Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

Exposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.

Glycosylated natural products such as bleomycin, neocarzinostatin, and calicheamicin γ 1 are efficient antitumor agents that cleave ds DNA by pathways that involve redox chemistry. In this paper we demonstrate the use of metalloderivatives of natural aminoglycosides as efficient DNA cleavage agents in the absence of external reducing agents. Kinetic characterization of DNA cleavage by copper neamine under Michaelis-Menten-"type" reaction conditions revealed a maximal reaction velocity V max ′ ) 0.031 min -1 , equivalent to a greater than 50-million-fold rate enhancement in DNA cleavage, when uncorrected for catalyst concentrations. Under true Michaelis conditions, a maximal reaction velocity V max ) 0.0595 min -1 was obtained (with k cat ) 5.95 × 10 -4 min -1 ), corresponding to a million-fold rate enhancement using micromolar concentrations of Cu 2+ -neamine. The specificity constants for DNA cleavage by copper neamine (k cat /K M ) 4.8 × 10 5 h -1 M -1 ) are 2 orders of magnitude greater than those reported elsewhere for synthetic compounds, at this time. Cleavage mediated by Cu 2+ -(kanamycin A) was found to be even more efficient. DNA cleavage was not inhibited by SOD, NaN 3 , DMSO, or EtOH, nor by handling under anaerobic conditions. The results of gel electrophoretic experiments provide clear evidence for a hydrolytic cleavage pathway with generation of 5′phosphate and 3′-hydroxyl termini.

Biomimetic hydrolysis of DNA or RNA is of increasing importance in biotechnology and medicine. Most natural nuclease enzymes that mediate such reactions utilize metal ion cofactors. Recent progress in the design of synthetic metallonucleases has included complexes of antibiotics, peptides, nucleic acids, and other organic ligands. In this article, we review a number of synthetic catalyst systems that have been developed to achieve efficient DNA hydrolysis. Methods to evaluate their catalytic efficiencies are critically discussed, and a prognosis for future work in this area is presented.

Citation for published item:uinnunenD r nne wF nd h rm D ik s nd gontrer sE oj sD vuis odrigo nd uD fei nd ellem nD ghl¤ oe nd reedh r D el v tt m nd pis herD tef n nd uh wliD veslie nd oheD tef n F nd fum D h niel nd t po'D hom s F nd h ughertyD enn vF nd wrsnyD nd ll tF @PHISA 9e novel in vitro method to model the f te of su ut neously dministered ioph rm euti ls nd sso i ted formul tion omponentsF9D tourn l of ontrolled rele seFD PIR F ppF WREIHPF Further information on publisher's website: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Movement of a biopharmaceutical from the injection chamber to the infinite sink compartment simulates the drug migration from the injection site and uptake by the blood and/or lymph capillaries. Here, we present an initial evaluation of the Scissor system using the ECM element hyaluronic acid and test formulations of insulin and four different monoclonal antibodies. Our findings suggest that Scissor can provide a tractable method to examine the potential fate of a biopharmaceutical formulation after its SC injection in humans and that this approach may provide a reliable and representative alternative to animal testing for the initial screening of SC formulations. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT