Characterization of maleuric acid derivatives on transgenic human monoclonal antibody due to post-secretional modifications in goat milk.

A fully human antibody to tumor necrosis factor-alpha was expressed in the mammary glands of transgenic goats. The goat expressed antibody (gAb) is heterogeneous and has several isoforms due to typical cellular post-translational modifications. In addition, one post-secretional modification on gAb was discovered by high-resolution cation exchange chromatography (CIEX). The presence of these variants in the final product was shown to be dependent upon the initial milk storage and traditional purification methodologies used. These observations allow for the development of new sample recovery and purification processes to eliminate these variants. Various enzymatic treatments were used to characterize different gAb heavy chain C-terminal lysine and sialic acid variants. In addition, an unknown derivative with the additional mass of 140 Da was found in transgenic gAb using mass spectrometry (MS). The modification sites were identified as the N-termini of gAb light chains and heavy chains using Q-TOF MS. Characterization of transgenic gAb isoforms was facilitated by utilizing different enzymes, CIEX and MS techniques. A maleuric acid modification on the N-terminal portion of gAb was shown to be consistent with the available data characterizing this new derivative of transgenic gAb isoforms in goat milk.

Characterization of noncovalent complexes of recombinant human monoclonal antibody and antigen using cation exchange, size exclusion chromatography, and BIAcore.

The binding of fully human monoclonal antibodies (MAbs) D2E7 and 2SD4 to their antigen, human tumor necrosis factor-alpha (TNFalpha), was investigated by BIAcore, cation exchange (CIEX), and size exclusion liquid chromatography (SEC) using ultraviolet and laser light scattering detectors. D2E7 has a higher affinity for TNFalpha than 2SD4 and the two antibodies (Abs) differ by 12 amino acids in the antigen (Ag) binding regions. A BIAcore biosensor instrument was used to determine the association, k(on) and dissociation, k(off), rate constants for the binding of TNFalpha to D2E7 and 2SD4. The HPLC methods were used to resolve and to study D2E7, 2SD4, and TNFalpha molecules and the noncovalent complexes of D2E7 and 2SD4 with TNFalpha. The CIEX method demonstrated that all D2E7 charged-variants bound TNFalpha equally well. There was no preferential binding for any one of D2E7 charged-variants to TNFalpha. D2E7 and 2SD4 Abs were resolved by the CIEX method. When a mixture of D2E7 and 2SD4 was mixed with excess TNFalpha, D2E7. TNFalpha complexes were formed before any 2SD4. TNFalpha complexes. Thus, the CIEX method was able to rank the affinities of the MAbs. D2E7 and TNFalpha formed complexes of 600-5000 kDa. The molecular weights of various D2E7. TNFalpha complexes were determined by a SEC method with light scattering (LS) and refractive index (RI) detectors. Upon overnight incubation, a 598-kDa complex emerged as the most stable and the only D2E7. TNFalpha complex. The molar ratio of D2E7 to TNFalpha in this complex was approximately 1:1. Based on molecular weights and the molar ratio, an immune complex, consisting of alternating three D2E7 and three TNFalpha molecules, is proposed as the most stable complex.

Homogeneous preparation of fluorescent-derivatized insulin and its application to competitive chromatographic immunoassays.

A homogeneously labeled insulin sample was prepared using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as the fluorescent-labeling reagent, and this was successfully applied to a chromatographic immunoassay. This labeled insulin was prepared by tagging all the three amino groups with AQC. Both CE and chromatographic immunoassay experiments indicated that the prepared insulin still kept its immunoaffinity to its antibody. It was observed that appropriate concentrations of acetonitrile (ACN) were efficient in lowering the quenching of the fluorescent signal of tagged insulin, in keeping the dilute, tagged insulin in solution, and in improving its peak shape during a chromatographic immunoassay. The tagged insulin was found to be 20-400 times more sensitive than native insulin detected under ultraviolet detection conditions. A competitive chromatographic immunoassay system was set up and calibrated. The system was used for analyses of an insulin-spiked urine sample, with a 96% recovery obtained.

Homogeneous fluorescent derivatization of large proteins.

A method of homogeneously derivatizing large proteins for highly sensitive analysis is described. Homogeneity of the derivative was realized by tagging all the free amino groups of proteins. With this method, alpha-chymotrypsinogen A, ovalbumin and bovine serum albumin were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Prior to the derivatization, all the proteins were reduced and alkylated. After reacting the resulting unfolded proteins with excessive amounts of AQC, the samples were analyzed with matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to determine the derivatization degree. The results indicated that all three proteins had been, or had almost been, fully derivatized. HPLC and CE were used for characterizing these protein derivatives. Under the optimized fluorescence detection conditions, the detectability of the tagged proteins was 2400-6200 times better than that detected at UV 280 nm, 170-300 times better than detected at UV 214 nm, and 150-420 times better than measured with their native fluorescence.

Femtomole peptide mapping by derivatization, high-performance liquid chromatography, and fluorescence detection.

A highly sensitive peptide mapping method using derivatization and fluorescence detection is described. Bovine cytochrome c was digested using a buffer compatible with the derivatization that followed. The derivatization was performed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The peptide mapping of the tagged digest was conducted with both HPLC and capillary LC (CLC) systems. A capillary LC-electrospray ionization mass spectrometer (MS) was set up for measuring the molecular weights of the tagged peptides. Optimization was made of the conditions used for digestion, derivatization, and mapping. MS measurements of the tagged peptides suggested that there was only one derivatization product produced from all peptides (except one) and that all the identified peptides were fully tagged. Peptide mapping of the tagged digest reviews a larger number of peptides, covering almost the entire sequence. Peptide mapping of a 20 fmol amount of tagged digest was readily performed with the CLC system. By using derivatization and fluorescence detection, the sensitivity of peptide mapping could be improved 2000 times compared to that observed with uv detection of untagged peptides.

Specific applications of capillary electrochromatography to biopolymers, including proteins, nucleic acids, peptide mapping, antibodies, and so forth.

Separation of biopolymers is an obvious application of capillary electrochromatography (CEC) technology, since speed and resolution should increase significantly over high-performance liquid chromatography (HPLC). All too often, HPLC chromatograms of polymers show poorly resolved envelopes of overlapping peaks from oligomers. The practical limitation of column length and pressure drop has hindered development of high resolution separations of many polymers in HPLC. However, this generally applies only to packed beds of small particles, and not to continuous (or monolithic) beds, as introduced by Hjerten et al. [S. Hjerten, Ind. Eng. Chem. Res. 38 (1999) 1205; S. Hjerten, C. Ericson, Y.-M. Li, R. Zhang, Biomed. Chromatogr. 12 (1998) 120; C. Ericson, S. Hjerten, Anal. Chem. 71 (1999) 1621; J.-L. Liao, N. Chen, C. Ericson, S. Hjerten, Anal. Chem. 68 (1996) 3468; S. Hjerten, A. Vegvari, T. Srichaiyo, H.-X. Zhang, C. Ericson, D. Eaker, J. Capillary. Elec. 5 (1998) 13; C. Ericson, J.-L. Liao, K. Nakazato, S. Hjerten, J. Chromatogr. A 767 (1997) 33; S. Hjerten, D. Eaker, K. Elenbring, C. Ericson, K. Kubo, J.-L. Liao, C.-M. Zeng, P.-A. Lidstrom, C. Lindh, A. Palm, T. Srichiayo, L. Valtcheva, R. Zhang, Jpn. J. Electroph. 39 (1995) 1]. Throughout this review we will refer to such packings as monolithic or continuous beds, but they are identical type packings, formed by the in situ polymerization in the capillary or column. CEC capillaries can be much longer, and contain smaller particles than is practical for HPLC. This improves resolution significantly. CEC is able to capitalize on existing mobile phase technology developed over 30 years to improve separations. The requirement that the mobile phase simultaneously promote the separation and mobile phase mobility needs to be considered. In RPLC, this dual role is not much of a problem. It may be much more important in other modes, particularly ion-exchange (IEC). As the field develops, it is becoming clear that CEC is not just a simple extension of HPLC. Instruments, column technology and operating optima are clearly different than HPLC. CEC will develop into its own unique field. Open tubular HPLC is almost precluded by the high pressures required for forcing liquids through 10 microm or smaller capillaries. Electroosmotic pumping (EOF) avoids the pressure constraints and provides better flow profiles. Compared to HPCE, the ability to interact with the stationary phase may enable separations that would be difficult with electrophoresis alone. Since the mobile phase can be less complex than micellar electrokinetic chromatography (MEKC), CEC also avoids the problem of high background signals from the micelle forming compounds. Thus CEC-MS (mass spectrometry) is expected to be even more powerful than HPCE-MS. The fortuitous, simultaneous development of matrix assisted laser desorption-time of flight MS (MALDI-TOF-MS) technology will enable extension of the mass range to above 100 000 Da. Lack of familiarity is the perhaps the largest liability of CEC compared to other techniques. This paper critically compares the state-of-the-art of CEC with HPLC and HPCE, with a particular emphasis on separation of biopolymers. The goal is to help the reader overcome the fear of the unknown, in this case, CEC.

Characterization of recombinant human monoclonal tissue necrosis factor-alpha antibody using cation-exchange HPLC and capillary isoelectric focusing.

Cation-exchange liquid chromatography (CIEX) and capillary isoelectric focusing (cIEF) methods have been developed for the routine analysis of a recombinant, human anti-tumor necrosis factor monoclonal antibody D2E7. Both of these methods can separate heavy-chain C-terminal variants of this antibody. Various enzymatic digestion methods have also been developed for the identification of the antibody C-termini lysine (Lys) variants. A comparison of conventional CIEX-HPLC and cIEF methods has been made for the analysis of antibodies. cIEF can also be used to determine the isoelectric points (pI) of antibody variants based on the use of internal pI standards. Different C-termini Lys variants have been separated and collected from the CIEX column and subsequently analyzed by cIEF and mass spectrometry.

Thermal stability studies of immunoglobulins using capillary isoelectric focusing and capillary zone electrophoretic methods.

The affinity of an antibody towards its antigen is highly specific to its conformation, in order to have optimal antibody-antigen interaction. The increase of temperature might cause changes in antibody conformations. The change of structure conformations may be reflected in the isoelectric points (pI values), peak shape and absorbance of the antibody. In this study, a monoclonal antibody was heated over a period of time. Capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) were used to monitor the change in the antibody. The longer the heating period, the lower the pI values were under cIEF conditions. CZE also showed changes in peak shapes and decreases in absorbance of the antibody with heating.

Separation and quantitation of monoclonal antibodies in cell growth medium using capillary zone electrophoresis.

IgG1 is separated from its impurities in cell growth medium under simple CZE conditions without specific sample pretreatment. Linearity, limit of quantitation, limit of detection, precision and accuracy for the method are demonstrated. The quantitation for IgG1 in the cell growth medium is obtained by generating a calibration curve and by using standard additions. This CE method can offer a good alternative to conventional HPLC methods. Attempts are also made to separate the heterogeneous species in monoclonal antibodies using both CZE and MECC.

Labeling reactions applicable to chromatography and electrophoresis of minute amounts of proteins.

Chromatography and electrophoresis have become extremely valuable and important methods for the separation, purification, detection and analysis of biopolymers and HPLC/HPCE may become the premier, preferable approaches for both qualitative and quantitative analyses of most proteins, especially from recombinant materials. This includes smaller peptides, polypeptides, proteins, antibodies and all types of protein or antibody-conjugates (antibody-enzyme, protein-fluorescent probe, antibody-drug and so forth). This entire Topical Issue of Journal of Chromatography emphasizes the application of chromatography and electrophoresis to protein analysis. This particular review deals with approaches to the selective tagging or labeling of proteins at trace (minute) levels, again using either chromatography or electrophoresis, with the emphasis on modern HPLC/HPCE methods and approaches. We discuss here both pre- and post-column labeling methods and reagents, techniques for realizing selective labeling of proteins or antibodies, applicable approaches to protein preconcentration in both HPLC and HPCE areas and in general, methods for improving (lowering) detection limits for proteins utilizing chemical or physical derivatization and/or preconcentration techniques. There are really two major goals or emphases in that which follows: (1) methods for selective labeling of proteins prior to or after HPLC/HPCE and (2) labeling of proteins at trace levels for improved separation-detection and lowered detection limits. We discuss here a large number of specific references related to both pre- and post-column/capillary derivatizations for proteins, as well as methods for improved detectability in both HPLC and HPCE by, for example, analyte preconcentration on a solid-phase extractor or membrane support, capillary isotachophoresis and other methods. Selective reactions or derivatizations on proteins refers to the ability to tag the protein at specific (e.g. reactive amino sites) in a controlled manner, with the products having the same number of tags all at the very same site or sites. The products are all the same species, having the same number of tags at the same locations on the protein. Selective reactions can also refer to the idea of tagging all of the protein sample at only a single, same site or at all available sites, homogeneously.

Characterization of antigen-antibody complexes by size-exclusion chromatography coupled with low-angle light-scattering photometry and viscometry.

In this paper, the molecular masses (M(r)s) of the complexes of monoclonal anti-BSA (antibody to bovine serum albumin) (clone: 33) and monomer BSA were determined on-line by using size-exclusion chromatography (SEC) coupled with a low-angle laser light-scattering (LALLS) detector and two concentration detectors, ultraviolet (UV) and refractive index (RI) (SEC-LALLS/UV/RI system). Also, the size and M(r)s of the complexes were evaluated by the SEC-LALLS/UV/viscometer (VISC) system. This study demonstrated that, for small size macromolecules, the combination of light scattering and viscosity detection was a suitable choice for determining their M(r)s and sizes.

High-performance capillary electrophoresis of a fermentation-derived cyclic peptide analog, animal growth promoter.

We have developed HPCE methods for the analysis of sulfomycin (trivial name) and related compounds (code name, crude material = U82127 = I), which is an animal growth promoter derived from a fermentation beer. The crude material, I, isolated from the fermentation consisted of more than 40 components which were not completely separated by conventional HPLC. Thus, as a complementary analysis method, we have optimized HPCE conditions for I using various capillaries including uncoated, coated, and packed using various buffers. The optimized HPCE conditions were obtained with an uncoated fused-silica capillary and a buffer that consisted of 30 mM Tris-tricine, 10 mM SDS, 10 mM NaCl and 20% MeOH, pH 8.0. Using these HPCE conditions, we were able to separate the one main component collected from the HPLC effluent into two or three components. In order to identify the main components of the fermentation product, an off-line HPLC-HPCE-MS analysis for I was performed. From the MALDI-TOF-MS results, the separated components collected from HPCE had different molecular masses. Four lots of I samples having different characteristics were also analyzed by HPCE to investigate lot-to-lot differences in peak profiles. The four lots of I were found to have very similar peak profiles. In this paper, I refers to the crude fermentation product and sulfomycin to the purified, major HPLC component of I.

Size-selective derivatizations with polymer immobilized reagents.

A reagent immobilized on a macroporous polymer support was prepared for size selective derivatizations. These derivatization reagents showed two distinct reaction zones. Sterically bulky analytes were denied access to some of the surface of the support, so that when reagent on the outer surface was exhausted from the support, only analytes that had fail access to all regions of the support could react. These reagents were applied to the derivatization of different amines in the presence of a high concentration of a bulky analyte, adamantanamine. Pore size measurements and determination of molecular dimensions also support a size-selective derivatization mechanism. The reagents were also applied to the derivatization of proteins. Proteins with several sites available for tagging shows a reduced number of products with size selective reagents, reflecting reactions only at sites accessible to the reagent.

HPCE methods for the identification and quantitation of antibodies, their conjugates and complexes.

We review here much of the existing literature that deals with analysis, resolution, characterization, and (at times) quantitation of antibodies in capillary electrophoresis modes. Each major mode of CE shown applicable to antibody analysis is described, along with the major applications of that mode for antibodies. Discussions are presented as to the mechanisms of antibody resolution in CE, interactions of various buffer components with the proteins leading to resolution, and methods of quantitation for antibodies. The literature is critically reviewed with regard to true application of CE for antibody analysis, limitations, information possible, information implied, and which samples have actually been assayed by CE modes. The literature is critically reviewed up to and including 1996, both for the scientific and commercial literature, especially vendor applications and real world applications possible.

Immunodetection approaches and high-performance immunoaffinity chromatography for an analogue of bovine growth hormone releasing factor at trace levels.

An indirect detection method using high-performance immunoaffinity chromatography (HPIAC) was used to measure low levels of an analogue of bovine Growth Hormone Releasing Factor (bGHRF). An antibody (Ab) labelled with alkaline phosphate (ALP) was incubated with the bGHRF analogue to perform a complex between the antigen (Ag) and the antibody-enzyme (Ab-En) conjugate. The complex was then injected onto a cartridge containing an immobilized Ag affinity support. Species which were not recognized by the affinity cartridge, i.e. eluted, were then directly combined, via a connecting tee, with a buffer containing a substrate. Incubation proceeded on-line, inside a knitted reactor coil, under conditions of constant flow. The subsequent generation of a fluorescently active substrate product was detected by conventional means. The assay described has a linear response region from 1.0 to 25 ng of the bGHRF analogue and a limit of detection of 0.60 ng (1.7 x 10(2) femtomole, 30 p.p.b.). This approach was compared against a method in the antigen/Ab-En complex was injected onto a immobilized Ab affinity cartridge to form an antibody-antigen conjugate sandwich and subsequent stop-flow incubation with substrate.

Immunochromatographic analysis of bovine growth hormone releasing factor involving reversed-phase high-performance liquid chromatography-immunodetection.

We have developed high-performance immunoaffinity chromatography (HPIAC) methods for the detection and quantitation of bovine growth hormone releasing factor (GHRF), which could also be applicable to its metabolites in biofluids. These approaches have involved a combination of IAC using immobilized antibody (Ab) to GHRF, together with reversed-phase high-performance liquid chromatography (RP-HPLC) separations of initially isolated and concentrated protein, followed by selective detection, involving on-line immunodetection (ID) schemes. ID methods involved HPIAC supports of the Ab, together with synthesized Ab-fluorescein isothiocyanate conjugates. We have demonstrated optimization methods for each step of the entire hyphenated technique (IAC-HPLC-ID), and then actually quantitated GHRF using this overall system. The minimum detectable concentration was about 1 ng/5 ml (200 ppt) with fluorescence detection (excitation wavelength, 490 nm; emission wavelength, 510-650 nm). We have also tested a single blind, spiked biological sample (bovine plasma), spiked with a known level of GHRF. Accuracy (7.4%) and precision (S.D. = +/- 22%) were quite acceptable for a double immunoassay method.

Perfusion immunoaffinity chromatography and its application in analysis and purification of biomolecules.

Immunoaffinity cartridges have been prepared by immobilizing the monoclonal antibody to bovine growth hormone (bGH) and bovine growth hormone releasing factor (bGRF) on the streptavidin-coated perfusion media. The cartridge-immobilized antibody to bGH has been used for the analysis of standard sample of recombinant bGH (r-bGH). The cartridge immobilized bGRF has been applied for purification of the C-and N-terminal antibodies and their conjugates with horseradish peroxidase. The purity of the antibody fractions purified by bGRF cartridges has been tested by capillary zone electrophoresis. It has been shown that the immunoaffinity cartridges prepared by immobilizing the antigen offer more biological specificity for the purification of antibody than the Protein G cartridges. The stability and life time of the immunoaffinity cartridges are mainly dependent on the stability of the biomolecules immobilized on them. Immunoaffinity cartridges based on the perfusion media offer us the tools for rapid analysis and purification of antigens or antibodies as well as for determination of the biological activity between them at a very low back pressure in the columns.

Capillary isoelectric focusing as a tool in the examination of antibodies, peptides and proteins of pharmaceutical interest.

This paper describes the recent history and development of capillary isoelectric focusing (cIEF), as it has evolved over the past 10 years forming a distinct mode of high-performance capillary electrophoresis (HPCE). The theory, equations, fundamentals and basics of cIEF are discussed and described, including modes of focusing and mobilization, coated vs uncoated capillaries, different detection schemes, resolutions possible, peak capacity possible and final commercialized approaches now available. Then, the applications of the technique are emphasized, as applied to smaller peptides, larger proteins and still larger antibodies and antibody-protein complexes. The emphasis has been on the application of capillary electromigration techniques in drug analysis. Throughout, attempts have been made to emphasize the potential applications and uses of cIEF methods, and how these might be successfully utilized in drug analysis and assays for larger biopolymers.

Characterization of immunochemical reaction for human growth hormone with its monoclonal antibody by perfusion protein G affinity chromatography and capillary zone electrophoresis.

An extremely rapid assay technique for antibody-antigen interaction using human growth hormone and its monoclonal antibody as an example has been developed by utilizing Protein G bound to perfusion chromatography matrices. The antibody and antigen were mixed and incubated at different molar ratios by keeping the concentration of antibody constant. The mixture of antibody and antigen solution was then injected onto the Protein G column. The complex of antibody and antigen formed in the sample solution was retained on the Protein G column as was the free antibody. The peak-area and height of the retained complex and antibody linearly increased with the molar concentration of the antigen when it was not in excess of the corresponding stoichiometric amount of antibody, however, those of the retained peak were constant when the concentration of antigen was in excess of that of the antibody. The validity of this method was confirmed by the results of capillary zone electrophoresis. The method developed cannot only be used to determine the biological activity between antibody and its antigen quickly, but also to determine the stoichiometry of immunological reactions between the antibody and antigen when a stable complex of them can be formed.

Electrochemical activity of 6-aminoquinolyl urea derivatives of amino acids and peptides. Application to high-performance liquid chromatography with electrochemical detection.

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (6-AQC) is a reagent used to increase the detection-sensitivity of amino acids and peptides in high-performance liquid chromatography with fluorescence detection. In this paper, the electrochemical characteristics of the derivatives of 6-AQC are described. Electrochemical detection of 6-AQC amino acids and peptide derivatives following reverse-phase HPLC are also reported. The response linearity of the derivatives on an amperometric detector was studied in the range of 5 pmol (0.5 mu M) to 2500 pmol (250 mu M). Approximately 2.5 pmol of the amino acid derivatives could be detected. The quantitative results of amino acids in plasma and a bovine serum albumin hydrolysate agreed well with values reported in the literature.