Salt-independent binding of antibodies from human serum to thiophilic heterocyclic ligands

Several thiophilic adsorbents with mercaptoheterocyclic ligands have been analyzed for their ability to bind human serum proteins in a salt-independent way. In contrast to 2-mercaptopyrimidine, 2-mercaptopyridine derived ligands show a group-selective binding of immunoglobulins and α₂-macroglobulin, not only in the presence of high concentrations of sodium sulphate but in buffers with low ionic strength. The binding is restricted to thiophilic gels obtained by coupling 2-mercaptopyridine to a vinylsulphone-activated matrix and is not achieved on epichlorohydrin-activated gels. A novel thiophilic ligand based on mercaptonicotinic acid, containing a carboxylic group together with the thiophilic pattern of thioaromatic adsorbents, is demonstrated to be useful as an alternative purification scheme for antibodies.     

Sulfone-aromatic ligands for thiophilic adsorption chromatography: purification of human and mouse immunoglobulins.

New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-O-CH2-CH2-SO2-CH2-CH2-X-Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300-1500%) was obtained by using 1.0-1.2 M ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.     

Thiophilic adsorption of immunoglobulins--analysis of conditions optimal for selective immobilization and purification

Immunoglobulins have been selected by their general affinity for adjacent sulfone-thioether sulfur groups as a useful model system for the characterization of thiophilic interaction chromatography. Mercaptoethanol coupled to divinylsulfone-activated agarose (thiophilic or T-gel) provided an affinity matrix for the efficient and reversible immobilization of the immunoglobulins. The adsorption/desorption process was investigated as a function of protein concentration, temperature, flow rate, and pH in different concentrations of ammonium sulfate. Immobilization of these proteins was (as a function of pH) found to be both dependent and independent of the adsorption-promoting effects of water-structure-forming salts. Buffer conditions are recommended for the selective adsorption of immunoglobulins from unfractionated human serum. These results indicate that thiophilic interaction chromatography provides a new and effective alternative for the immobilization and purification of immunoglobulins and other proteins under conditions known to preserve structure and biological activity.     

Investigations on the specificity of thiophilic interaction for monoclonal antibodies of different subclasses

A comparative study was carried out to investigate the influence of different mouse antibody subclasses on the chromatographic behaviour on thiophilic supports. Cell-free supernatants from different mouse-mouse hybridoma cultures in a standard medium were purified on thiophilic agarose and Fractogel EMD TA. The adsorption capacities and purification factors were monitored under optimised adsorption conditions. The different isotypes did not differ significantly regarding capacity of the thiophilic matrix, but the purity of the eluted antibody fractions was significantly lower for the IgG_2a subclass compared to all other murine antibodies. A significant copurification of proteins from cell culture supernatant with antibodies of the IgG_2a subclass indicated a restriction in the universal nature of thiophilic interaction.     

Novel heterocyclic ligands for the thiophilic purification of antibodies

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.     

Thiophilic paramagnetic particles as a batch separation medium for the purification of antibodies from various source materials

A preparation of thiophilic agarose-based paramagnetic particles (T-Gel) has been developed with physical characteristics (particle size and particle density) that facilitate its use as a batch separation medium suitable for the large-scale purification and isolation of immunoglobulins. The medium was used to extract immunoglobulins from a wide range of starting materials, including sera, ascites fluid, tissue culture medium, and whole blood. None of these starting materials required pretreatment such as clarification by centrifugation or filtration prior to antibody extraction. The antibody purity obtained using T-Gel compared well with that obtained using protein A agarose column chromatography. Yields were approximately 30 mg of immunoglobulins per milliliter of T-Gel, and little was required in the way of specialist equipment. The method is uncomplicated and involves a roll mix extraction overnight, followed by magnetic separation to facilitate supernatant removal and subsequent washing of the particles. Elution of bound antibodies was carried out at neutral pH to yield a concentration of immunoglobulins that was approximately 7 mg/ml. The method was found to be applicable to antibody purification from the blood serum of seven different mammalian species and for all immunoglobulin classes.     

Behaviour of human immunoglobulin G subclasses on thiophilic gels: comparison with hydrophobic interaction chromatography

We have used thiophilic and hydrophobic interaction chromatography in an attempt to obtain enriched human immunoglobulin G (IgG) subclasses from a therapeutic immunoglobulin preparation. Proteins were adsorbed on a thiophilic gel and on Phenyl-, Butyl-, or Octyl-Sepharose in 1 M ammonium sulphate. Elution with a decreasing salt gradient produced no marked subclass selectivity, except with Octyl-Sepharose, which yielded a poorly adsorbed fraction somewhat enriched in IgG2, representing ca. 20% of the total initial protein. Neither thiophilic nor hydrophobic interaction chromatography appear suitable for an efficient enrichment in subclasses, which all show a broad heterogeneity in their affinity for these columns. The influence of the starting salt concentration was also studied. With thiophilic gels, in the absence of ammonium sulphate, ca. 30% of the initial load was not adsorbed, and was found to be enriched in IgG2. At 2.5 and 5% ammonium sulphate, practically no adsorption occurred. At 7.5% ammonium sulphate, the non-adsorbed fraction was enriched in IgG3. With Phenyl-Sepharose, adsorption increased smoothly with the salt concentration. It is concluded that different forces come into play for adsorption on thiophilic gels at low and high salt concentration.     

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.     

Recombinant antibodies for the depletion of abundant proteins from human serum

The identification of biomarkers from serum or plasma is often hindered by a few proteins present at high concentrations, which may obscure less abundant proteins. Ideal serum depletion strategies would be flexible as regards the proteins to be removed, and would feature the use of reagents with long shelf-lives. In this article, we describe a novel protein depletion methodology based on the incubation of serum samples with phage-derived recombinant antibody fragments, which are able to bind to staphylococcal Protein A, and which carry a C-terminal peptide tag capable of streptavidin binding. The resulting protein-antibody complexes can be removed by simultaneous capture on Protein A and/or streptavidin resin. The depletion methodology was exemplified by the isolation of recombinant human mAb fragments specific to abundant human serum Ags and by the simultaneous depletion of albumin, immunoglobulins, alpha2-macroglobulin, hemoglobin, transferrin and haptoglobin. The depleted serum samples were analyzed by 2-DE and by gel-free MS-based methodologies, confirming the efficiency and selectivity of the depletion process. The methodology presented is modular in nature, since several recombinant antibodies can be combined in a single depletion experiment. Furthermore, antibodies do not have to be covalently coupled to a solid support facilitating long-term storage.     

Preparation of C60‐functionalized magnetic silica microspheres for the enrichment of low‐concentration peptides and proteins for MALDI‐TOF MS analysis

In this work, for the first time, a novel C60‐functionalized magnetic silica microsphere (designated C60‐f‐MS) was synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres. The resulting C60‐f‐MS microsphere has magnetite core and thin C60 modified silica shell, which endow them with useful magnetic responsivity and surface affinity toward low‐concentration peptides and proteins. As a result of their excellent magnetic property, the synthesized C60‐f‐MS microspheres can be easily separated from sample solution without ultracentrifuge. The C60‐f‐MS microspheres were successfully applied to the enrichment of low‐concentration peptides in tryptic protein digest and human urine via a MALDI‐TOF MS analysis. Moreover, they were demonstrated to have enrichment efficiency for low‐concentration proteins. Due to the novel materials maintaining excellent magnetic properties and admirable adsorption, the process of enrichment and desalting is very fast (only 5 min), convenient and efficient. As it has been demonstrated in the study, newly developed fullerene‐derivatized magnetic silica materials are superior to those already available in the market. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel C60‐f‐MS microspheres makes it a promising candidate for isolation of low‐concentration peptides and proteins even in complex biological samples such as serum, plasma, and urine or cell lysate.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Facile synthesis of C8-functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysis

In this study, novel C8-functionalized magnetic polymer microspheres were prepared by coating single submicron-sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8-functionalized magnetic silica (C8-f-M-S) microspheres exhibit well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8-f-M-S microspheres provide a convenient and efficient way for enrichment of low-abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI-TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8-f-M-S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine.     

Feasibility studies of large scale production of human anti-tetanus toxoid monoclonal antibodies

The feasibility of large scale production of human anti-tetanus toxoid monoclonal antibody for therapeutic use was evaluated using a human heterohybridoma. The effects of duration of subculture, transition from static to agitated culture conditions and the level of serum concentration were studied. The level of antibody secreted by the clone decreased with increasing length of subculture and decreasing serum concentration. The clone exhibited heterogeneity in expression of surface IgG after 2 or 7 weeks of subculture in static culture conditions irrespective of the serum concentration. However, a prolonged duration of subculture (9 weeks) in 3% serum medium had an effect on the expression of surface IgG both in static and agitated culture conditions. With respect to total (surface and intracellular) IgG, two distinct cell populations were observed. On long term subculture (9 weeks) in low serum medium (3% FCS), there was a decrease in the population which was the high synthesizer. In addition, when these cells were cultivated in agitated spinner flasks, a defect in secretion of antibodies was observed. Thus a general fall in the amount of antibody in the supernatant of agitated cultures was due to decrease in antibody synthesis as well as the defect in secretion of antibodies.     

Purification of individual tRNAs using a monoclonal anti-AMP antibody affinity column

A murine monoclonal anti-AMP antibody affinity matrix was used for isolation of individual species of amino acid transfer nucleic acids (tRNAs). The antibodies had been prepared using 5'-AMP covalently attached to bovine serum albumin as antigen and exhibited high affinity for 5'-AMP but greatly reduced affinity for 3'-AMP. Native uncharged tRNAs that terminate in a 5'-AMP group on the amino acid acceptor arm of the molecule bind tightly to the anti-AMP affinity matrix, whereas aminoacylated tRNAs are not retained. This allows separation of a particular tRNA species as its aminoacyl derivative from a complex mixture of uncharged tRNAs under very mild conditions.     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Thiophilic adsorption: a comparison of model protein behavior

A newly recognized type of protein-ligand interaction phenomenon has resulted in the preparation of simple, nonionic, and highly specific gel derivatives for selective adsorption chromatography. The essential structure of the immobilized ligand can be represented as agarose-CH2CH2SO2CH2CH2SCH2CH2OH, which was prepared by using mercaptoethanol to derivatize [0.9-1.0 mmol (g of dry gel)-1] divinyl sulfone activated agarose (thiophilic or T-gel). Proteins interacting with this ligand are provisionally termed "thiophilic" to recognize their affinity for the definitive sulfone-thioether constituents. To better understand the experimental variables affecting adsorption efficiency and selectivity, several well-characterized proteins with diverse physicochemical features have been evaluated for thiophilic properties. Thiophilic interaction chromatography was investigated as a function of pH as well as the type and concentration of water-structure-forming salts required to promote adsorption. The model proteins characterized varied distinctly in their individual thiophilic affinities. At acidic pH values, a salt-independent adsorption process was observed. Furthermore, a minimum in the salt-promoted thiophilic adsorption tendency at pH 5-6 was found, with varying magnitude, for each of the model proteins evaluated. Recovery of adsorbed proteins routinely varied from 90% to 100%. There does not appear as yet to be any easily recognized physicochemical property associated with either thiophilic or nonthiophilic behavior. These results suggest that thiophilic interaction chromatography is a process that utilizes a previously unrecognized protein-ligand interaction mechanism. We suggest that salt allows the protein into close proximity with the sulfone-thioether group where short-range forces are effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elimination of mycoplasma contaminants from cell cultures with animal serum

Repeated treatment with guinea pig or rabbit serum, but not with human serum, was found to eliminate mycoplasma contaminants from mammalian cell cultures as judged by staining with the fluorescent dye Hoechst 33258. Following treatment with rabbit serum and several passages, M. hyorhinis could not be detected by staining, isolation on agar, or specific immunofluorescence in a human prostate carcinoma cell line heavily contaminated with this organism. There was no evidence for the involvement of antimycoplasma antibodies in the bactericidal activity of rabbit serum. Mycoplasmacidal activity of rabbit serum was associated with a heat-labile component(s) which could be inactivated by incubation of the serum with goat antirabbit complement component C3.     

On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis

In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.     

C‐terminal lysine processing of human immunoglobulin G2 heavy chain in vivo

Although human IgG heavy chain genes encode a C‐terminal lysine, this residue is mostly absent from the endogenous antibodies isolated from serum. Some low but variable level of C‐terminal lysine is present on therapeutic antibodies expressed in mammalian cell culture systems. Here, we monitored the C‐terminal lysine processing of a recombinant human IgG2 antibody after intravenous injection into human subjects. Peptide mapping of the therapeutic antibody isolated from serum samples by affinity purification was used to quantify the C‐terminal lysine levels over time in vivo. The C‐terminal lysine residue was found to be rapidly lost in vivo with a half life of about an hour (62 min). In vivo C‐terminal lysine processing could be reproduced in vitro, but at a faster rate, by incubating in human serum. Pretreated serum, under conditions used to inactivate carboxypeptidase U, generated in vitro C‐terminal lysine processing rates that more closely matched those in vivo. Endogenous IgG, isolated from human blood, contained very low levels of C‐terminal lysine (～0.02%), consistent with the expected circulating half life of antibodies and the calculated C‐terminal lysine processing rate. Thus, the low residual IgG2 C‐terminal lysine is rapidly processed in vivo and such processing likely occurs on endogenous antibodies in circulation. Biotechnol. Bioeng. 2011;108: 404–412. © 2010 Wiley Periodicals, Inc.