Definition of a platform continuous capture scale down model and link to scale-up for monoclonal antibody clinical manufacturing

Definition and scale-up of a batch chromatography process is based on a few main variables such as linear velocity, column loading, and bed height, which are scaled proportionally to column volume. Continuous chromatography consists of multiple columns with column loading and washes/elution/regeneration occurring simultaneously. The definition of a small scale method for continuous chromatography can be extremely complex due to the extensive number of method variables. Limited knowledge exists for development of a scale down and up strategy for continuous chromatography. This abstract should provide some insight into case studies on integration of continuous operations and scale-up, which is one of the themes of the integrated continuous biomanufacturing (ICB) conference. This presentation will describe a strategy for definition of a platform continuous capture scale down model and scale-up pathway. The platform continuous capture step utilizes periodic counter-current chromatography (PCC) for operation of affinity chromatography in a semi-continuous manner. A scale down model for the PCC step was defined and simplified to the following three ranges of harvested cell culture fluid (HCCF) titers: ≤ 2 g/L, 2.5-8 g/L, and 8.5-13 g/L. For each of the three titer ranges, the following variable setpoints are changed based on the specific HCCF titer range: step linear velocity, number of columns, column size, and ΔUV. After these setpoints are inputted into the algorithm, PCC method variables, such as sample loading flowrate, loop time, number of loops and cycles, throughput (g/L/hr), and time cycle, will populate to finish the method design. This PCC scale down model was utilized to scale-up to a bioreactor range of 500-2000L. Quality results showed a good correlation between scale down model and scale-up data. Additional parameters for the 2000L scale-up run included assessment of cleaning and drug substance stability. The cleaning results of the continuous chromatography skid showed passing bioburden, endotoxin, and conductivity. Drug substance stability was also maintained for a year, which was the study duration. This data set proves the PCC small scale model data is representative of the scale-up quality results. In addition, targets such as skid cleanability and DS stability met specifications, which supports the scale-up package for implementation of a platform continuous capture step into a purification process for clinical mAb manufacturing

Vemurafenib-resistant BRAF-V600E-mutated melanoma is regressed by MEK-targeting drug trametinib, but not cobimetinib in a patient-derived orthotopic xenograft (PDOX) mouse model.

Melanoma is a recalcitrant disease. The present study used a patient-derived orthotopic xenograft (PDOX) model of melanoma to test sensitivity to three molecularly-targeted drugs and one standard chemotherapeutic. A BRAF-V600E-mutant melanoma obtained from the right chest wall of a patient was grown orthotopically in the right chest wall of nude mice to establish a PDOX model. Two weeks after implantation, 50 PDOX nude mice were divided into 5 groups: G1, control without treatment; G2, vemurafenib (VEM) (30 mg/kg); G3; temozolomide (TEM) (25 mg/kg); G4, trametinib (TRA) (0.3 mg/kg); and G5, cobimetinib (COB) (5 mg/kg). Each drug was administered orally, daily for 14 consecutive days. Tumor sizes were measured with calipers twice a week. On day 14 from initiation of treatment, TRA, an MEK inhibitor, was the only agent of the 4 tested that caused tumor regression (P < 0.001 at day 14). In contrast, another MEK inhibitor, COB, could slow but not arrest growth or cause regression of the melanoma. First-line therapy TEM could slow but not arrest tumor growth or cause regression. The patient in this study had a BRAF-V600E-mutant melanoma and would be considered to be a strong candidate for VEM as first-line therapy, since VEM targets this mutation. However, VEM was not effective. The PDOX model thus helped identify the very-high efficacy of TRA against the melanoma PDOX and is a promising drug for this patient. These results demonstrate the powerful precision of the PDOX model for cancer therapy, not achievable by genomic analysis alone

Dynamics in Stationary, Non-Globally Hyperbolic Spacetimes

Classically, the dynamics in a non-globally hyperbolic spacetime is ill posed. Previously, a prescription was given for defining dynamics in static spacetimes in terms of a second order operator acting on a Hilbert space defined on static slices. The present work extends this result by giving a similar prescription for defining dynamics in stationary spacetimes obeying certain mild assumptions. The prescription is defined in terms of a first order operator acting on a different Hilbert space from the one used in the static prescription. It preserves the important properties of the earlier one: the formal solution agrees with the Cauchy evolution within the domain of dependence, and smooth data of compact support always give rise to smooth solutions. In the static case, the first order formalism agrees with second order formalism (using specifically the Friedrichs extension). Applications to field quantization are also discussed.Comment: 18 pages, 1 figure, AMSLaTeX; v2: expanded discussion of field quantization, new Proposition 3.1, revised Theorem 4.2, corrected typos, and updated reference

Tumor-targeting Salmonella typhimurium A1-R combined with temozolomide regresses malignant melanoma with a BRAF-V600E mutation in a patient-derived orthotopic xenograft (PDOX) model.

Melanoma is a recalcitrant disease in need of transformative therapuetics. The present study used a patient-derived orthotopic xenograft (PDOX) nude-mouse model of melanoma with a BRAF-V600E mutation to determine the efficacy of temozolomide (TEM) combined with tumor-targeting Salmonella typhimurium A1-R. A melanoma obtained from the right chest wall of a patient was grown orthotopically in the right chest wall of nude mice to establish a PDOX model. Two weeks after implantation, 40 PDOX nude mice were divided into 4 groups: G1, control without treatment (n = 10); G2, TEM (25 mg/kg, administrated orally daily for 14 consecutive days, n = 10); G3, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); G4, TEM combined with S. typhimurium A1-R (25 mg/kg, administrated orally daily for 14 consecutive days and 5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, respectively, n = 10). Tumor sizes were measured with calipers twice a week. On day 14 from initiation of treatment, all treatments significantly inhibited tumor growth compared to untreated control (TEM: p < 0.0001; S. typhimurium A1-R: p < 0.0001; TEM combined with S. typhimurium A1-R: p < 0.0001). TEM combined with S. typhimurium A1-R was significantly more effective than either S. typhimurium A1-R (p = 0.0004) alone or TEM alone (p = 0.0017). TEM combined with S. typhimurium A1-R could regress the melanoma in the PDOX model and has important future clinical potential for melanoma patients

Combination treatment with recombinant methioninase enables temozolomide to arrest a BRAF V600E melanoma in a patient-derived orthotopic xenograft (PDOX) mouse model.

An excessive requirement for methionine termed methionine dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by methionine deprivation such as with recombinant methioninase (rMETase). The present study used a previously-established patient-derived orthotopic xenograft (PDOX) nude mouse model of BRAF V600E-mutant melanoma to determine the efficacy of rMETase in combination with a first-line melanoma drug, temozolomide (TEM). In the present study 40 melanoma PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n=10); TEM (25 mg/kg, oral 14 consecutive days, n=10); rMETase (100 units, intraperitoneal 14 consecutive days, n=10); combination TEM + rMETase (TEM: 25 mg/kg, oral rMETase: 100 units, intraperitoneal 14 consecutive days, n=10). All treatments inhibited tumor growth compared to untreated control (TEM: p=0.0081, rMETase: p=0.0037, TEM-rMETase: p=0.0024) on day 14 after initiation. However, the combination therapy of TEM and rMETase was significantly more efficacious than either mono-therapy (TEM: p=0.0051, rMETase: p=0.0051). The present study is the first demonstrating the efficacy of rMETase combination therapy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as melanoma, where rMETase may enhance first-line therapy

Recombinant methioninase (rMETase) is an effective therapeutic for BRAF-V600E-negative as well as -positive melanoma in patient-derived orthotopic xenograft (PDOX) mouse models.

Melanoma is a recalcitrant disease. Melanoma patients with the BRAF-V600E mutation have been treated with the drug vemurafenib (VEM) which targets this mutation. However, we previously showed that VEM is not very effective against a BRAF-V600E melanoma mutant in a patient-derived orthotopic xenograft (PDOX) model. In contrast, we demonstrated that recombinant methioninase (rMETase) which targets the general metabolic defect in cancer of methionine dependence, was effective against the BRAF-V600E mutant melanoma PDOX model. In the present study, we demonstrate that rMETase is effective against a BRAF-V600E-negative melanoma PDOX which we established. Forty BRAF-V600E-negative melanoma PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); temozolomide (TEM) (25 mg/kg, p.o., 14 consecutive days, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); TEM + rMETase (TEM: 25 mg/kg, p.o., rMETase: 100 units, i.p., 14 consecutive days, n = 10). All treatments inhibited tumor growth compared to untreated control (TEM: p = 0.0003, rMETase: p = 0.0006, TEM/rMETase: p = 0.0002) on day 14 after initiation. Combination therapy of TEM and rMETase was significantly more effective than either mono-therapy (TEM: p = 0.0113, rMETase: p = 0.0173). The present study shows that TEM combined with rMETase is effective for BRAF-V600E-negative melanoma PDOX similar to the BRAF-V600E-positive mutation melanoma. These results suggest rMETase in combination with first-line chemotherapy can be highly effective in both BRAF-V600E-negative as well as BRAF-V600E-positive melanoma and has clinical potential for this recalcitrant disease

Applications of a New Proposal for Solving the "Problem of Time" to Some Simple Quantum Cosmological Models

We apply a recent proposal for defining states and observables in quantum gravity to simple models. First, we consider a Klein-Gordon particle in an ex- ternal potential in Minkowski space and compare our proposal to the theory ob- tained by deparametrizing with respect to a time slicing prior to quantiza- tion. We show explicitly that the dynamics of the deparametrization approach depends on the time slicing. Our proposal yields a dynamics independent of the choice of time slicing at intermediate times but after the potential is turned off, the dynamics does not return to the free particle dynamics. Next we apply our proposal to the closed Robertson-Walker quantum cosmology with a massless scalar field with the size of the universe as our time variable, so the only dynamical variable is the scalar field. We show that the resulting theory has the semi-classical behavior up to the classical turning point from expansion to contraction, i.e., given a classical solution which expands for much longer than the Planck time, there is a quantum state whose dynamical evolution closely approximates this classical solution during the expansion. However, when the "time" gets larger than the classical maximum, the scalar field be- comes "frozen" at its value at the maximum expansion. We also obtain similar results in the Taub model. In an Appendix we derive the form of the Wheeler- DeWitt equation for the Bianchi models by performing a proper quantum reduc- tion of the momentum constraints; this equation differs from the usual one ob- tained by solving the momentum constraints classically, prior to quantization.Comment: 30 pages, LaTeX 3 figures (postscript file or hard copy) available upon request, BUTP-94/1

Volterra Distortions, Spinning Strings, and Cosmic Defects

Cosmic strings, as topological spacetime defects, show striking resemblance to defects in solid continua: distortions, which can be classified into disclinations and dislocations, are line-like defects characterized by a delta function-valued curvature and torsion distribution giving rise to rotational and translational holonomy. We exploit this analogy and investigate how distortions can be adapted in a systematic manner from solid state systems to Einstein-Cartan gravity. As distortions are efficiently described within the framework of a SO(3) {\rlap{\supset}\times}} T(3) gauge theory of solid continua with line defects, we are led in a straightforward way to a Poincar\'e gauge approach to gravity which is a natural framework for introducing the notion of distorted spacetimes. Constructing all ten possible distorted spacetimes, we recover, inter alia, the well-known exterior spacetime of a spin-polarized cosmic string as a special case of such a geometry. In a second step, we search for matter distributions which, in Einstein-Cartan gravity, act as sources of distorted spacetimes. The resulting solutions, appropriately matched to the distorted vacua, are cylindrically symmetric and are interpreted as spin-polarized cosmic strings and cosmic dislocations.Comment: 24 pages, LaTeX, 9 eps figures; remarks on energy conditions added, discussion extended, version to be published in Class. Quantum Gra

Neurologic Serious Adverse Events Associated with Nivolumab Plus Ipilimumab or Nivolumab Alone in Advanced Melanoma, Including a Case Series of Encephalitis

BackgroundDespite unprecedented efficacy across multiple tumor types, immune checkpoint inhibitor therapy is associated with a unique and wide spectrum of immune‐related adverse events (irAEs), including neurologic events ranging from mild headache to potentially life‐threatening encephalitis. Here, we summarize neurologic irAEs associated with nivolumab and ipilimumab melanoma treatment, present cases of treatment‐related encephalitis, and provide practical guidance on diagnosis and management.MethodsWe searched a Global Pharmacovigilance and Epidemiology database for neurologic irAEs reported over an 8‐year period in patients with advanced melanoma receiving nivolumab with or without ipilimumab from 12 studies sponsored by Bristol‐Myers Squibb. Serious neurologic irAEs were reviewed, and relationship to nivolumab or ipilimumab was assigned.ResultsIn our search of 3,763 patients, 35 patients (0.93%) presented with 43 serious neurologic irAEs, including neuropathy (n = 22), noninfective meningitis (n = 5), encephalitis (n = 6), neuromuscular disorders (n = 3), and nonspecific adverse events (n = 7). Study drug was discontinued (n = 20), interrupted (n = 8), or unchanged (n = 7). Most neurologic irAEs resolved (26/35 patients; 75%). Overall, median time to onset was 45 days (range 1–170) and to resolution was 32 days (2–809+). Median time to onset of encephalitis was 55.5 days (range 18–297); four cases resolved and one was fatal.ConclusionBoth oncologists and neurologists need to be aware of signs and symptoms of serious but uncommon neurologic irAEs associated with checkpoint inhibitors. Prompt diagnosis and management using an established algorithm are critical to minimize serious complications from these neurologic irAEs.Implications for PracticeWith increasing use of checkpoint inhibitors in cancer, practicing oncologists need to be aware of the potential risk of neurologic immune‐related adverse events and be able to provide prompt treatment of this uncommon, but potentially serious, class of adverse events. We summarize neurologic adverse events related to nivolumab alone or in combination with ipilimumab in patients with advanced melanoma from 12 studies and examine in depth 6 cases of encephalitis. We also provide input and guidance on the existing neurologic adverse events management algorithm for nivolumab and ipilimumab.Melanoma is a particularly immunogenic cancer, and immune checkpoint inhibitors have been extensively studied in this tumor type. This review focuses on the incidence of serious neurologic immune‐related adverse events, specifically encephalitis, in patients with advanced melanoma treated with nivolumab alone or in sequence or combination with ipilimumab. Practical guidance is provided for the diagnosis and management of treatment‐related encephalitis associated with nivolumab and ipilimumab.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139998/1/onco12130.pd

Particle detectors, geodesic motion, and the equivalence principle

It is shown that quantum particle detectors are not reliable probes of spacetime structure. In particular, they fail to distinguish between inertial and non-inertial motion in a general spacetime. To prove this, we consider detectors undergoing circular motion in an arbitrary static spherically symmetric spacetime, and give a necessary and sufficient condition for the response function to vanish when the field is in the static vacuum state. By examining two particular cases, we show that there is no relation, in general, between the vanishing of the response function and the fact that the detector motion is, or is not, geodesic. In static asymptotically flat spacetimes, however, all rotating detectors are excited in the static vacuum. Thus, in this particular case the static vacuum appears to be associated with a non-rotating frame. The implications of these results for the equivalence principle are considered. In particular, we discuss how to properly formulate the principle for particle detectors, and show that it is satisfied.Comment: 14 pages. Revised version, with corrections; added two references. Accepted for publication in Class. Quantum Gra