Biomedical subjects

S M Cramer

Publications and source records attributed to S M Cramer.

At least 19 recordsLinked to original sources

PubMed2005

The effect of multi-component adsorption on selectivity in ion exchange displacement systems.

This paper examines chemically selective displacement chromatography using affinity ranking plots, batch displacer screening experiments, column displacements, multi-component adsorption isotherms and spectroscopy. The affinity ranking plot indicated that the displacers, sucrose octasulfate (SOS) and tatrazine, should possess sufficient affinity to displace the proteins amyloglucosidase and apoferritin over a wide range of operating conditions. In addition, the plots indicated that the separation of these proteins by displacement chromatography would be extremely difficult. Further, the two proteins were shown to have very similar retention times under shallow linear gradient conditions. When batch displacement experiments were carried out, both tartrazine and SOS exhibited significant selectivity differences with respect to their ability to displace these two proteins, in contrast to the affinity ranking plot results. Column displacement experiments carried out with sucrose octasulfate agreed with the predictions of the affinity ranking plots, with both proteins being displaced but poorly resolved under several column displacement conditions. On the other hand, column displacement with tartrazine as the displacer resulted in the selective displacement and partial purification of apoferritin. Single- and multi-component isotherms of the proteins with or without the presence of displacers were determined and were used to help explain the selectivity reversals observed in the column and batch displacement experiments. In addition, fluorescence and CD spectra suggested that the displacers did not induce any structural changes to either of the proteins. The results in this paper indicate that multi-component adsorption behavior can be exploited for creating chemically selective displacement separations.

Adsorption↗

PubMed2002

Comparison of linear gradient and displacement separations in ion-exchange systems.

The linear gradient mode of chromatography is the most widely employed mode of operation in ion-exchange chromatographic separations. However, in recent years, the displacement mode has received considerable attention because of its promise of high throughput and high resolution. To enable a comparison of these two modes of chromatography, it is essential to identify the optimum operating conditions for each. We employed an iterative algorithm to carry out the necessary optimization. The Steric Mass Action model of ion-exchange chromatography is used in concert with the solid-film linear-driving force model to describe the chromatographic behavior of the solutes in these systems. The performances of displacement and gradient modes of chromatography are compared for different types of separation problems. It turns out that for "easy" separations, both the modes are equally effective. However, for challenging separations, the displacement mode is superior to the gradient mode. Our results shed significant light on the performance of gradient and displacement modes in protein ion-exchange systems.

Adsorption↗

PubMed2001

Prediction of protein retention in ion-exchange systems using molecular descriptors obtained from crystal structure.

In this paper, a novel approach is described for the a priori prediction of protein retention in ion exchange systems. Quantitative structure retention relationship (QSRR) models based on a genetic algorithm/partial least squares approach were developed using experimental chromatographic data in concert with molecular descriptors computed using protein crystal structures. The resulting QSRR models were well-correlated, with cross-validated r2 values of 0.938 and 0.907, and the predictive power of these models was demonstrated using proteins not included in the derivation of the models. Importantly, these models were able to predict selectivity reversals observed with two different stationary phase materials. To our knowledge, this is the first published example of predictive QSRR models of protein retention based on crystal structure data.

Chromatography, Ion Exchange↗

PubMed2001

Characterization and modeling of monolithic stationary phases: application to preparative chromatography.

A methodology for characterizing and modeling preparative separations on monolithic ion-exchange stationary phases is presented. A dimensionless group analysis was carried out to determine the relative importance of mass transfer and kinetic resistances on this stationary phase. In contrast to conventional beaded morphologies, the continuous bed stationary phase was found to possess enhanced mass transport properties resulting in kinetic resistance as the dominant non-ideality. Accordingly, a reaction-dispersive steric-mass action formalism was successfully utilized for simulating preparative displacement chromatography on this resin. Since kinetics were found to be important on this column morphology, mobile phase salt concentration was found to be an important variable during displacement chromatography on this stationary phase. An increase in the mobile phase salt concentration was found to significantly improve the displacement separation of a model protein mixture. The formalism presented in this paper provides a better understanding of preparative chromatography in monolithic resin systems and a means of simulating separations on this class of chromatographic stationary phases.

Adsorption↗

PubMed2001

Purification of an oligonucleotide at high column loading by high affinity, low-molecular-mass displacers.

The development of efficient techniques for large-scale oligonucleotide purification is of great interest due to the increased demand for antisense oligonucleotides as therapeutics as well as their use for target validation and gene functionalization. This paper describes the use of anion-exchange displacement chromatography for the purification of 20-mer phosphorothioate oligonucleotide from its closely related impurities using low-molecular-mass amaranth as the displacer. Experiments were carried out to examine the effect of the feed load on the performance of the displacement chromatography. In contrast to prior work, displacement chromatography was successfully scaled-up to high column loadings while maintaining high purity and yields. Experiments carried out on a Source 15Q column indicated that crude oligonucleotide loading as high as 39.2 mg/ml of column were readily processed, resulting in product recovery of 86% and purity of 92%. These results demonstrate that anion-exchange displacement chromatography can indeed be employed for large-scale oligonucleotide separations at high column loading.

Amaranth Dye↗

PubMed2000

Hydrophobic displacement chromatography of proteins.

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.

Biotechnology↗

PubMed2000

Optimization of ion-exchange displacement separations. I. Validation of an iterative scheme and its use as a methods development tool.

Displacement chromatography has been demonstrated to be a powerful, high-resolution preparative tool. The performance of displacement systems can be affected by a variety of factors such as the feed load, flow-rate, initial salt concentration and the displacer partition ratio. Thus, the optimization of displacement separations is a uniquely challenging problem. In this manuscript, an iterative optimization scheme has been presented whereby one can identify the optimum operating conditions for displacement separations at a given level of loading on a given resin material. The solid film linear driving force model has been employed in concert with the Steric Mass Action formalism of ion-exchange chromatography to describe the chromatographic behavior in these systems. Simple pulse techniques have been employed to estimate the transport parameters. The iterative scheme has been validated using a rigorous Feasible Sequential Quadratic Programming algorithm. Finally, the utility of the iterative optimization scheme as a methods development tool for displacement separations has been demonstrated for a difficult separation. The results indicate that the use of the optimization scheme leads to significantly better performance than standard rules of thumb.

Algorithms↗

PubMed2000

Optimization of ion-exchange displacement separations. II. Comparison of displacement separations on various ion-exchange resins.

A variety of stationary-phase materials are currently available for the chromatographic purification of biomolecules. However, the effect of various resin characteristics on the performance of displacement chromatography has not been studied in depth. In Part I, a novel iterative scheme was presented for the rapid optimization of displacement separations in ion-exchange systems. In this article, the optimization scheme is employed to identify the optimum operating conditions for displacement separations on various ion-exchange resin materials. In addition, the effect of different classes of separation problems (e.g., diverging, converging or parallel affinity lines) on the performance of displacement separations is also presented. The solid film linear driving force model is employed in concert with the Steric Mass Action isotherm to describe the chromatographic behavior in these systems. The results presented in this article provide insight into the effects of resin capacity and efficiency as well as the type of separation problem on the performance of various ion-exchange displacement systems.

Algorithms↗

PubMed1999

Characterization of recombinant human brain-derived neurotrophic factor variants.

The purpose of this work was the chemical characterization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli. This paper also addresses the question of the in vitro activity of these variants. Chemical characterization of the variants employed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing. The BDNF variants in this work have been designated by the order of their elution as observed from the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at positions 31 and 61, respectively. Peak 4, which is chromatographically a single peak, contained three variants. Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methionine at position 92. Peak 5 had norleucine at position 31 instead of methionine. All of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB receptor-ligand binding domain of the variants.

Alkylation↗

PubMed1999

A holistic approach to protein secondary structure characterization using amide I band Raman spectroscopy.

We have developed a holistic protein structure estimation technique using amide I band Raman spectroscopy. This technique combines the superposition of reference spectra for pure secondary structure elements with simultaneous aromatic, fluorescence, and solvent background subtraction, and is applicable to solution, suspension, and solid protein samples. A key component of this technique was the calculation of the reference spectra for ordered helix, unordered helix, and sheet, turns, and unordered structures from a series of well-characterized reference proteins. We accurately account for the overlap between the amide I and non-amide I regions and allow for different scattering efficiencies for different secondary structures. For hydrated samples, we allowed for the possibility that bound water spectra differ from the bulk water spectra. Our computed reference spectra compare well with previous experimental and theoretical results in the literature. We have demonstrated the use of these reference spectra for the estimation of secondary structures of proteins in solution, suspension, and dry solid forms. The agreement between our structure estimates and the corresponding determinations from X-ray crystallography is good.

Algorithms↗

PubMed1998

Structural characteristics of low-molecular-mass displacers for cation-exchange chromatography. II. Role of the stationary phase.

The relative efficacy of a variety of low-molecular-mass displacers was examined on three different stationary phase materials. Several homologous series of displacer molecules were evaluated on these ion-exchange resins using a displacer ranking plot based on the steric mass action model. The results demonstrate that while aromaticity and hydrophobicity can play a significant role in the affinity of displacer molecules on polymethacrylate based and hydrophilized polystyrene-divinylbenzene based materials, this effect is much less pronounced on an agarose based resin. The work presented in this paper demonstrates that different structural features of low-molecular-mass displacers can dominate their affinity on various stationary phase materials employed and provides rules of thumb for the design of high affinity, low-molecular-mass displacers for a variety of commercial cation-exchange materials.

Cation Exchange Resins↗

PubMed1998

Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants.

Removal of low level impurities that are closely related to the bioproduct is a commonly encountered challenge in the purification of biopharmaceuticals. These separations are typically carried out by using shallow linear salt gradients at relatively low column loadings, significantly limiting the throughput of the purification process. In this manuscript we examine the utility of displacement chromatography for the purification of recombinant human brain-derived neurotrophic factor, rHuBDNF. The utility of displacement chromatography is compared to gradient elution for the removal of variants of the rHuBDNF. The results demonstrate that displacement chromatography is capable of achieving high yields and purity at high column loadings. Displacements developed on 20 microns and 50 microns cation-exchange resins are shown to provide 8-fold and 4.5-fold increases in production rates, respectively, when compared to an existing linear gradient elution operation. These results demonstrate the efficacy of displacement chromatography for the purification of therapeutic proteins from complex feed streams.

Brain-Derived Neurotrophic Factor↗

PubMed1998

Structural characteristics of low-molecular-mass displacers for cation-exchange chromatography.

The relative efficacy of a variety of low-molecular-mass displacers was examined using a displacer ranking plot. This method enables an evaluation of the dynamic affinity of a variety of displacers over a range of operating conditions. Several homologous series of molecules were evaluated to provide insight into the effects of various structural features on displacer efficacy. The results indicate that linear flexible geometries may have advantages over branched or cyclic structures. Data also indicate that the spreading out of charges may increase affinity. The incorporation of aromatic moieties in these displacers, particularly near the surface of the molecules, appears to result in a dramatic increase in displacer affinity. The ability of several high-affinity low-molecular-mass displacers a very strongly bound cationic protein is also examined. The results confirm the predictions of the theory and indicate that it is indeed possible to displace highly bound macromolecules with low-molecular-mass dispatchers. The work presented in this paper indicates that non-specific interactions can be exploited for producing high-affinity low-molecular-mass displacers.

Algorithms↗

PubMed1997

Low-molecular-weight displacers for high-resolution protein separations.

The resolving power of displacement chromatography using low-molecular-weight displacers was investigated using a model mixture containing bovine and horse heart cytochrome c. The linear and nonlinear adsorption behavior of these two proteins was examined in cation-exchange chromatography and shown to be quite similar. Furthermore, an analysis of the dynamic affinity of these proteins indicated extremely similar affinities under displacement conditions. Despite the extreme similarities in the adsorption behavior, displacement chromatography using a protected amino acid displacer resulted in excellent separation of the proteins with both high yields and purity. These results indicate that displacement chromatography may be efficacious for a wide variety of difficult protein separation problems.

Adsorption↗

PubMed1994

Gibbs free energy of adsorption for biomolecules in ion-exchange systems.

The Gibbs free energy of adsorption (delta G0ads) was estimated for several amino acids, peptides and proteins in a cation-exchange system. Using the steric mass action formalism that describes biomolecular adsorption in ion-exchange systems, the delta G0ads was chromatographically determined under infinitely dilute conditions. The delta G0ads measured for seven globular proteins ranged from -5.7 to -13.9 kcal/mol. The average bond energy (defined as delta G0ads divided by the number of bonds formed between the protein and the surface) for these proteins varied from -1.1 to -1.7 kcal/mol. These bond energies were found to be comparable to the bond energies for lysine and arginine (-1.1 and -1.5 kcal/mol, respectively), the amino acids which primarily contribute to the cation-exchange of proteins. In contrast, an elevated average bond energy of -2.6 kcal/mol was observed for two peptides and protamine (a polypeptide) suggesting that synergistic binding may play a role for unstructured macromolecules, but not for globular proteins.

Absorption↗

PubMed1993

Preparative chromatography in biotechnology.

The field of preparative chromatography has experienced several important advances recently in both the structure of chromatographic materials and modes of chromatographic operation. This review presents recent applications of preparative chromatography in biotechnology, as well as novel stationary phase materials and engineering approaches to preparative chromatographic bioseparations.

Animals↗

PubMed1993

Preparative chromatographic purification of cyclosporine metabolites.

Polar and primary metabolites of cyclosporin A (CsA) have successfully been isolated by a novel separation protocol. An efficient, easy-to-scale-up chromatographic adsorption/desorption operation recovers polar and primary CsA metabolite pools from large volumes of urine; purified CsA metabolites are subsequently obtained by high-resolution preparative elution chromatography of the semipurified metabolite pools. Separations performed on a semipreparative scale [with a 250 x 9.4 mm (i.d.) reversed-phase HPLC column] yielded microgram quantities of CsA metabolites at > 97% purity, as determined by fast atom bombardment mass spectrometry. These separations also yielded two previously unreported CsA metabolites, similar to AM1A but with an additional hydroxylation. The yield of metabolites was increased to several milligrams by performing the separations with a preparative-scale [250 x 21.2 mm (i.d.)] reversed-phase column. The production rate of purified primary CsA metabolites was greatly increased by performing the separation with the preparative-scale column under conditions of severe mass overloading. In a single chromatographic run, we successfully isolated 11.0 and 5.0 mg of AM1 and AM1c, respectively, at a purity of > 97%. As expected, this increase in the yield of purified metabolites was accompanied by a decrease in the overall recovery. This separation scheme enables the rapid processing of large volumes of urine for isolation of the milligram quantities of CsA metabolites necessary to assess their biological activity. The procedure is applicable to small- or large-scale metabolite isolation and provides a ready source of purified metabolites for in vitro and whole-animal studies.

Amino Acid Sequence↗

PubMed1992

Assessment of the in vivo immunosuppressive activity of the major cyclosporine metabolite by leukemia allograft rejection.

AM1 (M17) is the major metabolite of cyclosporine found in the blood of human transplant recipients, and trough levels of this derivative exceed those of the parent compound approximately two-fold. Studies performed in vitro indicate that AM1 retains only 10-20% of the biological activity of the parent compound, but very little is known about its in vivo immunosuppressive effects. We therefore developed a rapid and sensitive method, based on the rejection of allogeneic L1210 (H-2d) leukemia cells by C57BL/6 (H-2b) mice, to assess the immunosuppressive activity of AM1 in vivo. Rejection of the leukemia allograft was determined by analyzing the spleens from mice injected intravenously with 10(5) L1210 cells for the presence of H-2Kd-positive cells by flow cytometry using an FITC-conjugated monoclonal anti-H-2Kd antibody. Nonimmunosuppressed mice rejected the allogeneic cells and survived indefinitely. Spleens from these mice were virtually free of H-2Kd-positive cells (0.51 +/- 0.21%) by day 7. In contrast, C57BL/6 mice treated with 10 mg/kg/day s.c. of CsA all died from the L1210 challenge (mean survival time of 9 +/- 1 days). Spleens from mice treated in this manner contained 11.02 +/- 3.31% H-2Kd-positive cells on day 7. There was a direct correlation between the dose of CsA administered (7.5-50 mg/kg/day) and the percentage of H-2Kd-positive cells in the spleen. We then compared the immunosuppressive activity of AM1 and CsA in this model. AM1 was purified from the urine of CsA-treated renal allograft recipients by a combination of preparative adsorption-desorption chromatography and preparative elution high-performance liquid chromatography. AM1 at a dose of 10 mg/kg/day exhibited no demonstrable immunosuppressive effect, and trough levels of AM1 on day 7 were only 36 +/- 4 ng/ml. Increasing the dose of AM1 to 50 mg/kg/day resulted in only 1.05 +/- 0.16% H-2Kd-positive cells in the spleens (P = NS) and a mean trough level of 221 +/- 27 ng/ml. In contrast, mice treated with 50 mg/kg/day of CsA exhibited 17.7 +/- 2.9% H-2Kd-positive cells in their spleens and a mean trough CsA level of 3036 +/- 277 ng/ml. The half-life of a single subcutaneous dose of 10 mg/kg of AM1 (4.6 hr) was significantly shorter than that of CsA (9.7 hr) in mice. Compared with CsA, the lack of immunosuppressive effect of AM1 in vivo therefore appears to be due to a combination of decreased immunosuppressive activity and increased rate of clearance in mice.

Animals↗