Rethinking clonality using modeling approaches

A combination of experimental procedures, imaging, and probability estimation are typically used as evidence of clonality for the manufacture of a biotherapeutic product. In situations where the totality of evidence is unavailable, establishing a high statistical probability for monoclonality can help strengthen the argument for clonality. In this study, the probability of clonality was re-examined for the limiting dilution method using a combination of experimental and modeling approaches. A limiting dilution experiment was performed using a 50:50 mixed population of GFP-and RFP-expressing cells and the plates were imaged over a span of two weeks. The imaged cells were scored for clonality and double checked with fluorescence imager. Among all wells that had single colony-like growth on day 14 and a single cell-like image on day 0, a fraction of the wells were confirmed to have two colors on day 14 by fluorescence imaging, indicating the singe cell-like day 0 images for these wells were false reads. Considering the possibility of having 2 or more cells with the same color in a particular well, we estimated the worst case total possible number of wells with 2 or more cells on day 0. Moreover, assuming a Poisson distribution for limiting dilution, the recovery rate of any single cell that grew into a visible colony by day 14 was estimated. Our modeling analysis indicated that only a fraction of the wells with \u3e2 cells on day 0 could grow into non-monoclonal colonies. If cells from any of the wells with single colony-like growth on day 14 and single cell-like image on day 0 were chosen as the final clone, the probability of monoclonality was estimated to be \u3e 95% with a 95% upper confidence limit

Limitations of subcloning as a tool to characterize homogeneity of a cell population

Cloning, or the derivation of a cell line from a single cell is a critical step in the generation of a manufacturing cell line. The expectation is that the process of cloning will result in a uniform and homogeneous cell line that will ensure robust product quality over the lifetime of the product. Regulatory guidelines require the sponsors provide assurance of clonality of the production cell line and when such evidence is not available, additional studies are required to further ensure consistent long-term manufacturing of the product. One approach to characterize homogeneity of a cell line is subclone analysis where clones are generated from the original cell line and an evaluation of their similarity is performed lines. To study the suitability of subclone analysis to provide additional assurance that a production cell line is clonally derived, an antibody producing CHO Master Cell Bank (MCB), which was cloned by a validated FACS method and with a clear documented day 0 image was characterized. Specifically, this MCB was subcloned and imaged to assure each of the subclones were derived from a single cell. A total of 46 subclones were analyzed for growth, productivity, product quality, as well as copy number and integration site analysis. Despite demonstration of clonality for both the MCB and the subclones, significant diversity in cell growth, protein productivity, and product quality attributes was observed between the 46 subclones. The diversity in protein productivity and quality were reproduced across bioreactor scales, suggesting that albeit different, the subclones were stable populations that varied from the parental clonal cell line. Additionally, while ~2-fold shifts in copy number were seen, no significant integration site changes were observed. Our data suggest subcloning induces changes (genetic or epigenetic) outside the region of the transgene which result in the subclones exhibiting a wide diversity in cell growth protein productivity, and product quality. Transcriptomic and genomic characterization studies are underway to further characterize the differences between subclones and the MCB. Importantly, the subclones do keep their individual characteristics as they mature and stabilize, suggesting that the resulting population that grows out of a single cell is stable but with unique properties. Overall, this work adds to the growing body of work on CHO cell plasticity and suggests that subcloning is not an effective approach to demonstrate homogeneity of a cell bank

Azimuthal anisotropy of charged jet production in root s(NN)=2.76 TeV Pb-Pb collisions

We present measurements of the azimuthal dependence of charged jet production in central and semi-central root s(NN) = 2.76 TeV Pb-Pb collisions with respect to the second harmonic event plane, quantified as nu(ch)(2) (jet). Jet finding is performed employing the anti-k(T) algorithm with a resolution parameter R = 0.2 using charged tracks from the ALICE tracking system. The contribution of the azimuthal anisotropy of the underlying event is taken into account event-by-event. The remaining (statistical) region-to-region fluctuations are removed on an ensemble basis by unfolding the jet spectra for different event plane orientations independently. Significant non-zero nu(ch)(2) (jet) is observed in semi-central collisions (30-50% centrality) for 20 <p(T)(ch) (jet) <90 GeV/c. The azimuthal dependence of the charged jet production is similar to the dependence observed for jets comprising both charged and neutral fragments, and compatible with measurements of the nu(2) of single charged particles at high p(T). Good agreement between the data and predictions from JEWEL, an event generator simulating parton shower evolution in the presence of a dense QCD medium, is found in semi-central collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Forward-central two-particle correlations in p-Pb collisions at root s(NN)=5.02 TeV

Two-particle angular correlations between trigger particles in the forward pseudorapidity range (2.5 2GeV/c. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B. V.Peer reviewe

Event-shape engineering for inclusive spectra and elliptic flow in Pb-Pb collisions at root(NN)-N-S=2.76 TeV

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Pseudorapidity and transverse-momentum distributions of charged particles in proton-proton collisions at root s=13 TeV

The pseudorapidity (eta) and transverse-momentum (p(T)) distributions of charged particles produced in proton-proton collisions are measured at the centre-of-mass energy root s = 13 TeV. The pseudorapidity distribution in vertical bar eta vertical bar <1.8 is reported for inelastic events and for events with at least one charged particle in vertical bar eta vertical bar <1. The pseudorapidity density of charged particles produced in the pseudorapidity region vertical bar eta vertical bar <0.5 is 5.31 +/- 0.18 and 6.46 +/- 0.19 for the two event classes, respectively. The transverse-momentum distribution of charged particles is measured in the range 0.15 <p(T) <20 GeV/c and vertical bar eta vertical bar <0.8 for events with at least one charged particle in vertical bar eta vertical bar <1. The evolution of the transverse momentum spectra of charged particles is also investigated as a function of event multiplicity. The results are compared with calculations from PYTHIA and EPOS Monte Carlo generators. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Elliptic flow of muons from heavy-flavour hadron decays at forward rapidity in Pb-Pb collisions at root s(NN)=2.76TeV

The elliptic flow, v(2), of muons from heavy-flavour hadron decays at forward rapidity (2.5 <y <4) is measured in Pb-Pb collisions at root s(NN)= 2.76TeVwith the ALICE detector at the LHC. The scalar product, two- and four-particle Q cumulants and Lee-Yang zeros methods are used. The dependence of the v(2) of muons from heavy-flavour hadron decays on the collision centrality, in the range 0-40%, and on transverse momentum, p(T), is studied in the interval 3 <p(T)<10 GeV/c. A positive v(2) is observed with the scalar product and two-particle Q cumulants in semi-central collisions (10-20% and 20-40% centrality classes) for the p(T) interval from 3 to about 5GeV/c with a significance larger than 3 sigma, based on the combination of statistical and systematic uncertainties. The v(2) magnitude tends to decrease towards more central collisions and with increasing pT. It becomes compatible with zero in the interval 6 <p(T)<10 GeV/c. The results are compared to models describing the interaction of heavy quarks and open heavy-flavour hadrons with the high-density medium formed in high-energy heavy-ion collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V.Peer reviewe

Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at root s(NN)=2.76TeV

Peer reviewe