Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis.

The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition. Biophysical properties like surface charge or hydrophobicity, and binding to surrogates for high abundant off-targets (e.g., baculovirus particles, Chinese hamster ovary cell membrane proteins) were proposed to be responsible for these differences. Here, we used heparin chromatography to separate a polyclonal mix of endogenous human IgGs (IVIG) into fractions that differ in their PK properties. Heparin was chosen as a surrogate for highly negatively charged glycocalyx components on endothelial cells, which are among the main contributors to nonspecific clearance. By directly correlating heparin retention time with clearance, we identified heparin chromatography as a tool to assess differences in unspecific cell-surface interaction and the likelihood for increased pinocytotic uptake and degradation. Building on these results, we combined predictors for FcRn-mediated recycling and cell-surface interaction. The combination of heparin and FcRn chromatography allow identification of antibodies with abnormal PK by mimicking the major root causes for fast, non-target-mediated, clearance of therapeutic, Fc-containing proteins.

Validation of short-pulse-laser-based measurement setup for absolute spectral irradiance responsivity calibration.

This paper describes the validation process of mode-locked lasers in the "tunable lasers in photometry" (TULIP) setup at Physikalisch-Technische Bundesanstalt (PTB) regarding spectral irradiance responsivity calibrations. Validation has been carried out in the visible spectral range, 400-700 nm, with two different photometer heads and in the long wavelength range, 690-780 nm, with a filtered radiometer. A comparison of the results against those from two different validated measurement setups has been carried out for validation. For the visible spectral range, the comparison is conducted against the data obtained from a lamp-based monochromator setup for spectral irradiance responsivity calibrations and against the photometric values (integral quantity) measured at the photometric bench setup of PTB. For the long wavelength range, comparisons against results from two different lamp-based monochromator measurement setups were made. Additionally, the effect of different radiation bandwidths on interference oscillations has been determined for a filter radiometer without a diffuser. A procedure for the determination of the optimum bandwidth of the setup for the respective measurement device is presented.

Spectral calibration of radiometric detectors using tunable laser sources.

This paper describes the analysis of laser-based responsivity measurements using the Tunable Lasers in Photometry setup at the Physikalisch-Technische Bundesanstalt. An approach based on digital signal analysis is proposed to remove interference-caused oscillations in highly resolved spectral data from laser-based measurements, yielding an improved reproducibility and comparability of results. Digital filters are used to selectively suppress the frequency components of interference fringes visible in the measurement data. We describe the algorithm used and discuss the associated uncertainty components of laser-based measurements. Finally, we give examples of the calibration of different detectors with and without interference effects.