Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Sensitive Analysis of Residual Protein Tat Bh1-101 in Lentiviral Vectors for Gene Therapy.

Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL.

Highly Sensitive SDS Capillary Gel Electrophoresis with Sample Stacking Requiring Only Nanograms of Adeno-Associated Virus Capsid Proteins.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been the method of choice in the past decades for size-based protein analysis. However, in general it requires the protein concentration in mg/mL level and thus is not practical for trace level protein analysis, not to mention the lengthy labor-intensive procedures. The SDS capillary gel electrophoresis (SDS CGE) method reported herein requires only nanogram-sized proteins loaded onto the autosampler. A sample stacking technique (e.g., head-column field-amplified sample stacking (HC FASS)) was employed, providing three orders of magnitude sensitivity enhancement compared to conventional SDS CGE. This method has been used routinely in purity analysis and characterization of adeno-associated virus (AAV) intermediates and finished gene therapeutics of AAV vectors. The sensitivity achieved is comparable to the currently most sensitive size-based protein assay silver-stained SDS PAGE. The highly sensitive sample stacking SDS CGE can be used for other types of proteins as well.

Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins.

Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.

Identification of carboxyl-terminal peptide fragments of parathyroid hormone in human plasma at low-picomolar levels by mass spectrometry.

For decades, researchers have tried to identify the primary structures of circulating carboxyl-terminal parathyroid hormone (C-PTH) peptide fragments that may be present at only picomolar levels in human plasma. Although immunoassays and radiosequencing techniques have provided valuable fragment characterizations, no analysis has successfully determined their exact primary structures. In this work, for the first time, four human C-PTH peptide fragments, hPTH(34-84), hPTH(37-84), hPTH(38-84), and hPTH(45-84), have been identified from human plasma using MS-based methods. C-PTH peptide fragments were isolated from plasma samples by immunoaffinity extraction. The eluate was analyzed by capillary LC fractionation followed by MALDI-TOF-MS or by on-line coupling of nano-LC with ESI-TOF-MS. Both the MALDI- and the ESI-based approaches were capable of detecting C-PTH peptide fragments in human plasma at <10 pmol/L. The MALDI-TOF approach was effective in preliminary searches for C-PTH peptide fragments, but the use of high laser power limited the resolution necessary for accurate C-PTH peptide identification. The high mass resolution (10,000) and accuracy (10 ppm) attained by the ESI-TOF approach enabled unambiguous identification of these peptides. The four C-PTH peptide fragments identified in plasma samples from patients with chronic renal insufficiency were also found in the plasma of healthy women receiving recombinant human PTH either by subcutaneous injection or by intravenous infusion. This newly developed analytical capability should greatly enhance the understanding of PTH metabolism and parathyroid gland function.

High-speed free-flow electrophoresis on chip.

A microfluidic device has been developed for continuous separation in free-flow electrophoresis (FFE) mode. A mixture of two fluorescent reagents is separated into two component streams in 75 ms using a sample flow rate of 2 nL/s. The residence time of sample in the whole separation compartment is 2 s. The separation bed volume is 0.2 microL. The chip has also been used for free-flow electrophoresis of fluorescein-5-isothiocyanate-labeled amino acids in both aqueous and binary media. The short residence time and small sample flow rate make the FFE chip feasible for on-line monitoring on production lines and other chemical or biochemical processes. The in-house-made chip was composed of a plain glass substrate of 1.5-mm thickness and a PDMS layer of 0.3-mm thickness with micromachined channels. The channel design presented in this paper is versatile. With the same kind of PDMS substrates, chips for various purposes can be made depending on the locations of the reservoirs, which are cut out on the PDMS substrate. The results presented verify the scaling laws and allow prediction of FFE performances comparable to what is now state of the art on capillary electrophoresis chips.

In-column field-amplified sample stacking of biogenic amines on microfabricated electrophoresis devices.

A novel method for performing in-column field-amplified sample stacking (FASS) in chip-based electrophoretic systems is presented. The methodology involves the use of a narrow sample channel (NSC) injector. NSC injectors allow sample plugs to be introduced directly into the separation channel, and subsequent stacking and separation can proceed without any need for leakage control. More importantly, stacking and separation occur in a single step negating the requirement for complex channel geometries and voltage switching to control sample plugs during the stacking procedure. The chip is composed of six paralleled systems. Using the NSC injector design, the number of reservoirs in the multiplexed chip is reduced to N + 2, where N is the number of paralleled systems. This design feature radically reduces the complexity in chip structures and associated chip operation. The approach is applied to the analysis of fluorescently labelled biogenic amines affording detection at concentrations down to 20 pM.

Sub-second isoelectric focusing in free flow using a microfluidic device.

Using a microfabricated chip with a bed volume of 0.2 microL we demonstrate the validity of the scaling laws for molecular mass transport of isoelectric focusing (IEF) in free flow. Nano- or microlitre sample volumes can be concentrated within 430 ms by a factor of up to 400. These very fast performances make the chip applicable to proteomic analysis and for continuous monitoring of biochemical processes.