Purification of monoclonal antibodies by hydroxylapatite HPLC and size exclusion HPLC

Monoclonal antibodies of both the IgG and the IgM type were purified by hydroxylapatite HPLC (HA-HPLC) under very mild conditions. The IgM type antibodies, which were isolated from ascites fluid and separated from other proteins also by means of size exclusion HPLC. It was shown that the most frequently observed disadvantage of HA-HPLC, that is the relative short life of the columns (P. Steffen (1989) GIT Fachz. Lab., Suppl. 3/89 (Chromatogr.), 50-90), is due to microbial contamination rather than lower mechanical stability. In order to monitor column performance, a test was developed based on the use of standard proteins under isocratic separation conditions. This allows a direct comparison between the respective performances of columns made from different materials, hydroxylapatite or fluoroapatite, from different sources and with different particle sizes. A problem which often occurs with HA-HPLC in the case of IgM antibody isolation, namely precipitation of the antibodies at low salt concentrations at the beginning of a chromatographic run, was avoided by adding sodium chloride to both separation buffers.     

Immunological evidence for a P2 protamine precursor in mature rat sperm.

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.     

Selenoprotein P analysis in human plasma

Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.     

Gene synthesis, expression in Escherichia coli and purification of immunoreactive human insulin-like growth factors I and II. Application of a modified HPLC separation technique for hydrophobic proteins

We have expressed synthetic genes encoding human insulin-like growth factors I and II in large quantities in Escherichia coli as fusion proteins with the 300 N-terminal amino acids of the E. coli trpE gene product. The resulting hybrid proteins were purified from the insoluble fraction of crude bacterial lysates using a rapid HPLC separation procedure employing a C8 reversed-phase column and a gradient of 2-propanol in formic acid. With the procedure we obtained in volatile solvents highly purified fusion proteins that were used for further biochemical and immunological procedures. Here we describe biochemical characteristics of the bacterially expressed fusion proteins and demonstrate that these proteins share substantial antigenic properties with the native polypeptides allowing the establishment of highly specific monoclonal antibodies.     

An avidin-biotin solid phase ELISA for femtomole isopentenyladenine and isopentenyladenosine measurements in HPLC purified plant extracts

A solid phase enzyme immunoassay was developed for isopentenyladenine (iP) and isopentenyladenosine (iPA) quantitation in HPLC purified plant extracts. It was performed on antigen-coated microtitration plates on which bound antibodies were indirectly labeled by the means of a biotinylated goat anti-rabbit antibody and an avidin-alkaline phosphatase conjugate. Less than 3 femtomoles of iP or iPA were easily detected and the measuring range extended from 3 femtomole to 1 picomole. The reproducibility has been tested and intra- and interassay variations did not exceed 5.0%. The specificity of iPA antibodies was good, as determined by cross-reactivity measurements with other adenylic compounds. The specificity of the measurements for iP and iPA was demonstrated by analysis of the immunoreactivity of fractions obtained by HPLC separation of a methanolic tobacco leaf extract.     

HPLC techniques for proteomics analysis--a short overview of latest developments.

Due to the complex nature of the proteome, instrumentation and methods development for sample cleanup, fractionation, preconcentration, chromatographic separation and detection becomes urgent for the identification of peptides and proteins. Newly developed techniques and equipment for separation and detection, such as nano-HPLC and multidimensional HPLC for protein and peptide separation, enabled proteomics to experience dynamic growth during the past few years. In any proteomic analysis the most important and sometimes most difficult task is the separation of the complex mixture of proteins or peptides. This review describes some aspects and limitations of HPLC, both multidimensional and one-dimensional, in proteomics research without attempting to discuss all available HPLC methods, which would need far more space than available here.     

Recombinant human insulin V Optimization of the reversed-phase high-performance liquid chromatographic separation

The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (μ). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.     

Alternative workflows for plant proteomic analysis

High-throughput separations are intrinsic to the detection and analysis of peptides and proteins by mass spectrometry (MS). Together, efficient separation and MS can lead to the identification of thousands of proteins in a sample, cell or tissue and help build proteome maps that can be used to define a cell type or cellular state. Although 2D gels have been successfully used to separate proteins for subsequent MS analysis, alternative separation efficiencies and, consequently deeper results could be obtained with HPLC or other separation techniques that improve throughput. This highlight is aimed toward plant scientists who have special separation needs due to the nature of plant cells and who could benefit from knowing options and requirements for adopting alternative separation protocols. Through the various sample processing and protein separation strategies, plant biologists should be able to improve the quality of their proteomic reference maps and gain new information about the proteins that define plant cells.     

Antikinetochore antibodies interfere with prometaphase but not anaphase chromosome movement in living PtK2 cells.

Injection of CREST antikinetochore antiserum (AKA) containing antibodies to the kinetochore into living prometaphase PtK2 cells decreased chromosome velocity to near zero. Injection of either phosphate-buffered saline or CREST antiserum without antikinetochore antibodies (antikinetochore negative: AKN) had no effect on prometaphase oscillations. AKA antiserum injected into anaphase cells at the beginning of chromatid separation had no effect on anaphase chromosome velocity, spindle elongation, or cytokinesis. Visible binding of antikinetochore antibodies in prometaphase cells at room temperature occurred between 5 and 15 minutes after injection. Anaphase cells injected at the beginning of chromatid separation had bound antibody at the end of anaphase. AKA antiserum recognizes in Western blots proteins associated with the primary constriction: CENP-B, -C, and -D, as reported by other workers. The control antiserum, AKN, does not recognize these proteins. These results imply that the antigens recognized by CREST antibodies are important for chromosome movement. Whether or not these antigens are themselves motor molecules cannot be addressed by the present data. In addition, the results suggest that these antigens are not involved in an important way in anaphase movement.     

Automated sample introduction for an imaged capillary isoelectric focusing instrument via high-performance liquid chromatography sampling devices

Sample introduction of an imaged capillary isoelectric focusing (cIEF) instrument is fully automated by using commercially available high-performance liquid chromatography (HPLC) injection valves and autosamplers. Sample carryover can be controlled to under 1% when the valve and separation column are washed for 1 min between sample runs. The standard deviation of peak areas for 20 injections is 3.5%, which includes deviations created by the absorption imaging detector and the isoelectric focusing process inside the 75 μm I.D. column. Sample throughput is up to 10 samples per hour. The instrument has been applied to fast analysis of many proteins including monoclonal antibodies.     

Principles of the design and operational considerations of large scale high performance liquid chromatography (HPLC) systems for proteins and peptides purification.

This review introduces concepts of design of large scale HPLC systems for purification of proteins and peptides. It is addressed to users of large scale HPLC systems to aid in system selection and help in customizing the design. Major techniques used for large scale HPLC purification of proteins and peptides are briefly reviewed. Engineering aspects of system design are discussed in detail. The review of selected relevant literature is provided as well as author's experience with the existing designs and his own systems. Commercial publications have been used in preparation of this review but only the most significant are listed as examples. The design process for any new system should be related to the performance of existing systems, if possible of a large scale. Laboratory data are also very useful in aiding the design process since they provide a lead, as to which chromatography techniques may succeed in providing required separation levels. The expertise needed for system design and operation comes from many areas: protein and peptide chemistry, chromatographic theory, mass transfer and hydrodynamics, machine design and material science. All these factors have to be blended together during the system design process. The controls must ensure flexibility in adapting to changing system configuration, depending on the operational requirements for various products. Extensive interfacing with the operator during the process run is essential. This work is focused mostly on system design and operation for reversed-phase chromatography and hydrophobic interaction chromatography, but it also covers aspects associated with other chromatographic techniques. The reviewed design principles would also apply to design other than HPLC large scale chromatography systems for biotechnology and pharmaceutical operations.     

Ribosomal protein interactions in yeast. Protein L15 forms a complex with the acidic proteins

Protein L15 from Saccharomyces cerevisiae ribosomes has been shown to interact in solution with acidic ribosomal proteins L44, L44' and L45 by different methods. Thus, the presence of the acidic proteins changes the elution characteristics of protein L15 from CM-cellulose and DEAE-cellulose columns and from reverse-phase HPLC columns. Moreover, immunoprecipitation using anti-L15 specific monoclonal antibodies coprecipitates the acidic proteins, too. Conversely, antibodies raised against the acidic proteins immunoprecipitate protein L15. This coprecipitation seems to be specific since it does not involve other ribosomal proteins present in the sample. Similarly, plastic-adsorbed antibodies specific for one of the components in the L15--acidic-protein complex are able to retain the other component of the complex but cannot bind unrelated proteins. Moreover, protein L15 can be chemically cross-linked to the acidic proteins in solution. These results indicate that protein L15 might be equivalent to bacterial ribosomal protein L10 in forming a complex with the acidic proteins. Since, on the other hand, protein L15 has been shown to be immunologically related to bacterial protein L11 [Juan Vidales et al. (1983) Eur. J. Biochem. 136, 276-281] and to interact with the same region of the large ribosomal RNA as does protein L11 [El-Baradi et al. (1987) J. Mol. Biol. 195, 909-917], these results suggest strongly that protein L15 plays the same role in the yeast ribosome as proteins L10 and L11 do in the bacterial particles.     

Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.     

Low-mass proteome analysis based on liquid chromatography fractionation,nanoliter protein concentration/digestion,and microspot matrix-assisted laser desorption ionization mass spectrometry

HPLC fractionation combined with mass spectrometry can become a powerful tool for analyzing the proteome in the mass range below 15 kDa where efficient protein separation by gel electrophoresis can be difficult. For sensitive and high-resolution separation of the low-mass proteome, the use of analytical rather than preparative HPLC columns is preferred. However, individual fractions collected by a conventional HPLC separation usually contain a small amount of proteins whose concentrations may not be sufficiently high for subsequent enzyme digestion and protein identification by mass spectrometry. In this work, we present a high sensitivity nanoliter sample handling technique to analyze proteins fractionated by HPLC. In this technique, an individual HPLC fraction in hundreds of microliter volume is pre-concentrated to several microliters. About 700 pl of the pre-concentrated fraction is then drawn into a 20-microm I.D. capillary and dried in a small region near the capillary's entrance. This process can be repeated many times to concentrate a sufficient amount of protein to the small region of the capillary. After protein concentration, protein digestion is achieved by drawing 1 nl of chemical or enzymatic reagent into the capillary and placing it in the same region where the dried protein sits. The resulting peptides are then deposited onto a microspot in a MALDI probe for mass analysis. The performance of this technique is demonstrated with the use of a standard protein solution. This technique is applied to the identification of low-mass proteins separated by HPLC from a complex mixture of an E. coli extract.     

Proteome analysis of a rat liver nuclear insoluble protein fraction and localization of a novel protein, ISP36, to compartments in the interchromatin space

A rat liver nuclear insoluble protein fraction was analyzed to investigate candidate proteins participating in nuclear architecture formation. Proteins were subjected to two-dimensional separation by reversed-phase HPLC in 60% formic acid and SDS/PAGE. The method produced good resolution of insoluble proteins. One hundred and thirty-eight proteins were separated, and 28 of these were identified. The identified proteins included one novel protein, seven known nuclear proteins and 12 known nuclear matrix proteins. The novel 36 kDa protein was further investigated for its subnuclear localization. The human ortholog of the protein was expressed in Escherichia coli and antibodies were raised against the recombinant protein. Exclusive localization of the protein to the nuclear insoluble protein fraction was confirmed by cell fractionation followed by immunoblotting. Immunostaining of mouse C3H cells suggested that the 36 kDa protein was a constituent of an insoluble macromolecular complex spread throughout the interchromatin space of the nucleus. The protein was designated 'interchromatin space protein of 36 kDa', ISP36.     

A radiochemical study of irreversible adsorption of proteins on reversed-phase chromatographic packing materials

A radiochemical study of the irreversible adsorption of proteins on commercial reversed-phase HPLC packing materials is reported. The conditions of study are similar to those used in HPLC separation of protein. The effects of the amount and contact time of two proteins, ovalbumin and cytochrome c, are reported. Additional results include the effect of column pretreatment with protein, silanophilic mobile-phase blocking agent, and type of packing material on the extent of irreversible adsorption. The loss process is shown to be at least biphasic and the mechanisms of loss distinct for different proteins.     

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.     

Isolation of intact proteins from acid‐degradable polyacrylamide gel

Elucidating native structure–function relationships of proteins identified using PAGE has been impeded by limitations in the isolation of intact proteins from the gel. By hydrolyzing polyacrylamide gel band under mildly acidic conditions rather than digesting entrapped proteins ～70% of a large native protein, mouse IgG1 (molecular weight 150 kDa), was isolated. Further analysis indicated that the isolated antibodies had preserved specific binding capability to target antigens as well as intact molecular weights. This new technology may contribute to functional proteomic studies through the isolation of proteins in their native state after PAGE, and other technologies requiring simultaneous separation and isolation of other macromolecules and complexes.