Analysis of recombinant adenoviruses using an integrated microfluidic chip-based system.

The use of recombinant virus for gene therapy requires rigorous quality control methods to ensure that the viral vector preparations are functional and safe. A viral identity test is performed in which the viral payload, or transgene, is PCR amplified, followed by digestion with restriction enzymes that yields a characteristic "fingerprint." These DNA fragments are typically analyzed by agarose gel electrophoresis. The ethidium bromide-stained gels are photographed or scanned and the results are sufficient for a qualitative or semiquantitative identity confirmation of the viral product. We have investigated the use of an integrated microfluidic chip-based system as a new tool in the quality control testing of a recombinant, adenoviral, gene therapy product. The chip-based method was found to be very sensitive, requiring 100-fold less sample and only one-third the time compared to the agarose gel method. The automated data analysis sizes and quantitates the DNA fragments, thus yielding a more thorough, reproducible, sensitive, and rapid analysis.

Approaches to functional genomics: potential of matrix-assisted laser desorption ionization--time of flight mass spectrometry combined with separation methods for the analysis of DNA in biological samples.

MALDI-TOF MS has potential as a valuable technique in DNA mapping studies and may well be complementary to other approaches to DNA analysis such as gel electrophoresis and sequencing. This study used 2,6-dihydroxyacetophenone (DHAP) mixed with diammonium hydrogen citrate (DAHC) as the matrix. In addition, recent technical advances such as time lag focussing (TLF) and better selection of matrices (such as 3-hydroxypicolinic acid (3 HPA) and picolinic acid (PA)) extended the range of DNA fragments that can be studied by this approach. The following samples were investigated: Poly-T mixture (dT 15, 19, 20, 25, 74 and 75), plasmid pBR322 derived oligonucleotides (10, 11, 12, 13, 14, 15, 19, 20 and 50 nucleotides long) and DNA fragments of 25, 36 and 37 base pairs corresponding to a fragment in the restriction map for the gene corresponding to the hexon protein of Adenovirus 2 and 5. The results were contrasted with similar analyses performed by ion-paired reversed-phase HPLC coupled to on-line electrospray mass spectrometry.

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome.

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2 x 10(8) particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.

Effect of electric field on liquid chromatographic separation of peptide digests. Combining capillary separation techniques.

A system is described which allows operation of a range of capillary based liquid phase separations including capillary electrophoresis, isocratic and gradient capillary electrochromatography, isocratic and gradient capillary liquid chromatography and electrically assisted gradient capillary liquid chromatography. The system was coupled to electrospray ionization mass spectrometry in the electrically assisted capillary liquid chromatography mode to investigate the effect of applied voltage on the selectivity in peptide mapping separations. Analyses were performed on tryptic digests of recombinant human growth hormone and tissue plasminogen activator. The results show a small but useful effect on selectivity that can be used to fine tune specific separations.

Application of new analytical technology to the production of a "well-characterized biological".

The role of new analytical technology in the development of the concept of a "Well Characterized Biological" is to provide suitable methodology that allows the characterization of even the most complex protein sample so that a consistent manufacturing process can be established. Glycoproteins are among the most challenging of products to characterize because of extreme sample microheterogeneity due to the carbohydrate moieties. As an example of the appropriate use of new analytical technology this review will examine the steps necessary to demonstrate that a glycoprotein is a "Well Characterized Biological". The key to characterization of complex protein samples lies in the use of appropriate combinations of the different methods that analyse the sample from substantially orthogonal and independent directions. An important advantage of capillary electrophoresis (HPCE) in this application is the complementarily of the technique with reversed phase HPLC (RPLC). Thus mixtures of variants of a polypeptide that are difficult to separate by RPLC can often be readily resolved by HPCE. Both separation techniques are well suited to the analysis of peptide maps, although RPLC is particularly powerful when used in combination with on-line electrospray mass spectrometry (ESI-MS) which allows for the effective ionization and detection of even high MW glycopeptides. In this sense the ESI-MS is an ideal detector for on-line mass detection after a RPLC separation of medium MW fragments (300 to 6000 emu) that are typically present in an enzyme digest of a protein. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOFMS) is a valuable technique for off-line characterization of CE fractions due to the high sensitivity of the method and its tolerance of samples with moderate levels of salt. The development of an effective protocol for the analysis of glycoform populations of intact glycoproteins by a combination of HPCE and off-line MALDI-TOFMS wil be demonstrated by the successful analysis of two highly heterogeneous glycoproteins, ovalbumin and Desmodus Salivary Plasminogen Activator (DSPA).

A rapid and sensitive procedure for the micro-purification and subsequent characterization of peptides and protein samples by N-terminal sequencing and matrix assisted laser desorption ionization time of flight mass spectrometry.

The characterization of the proteome, a key activity in the post-genome era, is made extremely challenging by the microheterogeneity introduced by post translational modifications such as glycosylation in the diverse set of proteins expressed in a cellular system. High resolution separation systems, such as 2D-gel electrophoresis and more recently liquid chromatography (LC) have been used to fractionate these complex mixtures, however, subsequent mass analysis is hindered by the low level of the purified components. Off-line coupling of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS) is an attractive technique for the analysis of such samples, but suffers from sensitivity to the degree of salt contamination that is unavoidable in the isolation of low level protein samples from biological extracts. In this publication we will report on a novel application of a commercially available system for the micro-purification of peptides and proteins. In this procedure micro-columns (normally used for sequencing of electroblotted samples) were used to rapidly purify protein digests or crude extracts of proteins in sufficient amounts for further analyses by protein sequencing and MALDI-TOF/MS. To demonstrate the applicability of these techniques we isolated and performed structural analysis of the following samples: a high-mannose glycopeptide isolated from a digest of the glycoprotein rt-PA, a poly-His tagged recombinant DNA-binding protein isolated by Ni2+-chelating agarose and a polyclonal antibody sample.

The use of multidimensional liquid-phase separations and mass spectrometry for the detailed characterization of posttranslational modifications in glycoproteins.

The goal of characterization of the proteome, while challenging in itself, is further complicated by the microheterogeneity introduced by posttranslational modifications such as glycosylation. A combination of liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) offers the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. In the current work, the combination of liquid-phase separations and mass spectrometry is demonstrated through the on-line coupling of electrospray ionization mass spectrometry (ESI-MS) and off-line coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). LC/ESI-MS yields real-time results while maintaining the separation obtained from the LC analysis. CE/MALDI TOF-MS offers high-mass detection and extremely low detection limits. The unique separation selectivity of CE relative to reversed-phase HPLC separations of the members of a glycopeptide family was used to develop an integrated multidimensional analysis achieved by the off-line coupling of LC, CE, and MALDI TOF-MS. To demonstrate the applicability of these techniques to the characterization of the heterogeneity of posttranslational modifications present in glycoproteins, we will report on the study of the glycoforms present in a N-linked site in a single-chain plasminogen activator (DSPAα1).

Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.

Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry.

A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.

Application of multidimensional affinity high-performance liquid chromatography and electrospray ionization liquid chromatography-mass spectrometry to the characterization of glycosylation in single-chain plasminogen activator. Initial results.

Preliminary results are presented using a combination of affinity chromatography, reversed-phase HPLC and electrospray ionization mass spectrometry to produced peptide maps for N-linked, O-linked and non-glycosylated peptides from an endoproteinase LysC digest of DSPA alpha 1, a recombinant DNA derived glycoprotein. Although the system was used to identify a number of major N-linked structures, notably complex biantennary structures attached to asparagine 362, no O-linked glycopeptides from the possible 4 attachment sites were identified. The system did, however, demonstrate the feasibility of the approach and the applicability of the instrumental system.

Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator.

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.

Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator.

The analysis of recombinant Desmodus salivary plasminogen activator (DSPA alpha 1), a heterogeneous glycoprotein, is demonstrated through the use of high-performance liquid chromatography (HPLC), high-performance capillary electrophoresis (HPCE), liquid chromatography-electrospray mass spectrometry (LC--ES-MS), and matrix-assisted laser desorption ionization--time of flight mass spectrometry (MALDI--TOF-MS). The proteins is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein, and fractions from the proteolytic digest. A method for "on-column" collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI--TOF-MS is shown.

Affinity purification and microcharacterization of recombinant DNA-derived human growth hormone isolated from an in vivo model.

A procedure has been developed for the isolation and purification of trace amounts of unlabeled proteins from biological solutions. Using a combination of affinity chromatography and reversed-phase HPLC, microgram amounts of recombinant DNA-derived human growth hormone (rhGH) were purified from an in vivo rat model. Microcharacterization techniques were developed, and picomole amounts of the recovered protein were digested with trypsin and characterized using capillary HPLC peptide mapping. The described procedures were used to study the chemical changes that occur in rhGH following intravenous administration. The study demonstrated that both deamidation and oxidation can occur in vivo, although the former would occur to a significant extent only in proteins with an extended half-life.

Application of capillary high-performance liquid chromatography to biotechnology, with reference to the analysis of recombinant DNA-derived human growth hormone.

Using capillary HPLC, femtomole amounts of recombinant DNA-derived human growth hormone (rhGH) have been successfully detected from solutions at nanomolar concentrations. The separation used capillaries of 15 cm x 320 microns I.D. and detection was with a UV absorbance detector containing a capillary Z-shaped flow-cell. A sample of rhGH that was recovered from rat serum was analyzed by capillary reversed-phase HPLC, using both acidic- and neutral-pH mobile phases, as well as by capillary ion-exchange chromatography. When compared to HPLC separations performed at flow-rates of 1 ml/min, the sensitivity of the detection was increased 200 times, without any loss in resolution. Sub-microgram amounts of rhGH were also analyzed by tryptic mapping using capillary HPLC and peptides were identified by capillary LC-MS.

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. I. Optimizing the separation of a growth hormone tryptic digest.

Peptide and protein samples are often complex mixtures that contain a number of individual compounds. The initial HPLC separation of such samples typically results in the poor resolution of one or more band pairs. Various means have been suggested for varying separation selectivity so as to minimize this problem. In this study of a tryptic digest of recombinant human growth hormone, the simultaneous variation of temperature and gradient steepness was found to be a convenient and effective means of varying selectivity and optimizing the separation. The use of computer simulation greatly facilitated this investigation.

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. II. Selectivity effects observed in the separation of several peptide and protein mixtures.

Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution. The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.

Diketopiperazine formation and N-terminal degradation in recombinant human growth hormone.

A new degradation process has been identified that occurs in recombinant DNA-derived human growth hormone. Non-enzymatic cyclization of the first two amino acids from the N-terminus and subsequent cleavage results in the formation of a diketopiperazine and a truncated variant of rhGH. The truncated protein was separated using hydrophobic interaction chromatography and identified as desPhe1Pro2-rhGH using N-terminal sequence analysis, tryptic mapping, and mass spectrometry.

Characterization of humanized anti-TAC, an antibody directed against the interleukin 2 receptor, using electrospray ionization mass spectrometry by direct infusion, LC/MS, and MS/MS.

Characterization of a humanized monoclonal antibody (Hu-anti-TAC) directed against a surface protein expressed on T-lymphocytes was performed with an electrospray mass spectrometer. Capillary reversed-phase liquid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along with tandem MS/MS analysis to confirm the sequence and to determine the sources of heterogeneity in Hu-anti-TAC. The MS analysis was performed on disulfide-reduced and trypsin-digested samples of the antibody. Two forms of diantennary carbohydrate structures were identified and found to be consistent with those reported for the human IgG1 framework. The analysis demonstrated that the N-terminus was modified by conversion of a glutamine residue to pyroglutamic acid. Another source of heterogeneity was the partial removal of the C-terminal lysine residue and was confirmed by mass calculations of tryptic peptides followed by MS/MS sequencing. This study demonstrates that the high sensitivity of electrospray mass spectrometry when combined with capillary chromatography can allow detailed characterization of microgram samples of high molecular weight proteins such as antibodies.

Analytical considerations in the development of protein purification processes.

Successful and rapid process development requires the availability of suitable analytical methods at all stages of the development cycle (see Tables 2 and 3). The methods should be selected first to ensure the identity, potency, and purity of the product and second to facilitate the process development. Some assays are particularly valuable early in the recovery process (and early in the development cycle) because they are typically robust and can be used with crude samples that contain many components. Popular examples include SDS-PAGE, IEF, and immunoassay, which allow the process scientist simply to monitor the yield and removal of impurities on a broad scale. Each step should be monitored with a biological assay, so that the specific activity of the target protein can be followed and steps that cause a loss of activity can be quickly detected. At the end of the recovery process (and development cycle) more sophisticated analytical methods, for example RP-HPLC and mass spectrometry, are used to detect and characterize variants of the desired product. Highly process-specific assays, such as host cell impurity ELISAs, are developed at this point, and the key methods for process monitoring and final product characterization are selected. While each process development project is distinct, in all cases the use of well-validated complementary analytical methods will provide greater assurance of a timely and successful project.