deficit/hyperactivity disorder.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) affects almost 2.4 million US children. Because American Academy of Pediatrics guidelines for ADHD recommend use of standardized diagnostic instruments, regular follow-up and the chronic care model, this pilot project sought to implement and assess an electronic registry of patients with ADHD combined with care coordination by a planned care team. METHODS: This quality improvement project was structured with 2 intervention and 2 control clinics to facilitate evaluation of the use of a planned care system for management of ADHD. Care teams included a pediatrician, nurse, medical assistant, and care coordinator and tracked patients using an electronic registry with data drawn from the EMR. Clinical work flows were pilot tested to facilitate use of the Vanderbilt scales and their incorporation into the EMR at intervention sites. Outcome measures included 2 recommended clinical follow-ups based on HEDIS measures as well as use of the Vanderbilt rating scales. Initiation phase measure was for follow-up after initiating medication, while the continuation phase measure was for subsequent follow-up during the first year of treatment. Measures were monitored during the project year and then also in the ensuing period of spread of the intervention to other sites. RESULTS: Although the modified HEDIS initiation phase measure for patients newly on medication remained static at approximately 50% throughout the project period, the continuation phase measure showed improvement from 35% at baseline to 45% at the end of the project assessment year, a 29% increase. Follow-up for patients stable on medications also remained unchanged during the project period, but during subsequent spreading of the intervention to nonproject sites, follow-up of these patients improved to over 90%. In adjusted analyses, patients with ADHD at intervention sites were over 2 times more likely than patients at control sites to have had a Vanderbilt score documented in their records. CONCLUSIONS: The project achieved modest improvements in the diagnostic and treatment process for patients with ADHD. The use of a planned care system and electronic patient registry shows promise for improving the diagnosis and treatment process for patients with ADHD.

Comparison of predicted extinction coefficients of monoclonal antibodies with experimental values as measured by the Edelhoch method.

Pace et al. (1995) [1] recommended an equation used to predict extinction coefficient of a protein. However, no antibody data was included in the development of this equation. The main objective of this study was to therefore investigate how the predicted value of the extinction coefficient is comparable to the experimentally determined extinction coefficient of antibodies measured by the Edelhoch method. We have measured the extinction coefficients (ɛ) of 13 IgG1 monoclonal antibodies (mAbs) in phosphate buffer at pH 7.2. The maximum variability in the experimentally measured extinction coefficient of a given mAb molecule was found to be about 2%. Experimentally determined extinction coefficients of all mAbs were found to be lower than the predicted value, with the maximum difference found to being 4.7%. The highest and lowest values of experimental extinction coefficient among the thirteen IgG1 monoclonal antibodies obtained were 230525.9M(-1)cm(-1) (i.e. 1.55(mg/ml)(-1)cm(-1)) and 191,411.6M(-1)cm(-1) (i.e. 1.29(mg/ml)(-1)cm(-1)). A difference of <3% (with respect to mean value) was observed between the experimental and predicted values of the extinction coefficient. A comprehensive analysis and interpretation of the comparison of the predicted and experimentally determined extinction coefficient by the Edelhoch method is discussed in terms of structural characterization and accessible surface area (ASA).

Referral and follow-up after mental health screening in commercially insured adolescents.

PURPOSE: Although mental health screening is recommended for adolescents, little is known about the predictors of referral to mental health services or engagement in treatment. We examined predictors of mental health referral from primary care and service use for commercially insured youth who had been screened using the Pediatric Symptom Checklist or Youth-Pediatric Symptom Checklist. METHODS: A retrospective chart review was conducted of commercially insured patients 14-17 years of age who were newly identified by the Pediatric Symptom Checklist or Youth-Pediatric Symptom Checklist at a well-child visit. Comparisons were made with propensity-matched negative adolescents meeting the same criteria. Bivariate analyses were conducted to examine differences between positives and negatives and between referred and nonreferred positives. Logistic regression analyses were performed to assess predictors of mental health referral for positive youth. RESULTS: Medical records of 117 positive and 110 negative youth were examined. Compared with negative youth, positive youth were significantly more likely to be referred for mental health treatment (p < .0001) and receive specialty mental health services (p < .0001). Of the positives, 54% were referred for mental health care and 67% of them accepted. However, only 18% completed a face-to-face mental health visit in the next 180 days. Pediatric Symptom Checklist score (odds ratio, 1.21; confidence interval, 1.03-1.42), parental or personal concern (odds ratio, 10.87; confidence interval, 2.70-43.76), and having depressive symptoms (odds ratio, 9.18; confidence interval, 1.49-56.60) were predictive of referral. CONCLUSIONS: Despite identification after behavioral health screening, limited treatment engagement by referred patients persists. Primary care physicians and mental health specialists must enhance their efforts to engage and monitor identified patients.

Vice-presidential behavior in a disability crisis. The case of Thomas R. Marshall.

Vice President Thomas R. Marshall has been criticized for not acting more aggressively to exercise presidential powers and duties after President Woodrow Wilson suffered a stroke in October 1919 which compromised his ability to discharge his office for much of the remainder of his term. Yet Marshall faced formidable constraints in the constitutional, political, institutional, and factual context in which he operated. This paper examines these constraints on Marshall's political behavior. His conduct becomes understandable when viewed in the context of those inhibiting factors. The paper also considers the impact of the presidential inability provisions of the subsequently ratified Twenty-Fifth Amendment which renowned Wilson scholar Arthur Link suggested would have made no difference. While questioning the practicality of that counter-factual, the paper argues that the Amendment would have been helpful but suggests that a Wilson-like situation, if one could be imagined in modern times, could present a relatively taxing challenge to our constitutional system.

Pediatric provider processes for behavioral health screening, decision making, and referral in sites with colocated mental health services.

OBJECTIVE: Validated behavioral health (BH) screens are recommended for use at well-child visits. This study aimed to explore how pediatricians experience and use these screens for subsequent care decisions in primary care. METHODS: The study took place at 4 safety net health centers. Fourteen interviews were conducted with pediatricians who were mandated to use validated BH screens at well-child visits. Interview questions focused on key domains, including clinic BH context, screening processes, assessment of screening scores, and decision making about referral to mental health services. Qualitative analysis used the Framework Approach. RESULTS: A variety of themes emerged: BH screens were well accepted and valued for the way they facilitated discussion of mental health issues. However, screening results were not always used in the way that instrument designers intended. Providers' beliefs about the face validity of the instruments, and their observations about performance of instruments, led to discounting scored results. As a result, clinical decisions were made based on a variety of evidence, including individual item responses, parent or patient concerns, and perceived readiness for treatment. Additionally, providers, although interested in expanding their mental health discussions, perceived a lack of time and of their own skills to be major obstacles in this pursuit. CONCLUSIONS: Screens act as important prompts to stimulate discussion of BH problems, but their actual scored results play a variable role in problem identification and treatment decisions. Modifications to scheduling policies, additional provider training, and enhanced collaboration with mental health professionals could support better BH integration in pediatric primary care.

Changing trends in inpatient care for psychiatrically hospitalized youth: 1991-2008.

Research has focused on changes in the psychiatric treatment of youth in outpatient settings, but less is known about trends in inpatient care. This study documents changes in the lengths of stay (LOS), clinical profiles of youth, and medication use within an inpatient setting in Massachusetts between 1991 and 2008. A chart review of 233 medical records of psychiatrically hospitalized youth was conducted at three points in time (1991, 1998, and 2008). Sample includes youth between ages 4 and 18. Clinical data, including LOS, diagnoses and other clinical variables, and number and type of medications prescribed were compared across sample years. Findings indicate a significant decrease in the LOS coupled with a concurrent increase in psychotropic medication use between each successive sample year. The prescription of anti-psychotic medications, in particular, increased significantly. On clinical indices, findings show that there was an increase in the diagnosis of bipolar spectrum disorders and a concurrent decrease in unipolar diagnoses in the 2008 sample. Attention-deficit and developmental disorders showed little change. Trauma-related disorders were significantly less frequently diagnosed in 2008. Children hospitalized in 1998 and 2008 had more prior hospitalizations and presented with greater acuity than those in the 1991 sample. Results highlight important changes that have occurred in child/adolescent inpatient settings over the past two decades. Data suggest that these changes have not resulted in decreased rates of inpatient hospitalization for youth with more severe psychiatric disorders.

Effect of polysorbate 80 concentration on thermal and photostability of a monoclonal antibody.

Polysorbate 80 is widely used in protein formulations to protect protein against agitation-induced aggregation. In this study, we address concerns about residual peroxide present in Polysorbate 80 on protein stability. Residual peroxide may oxidize active pharmaceutical ingredients leading to reduced stability and may ultimately lead to lower potency and efficacy. The effect of Polysorbate 80 concentration on thermal and photostability of monoclonal antibody of the IgG1 subclass (MAb1) was evaluated at Polysorbate 80 concentrations ranging from 0.00% to 1.00% (w/v). MAb1 samples at 5 mg/mL with various Polysorbate 80 concentrations were subjected to accelerated thermal stress by incubation at 25°C, 40°C, and 50°C for a period of 4 weeks and light stress per ICH guideline Q1B, option 1. Our results show that Polysorbate 80 concentration of 1.00% (w/v) adversely affected thermal and photostability of MAb1. This study demonstrates the importance of carefully choosing Polysorbate 80 concentration in protein formulations to prevent destabilizing effect of Polysorbate 80 on thermal and photostability.

Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines.

Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants' levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment.

Principles and applications of selective biophysical methods for characterization and comparability assessment of a monoclonal antibody.

The strategy for a comparability assessment is developed on a hierarchical risk-based approach. Critical analysis of physicochemical and biological characterization assays is essential for the development of a good comparability protocol. Therefore, selection and sensitivity of these assays is very important. This article discusses a case study to evaluate the sensitivity of various methods in a comparability assessment of three lots of an IgG1 monoclonal antibody (mAb). Analysis with eighteen methods demonstrated that only six of the methods were sensitive enough to show a measurable difference of comparability under accelerated conditions (40°C). Samples stored at 4°C were found to be comparable by all methods. A brief comparison of the results of biochemical and functional assays with biophysical analysis is discussed. Basic principles, applications, strength, and limitations of different biophysical methods are also discussed here.

Effect of polysorbate 80 quality on photostability of a monoclonal antibody.

Polysorbate 80 is one of the key components of protein formulations. It primarily inhibits interfacial damage of the protein molecule due to mechanical stress during shipping and handling. However, polysorbate 80 also affects the formulation photostability. Exposure to light of polysorbate 80 aqueous solution results in peroxide generation, which in turn may result in oxidation of the susceptible amino acid residues in the protein molecule. The purpose of this study was to determine if the photostability of our proprietary IgG(1) monoclonal antibody formulation containing polysorbate 80 is affected by the quality (grade/vendor) of polysorbate 80. Following four types of polysorbate 80 were tested: (1) Polysorbate 80 Super-Refined, Mallinckrodt Baker, (2) Polysorbate 80 NF, Mallinckrodt Baker, (3) Polysorbate 80 NF, EMD Chemicals, and (4) Ultra-pure Polysorbate 80 (HX), NOF Corporation. The samples were exposed to light as per ICH guidelines Q1B. The results of the study show that photostability of the antibody formulation is indeed affected by the quality of polysorbate 80. This study underscores the importance of carefully choosing the quality of polysorbate 80 to ensure the robustness of formulation.

Robustness index score: a new stability parameter for designing robustness into biologic formulations.

Formulation robustness is critical to product quality. A challenge in designing robustness into biologic formulations is the absence of one global parameter, which allows equitable comparison across formulations and quality measures. The new measure introduced here derived from accelerated testing data allows for the design of globally optimal robust formulations. The measure is easy to understand and also numerically and computationally stable when performing optimizations. The measure termed here as robustness index (RobX) score is a relative measure allowing for global optimization across multiple critical quality attributes. Described here are the calculation methods, a discussion of the measure characteristics, and a brief example of using RobX to find a global optimal formulation across multiple quality characteristics within the framework of a mixture experimental design. The data presented are actual stability data generated during a formulation experimental design for a monoclonal antibody. We anticipate this approach will provide a good general measure of drug stability and will help to accelerate the drug development process. We believe it will improve formulation robustness, long-term stability, and the delivery of quality drug products to the patient.