Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE-ESI-MS/MS system.

We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic-labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope-labeled lysine or arginine. The heavy-labeled peptides were spiked in the HCP digests at known concentrations. After CZE-ESI-MS/MS analysis, the peaks of native and isotopic-labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic-labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R(2) values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 µg sample of digested HCPs. The target proteins were present at the 7-30 ppt level in the purified HCP samples.

A global benchmark study using affinity-based biosensors.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Fetal growth in women managed with insulin pump therapy compared to conventional insulin.

OBJECTIVE: Fetal hyperinsulinaemia secondary to maternal hyperglycaemia is considered to be the driving force behind excessive fetal growth. We hypothesised that insulin pump therapy (continuous subcutaneous insulin infusion, CSII) would improve maternal glycaemic control and normalise fetal growth parameters. To this end, this study compares maternal glycaemic control and fetal growth of women receiving insulin pump therapy with those receiving conventional insulin therapy. STUDY DESIGN: Prospective non-randomised study of 42 women with pre-existing diabetes attending a joint obstetric diabetic clinic. Each woman was offered the choice of commencing insulin pump therapy or remaining on a conventional insulin regime. Estimated fetal weight and fetal growth velocity were calculated from routinely collected third trimester ultrasound biometry and expressed as standard deviation (Z) scores. RESULTS: Eighteen women commenced insulin pump therapy. There was no difference in pre-conception glycosylated haemoglobin A1c concentrations (HbA1c) between pump and conventional therapy groups (mean HbA1c 7.62 versus 8.01; p=0.49) or third trimester glycaemic control (mean HbA1c 6.63 versus 6.44; p=0.51). Women using pump therapy had similar mean growth velocity Z scores (1.5 versus 1.36; p=0.83), similar mean estimated fetal weight Z scores prior to delivery (2.80 versus 2.16; p=0.16) and similar mean birthweight Z scores (2.09 versus 2.00; p=0.86) compared to women using conventional insulin therapy. CONCLUSION: This small, non-randomised study suggests that the use of insulin pump therapy offers no benefit in terms of normalising fetal growth velocity, fetal size, birthweight or improving maternal glycaemic control compared to conventional insulin therapy.

Can ultrasound fetal biometry predict fetal hyperinsulinaemia at delivery in pregnancy complicated by maternal diabetes?

OBJECTIVE: The aim of this study was to determine whether there is an association between ultrasound fetal biometry and amniotic fluid insulin levels at delivery in women with pre-existing diabetes or impaired glucose tolerance in pregnancy. STUDY DESIGN: This retrospective cohort study identified 93 women who had amniotic fluid insulin levels measured at time of delivery. Standardised estimated fetal weight and fetal growth velocity were calculated from serial third trimester fetal ultrasound measurements. RESULTS: Women with pre-existing diabetes had significantly greater mean growth velocity [1.39 (95% CI: 0.43-2.23) versus 0.39 (95% CI: -01.7-0.95); p=0.04], significantly greater mean estimated fetal weight (EFW) Z score prior to delivery [2.36 (95% CI: 1.82-2.9) versus 1.38 (95% CI: 1.02-1.74); p=0.002] and greater mean birthweight centile [82 (95% CI: 0.74-0.89) versus 67 (95% CI: 58-76); p=0.02] than those with GDM/IGT. Amniotic fluid insulin levels demonstrated a similar significant difference between the pre-existing and GDM/IGT groups [20.5 (95% CI: 12.9-28.1) versus 8.5 (95% CI: 5.4-11.7); p=0.001]. An association between fetal growth and size and amniotic fluid insulin was observed in women with pre-existing diabetes. Positive likelihood ratios were 1.67 and 2.08, respectively, for the prediction of liquor insulin greater than the 95th centile in women with pre-existing diabetes. CONCLUSION: Ultrasound measures of fetal size and growth used in this study are not sufficiently accurate to predict those infants likely to be at risk from the adverse effects of fetal hyperinsulinaemia.

A rapid cIEF-ESI-MS/MS method for host cell protein analysis of a recombinant human monoclonal antibody.

A rapid and reproducible system that couples capillary isoelectric focusing to a high-resolution mass spectrometer was developed for on-line analysis and identification of protein digests. Magnetic microsphere-based immobilized trypsin was used for protein digestion to reduce the digestion time to 10 min, with a total analysis time of 4h. A three-protein-mixture (myoglobin, BSA, cytochrome c) with a molarity ratio of 1:10:50 was successfully digested and identified. This system was also used to analyze host cell protein impurities in a recombinant humanized monoclonal antibody product in which the sample was product-depleted using affinity capture on protein A/protein L columns prior to analysis. A database search identified 37 host cell proteins with peptide and protein identity probability greater than 0.9.