Martin Vanderlaan - Academia.edu (original) (raw)

Papers by Martin Vanderlaan

Electrophoresis, 1999

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical pr... more Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension. The map is composed of over 1000 silver-stained protein spots. Protein identification is proceeding using a combination of immunostaining, NH2-terminal sequencing, and mass spectrometric analyses. Among the proteins so far identified are glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), galectin-1, and several heat-shock proteins. The goal is to generate a database which emphasizes those proteins most relevant to the use of CHO cells as a host for recombinant protein expression.

Journal of Chromatography a, Aug 24, 2001

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase sep... more An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Http Dx Doi Org 10 1080 02648725 2001 10648017, Apr 15, 2013

Biotechnology and applied biochemistry, 1999

We describe the performance characteristics of five Protein A affinity-chromatography sorbents (S... more We describe the performance characteristics of five Protein A affinity-chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host-cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.

Journal of Chromatography B: Biomedical Sciences and Applications, 1999

The development of an automated, dual column assay to quantitate and recover the glycoprotein, tu... more The development of an automated, dual column assay to quantitate and recover the glycoprotein, tumor necrosis factor receptor immunoadhesin (TNFr-IgG) from monkey plasma, human serum, cell culture fluid and buffer samples is described. A combination of immunoaffinity and reversed-phase chromatographies are used. The targeted protein was captured using an anti-TNFr-1 monoclonal antibody immobilized on POROS resin. After non-specific adsorption had been reduced, the affinity column was placed in-line with a reversed-phase column and eluted with dilute acid. The reversed-phase column was subsequently eluted with an acetonitrile gradient and the TNFr-IgG collected and quantitated by comparison with peak areas of similarly treated standards. Detection was performed by measurement of absorbance at 214 nm. The dynamic range is from 0.5-15 microg total sample. Samples were quantitated and recovered from monkey and human pharmacokinetics samples, as well as from cell culture fluid and buffers. The lowest concentrations assayed were 100 ng ml(-1). Quantitation is reproducible, with a coefficient of variation of 2%. The procedure was used to develop a pharmacokinetic profile for the clearance of TNFr-IgG in humans and cynomolgus monkeys. Sufficient material was recovered such that the glycoforms could be identified. Additionally it has been used for process monitoring. The results compared favorably with data generated by ELISA. Optimization of the method and results are presented.

Cytometry, 1984

Papanicolaou-stained bladder cells were scanned on the ACUity microscope system in the Biomedical... more Papanicolaou-stained bladder cells were scanned on the ACUity microscope system in the Biomedical Sciences Division of Lawrence Livermore National Laboratory. The hardware and software components of ACUity have been described elsewhere (211, so only a brief description will be given here. A Leitz Ortholux microscope, configured for absorption microscopy, was used with a 1OOx oil-immersion objective (NA = 1.32) '

Biotechnology and Bioengineering, 2014

Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant p... more Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. A multi-analyte enzyme-linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti-HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non-immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution-dependent non-linearity with multiproduct HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co-purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low-level HCPs is also outlined, with consideration of clinical information.

PROTEOMICS, 2003

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human gr... more Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin  ) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.

Journal of Wood Chemistry and Technology, 1996

A prototype solvolytic lignin has been fractionated into three fractions by successive extraction... more A prototype solvolytic lignin has been fractionated into three fractions by successive extraction with organic solvents of increasing hydrogen-bonding capacity. A comparison between the fractions and the starting lignin has been made in terms of methoxyl group content, fractionation yields, molecular weight distribution, alkaline nitrobenzene oxidation, and H and C NMR spectroscopy. The fractionation procedure was found to isolate fractions

Journal of Immunological Methods, 1985

A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids... more A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or lgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.

Journal of Chromatography A, 2001

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase sep... more An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Electrophoresis, 1999

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical pr... more Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension. The map is composed of over 1000 silver-stained protein spots. Protein identification is proceeding using a combination of immunostaining, NH2-terminal sequencing, and mass spectrometric analyses. Among the proteins so far identified are glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), galectin-1, and several heat-shock proteins. The goal is to generate a database which emphasizes those proteins most relevant to the use of CHO cells as a host for recombinant protein expression.

Biomass and Bioenergy, 1997

Polyurethanes have been prepared from a prototype organosolv lignin by solution casting with poly... more Polyurethanes have been prepared from a prototype organosolv lignin by solution casting with polyethylene glycol (PEG) and polymeric methyl-diisocyanate (MDI). The effects of lignin composition and NCOjOH ratio on the mechanical properties and swelling behaviour of the derived polyurethane films were investigated. At low lignin content, flexible but weak polyurethanes were produced regardless of the NCO/OH ratio. Rather tough polyurethanes were prepared at intermediate NCOjOH ratios and lignin contents of 15-25 wt%. At lignin contents of 30 wt% or above, hard and brittle films were obtained irrespective of the NCO/OH ratio employed. Hard and glassy polyurethanes were produced at NCOjOH ratios of I.8 or greater which were not testable. 0 1997 Published by Elsevier Science Ltd.

The American Journal of Pathology, 1998

Journal of Agricultural and Food Chemistry, 1993

A series of monoclonal antibodies were generated that can bind dimetridazole, a nitroimidazole dr... more A series of monoclonal antibodies were generated that can bind dimetridazole, a nitroimidazole drug used in veterinary medicine. A competition enzyme-linked immunosorption assay (cELISA) is described and is used to characterize the binding of these antibodies to a number of nitroimidazole drugs. The 50% inhibition of control occurred for the most sensitive of these antibodies at 12 ng/mL for dimetridazole, 75 ng/mL for hydroxydimetridazole, 25 ng/mL for ipronidazole, 2000 ng/mL for hydroxyipronidazole, and 20 000 ng/mL for metronidazole. An extraction method for these nitroimidazoles that is compatible with the immunoassay is described. Using this method, as little as 1 ng of dimetridazole could be detected in turkey muscle. Eds.; ACS Symposium Series 451; American Chemical Society: Washington, DC, 1991; pp 108-123. Stone, L. R.; Lawrence, J. W.; Hobson, D. L. GLC Determination of l-methyl-2-hydroxymethyl-5-nitroimidazole in Swine Tissue.

Electrophoresis, 1999

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical pr... more Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension. The map is composed of over 1000 silver-stained protein spots. Protein identification is proceeding using a combination of immunostaining, NH2-terminal sequencing, and mass spectrometric analyses. Among the proteins so far identified are glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), galectin-1, and several heat-shock proteins. The goal is to generate a database which emphasizes those proteins most relevant to the use of CHO cells as a host for recombinant protein expression.

Journal of Chromatography a, Aug 24, 2001

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase sep... more An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Http Dx Doi Org 10 1080 02648725 2001 10648017, Apr 15, 2013

Biotechnology and applied biochemistry, 1999

We describe the performance characteristics of five Protein A affinity-chromatography sorbents (S... more We describe the performance characteristics of five Protein A affinity-chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host-cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.

Journal of Chromatography B: Biomedical Sciences and Applications, 1999

The development of an automated, dual column assay to quantitate and recover the glycoprotein, tu... more The development of an automated, dual column assay to quantitate and recover the glycoprotein, tumor necrosis factor receptor immunoadhesin (TNFr-IgG) from monkey plasma, human serum, cell culture fluid and buffer samples is described. A combination of immunoaffinity and reversed-phase chromatographies are used. The targeted protein was captured using an anti-TNFr-1 monoclonal antibody immobilized on POROS resin. After non-specific adsorption had been reduced, the affinity column was placed in-line with a reversed-phase column and eluted with dilute acid. The reversed-phase column was subsequently eluted with an acetonitrile gradient and the TNFr-IgG collected and quantitated by comparison with peak areas of similarly treated standards. Detection was performed by measurement of absorbance at 214 nm. The dynamic range is from 0.5-15 microg total sample. Samples were quantitated and recovered from monkey and human pharmacokinetics samples, as well as from cell culture fluid and buffers. The lowest concentrations assayed were 100 ng ml(-1). Quantitation is reproducible, with a coefficient of variation of 2%. The procedure was used to develop a pharmacokinetic profile for the clearance of TNFr-IgG in humans and cynomolgus monkeys. Sufficient material was recovered such that the glycoforms could be identified. Additionally it has been used for process monitoring. The results compared favorably with data generated by ELISA. Optimization of the method and results are presented.

Cytometry, 1984

Papanicolaou-stained bladder cells were scanned on the ACUity microscope system in the Biomedical... more Papanicolaou-stained bladder cells were scanned on the ACUity microscope system in the Biomedical Sciences Division of Lawrence Livermore National Laboratory. The hardware and software components of ACUity have been described elsewhere (211, so only a brief description will be given here. A Leitz Ortholux microscope, configured for absorption microscopy, was used with a 1OOx oil-immersion objective (NA = 1.32) '

Biotechnology and Bioengineering, 2014

Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant p... more Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. A multi-analyte enzyme-linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti-HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non-immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution-dependent non-linearity with multiproduct HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co-purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low-level HCPs is also outlined, with consideration of clinical information.

PROTEOMICS, 2003

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human gr... more Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin  ) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.

Journal of Wood Chemistry and Technology, 1996

A prototype solvolytic lignin has been fractionated into three fractions by successive extraction... more A prototype solvolytic lignin has been fractionated into three fractions by successive extraction with organic solvents of increasing hydrogen-bonding capacity. A comparison between the fractions and the starting lignin has been made in terms of methoxyl group content, fractionation yields, molecular weight distribution, alkaline nitrobenzene oxidation, and H and C NMR spectroscopy. The fractionation procedure was found to isolate fractions

Journal of Immunological Methods, 1985

A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids... more A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or lgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.

Journal of Chromatography A, 2001

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase sep... more An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Electrophoresis, 1999

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical pr... more Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension. The map is composed of over 1000 silver-stained protein spots. Protein identification is proceeding using a combination of immunostaining, NH2-terminal sequencing, and mass spectrometric analyses. Among the proteins so far identified are glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), galectin-1, and several heat-shock proteins. The goal is to generate a database which emphasizes those proteins most relevant to the use of CHO cells as a host for recombinant protein expression.

Biomass and Bioenergy, 1997

Polyurethanes have been prepared from a prototype organosolv lignin by solution casting with poly... more Polyurethanes have been prepared from a prototype organosolv lignin by solution casting with polyethylene glycol (PEG) and polymeric methyl-diisocyanate (MDI). The effects of lignin composition and NCOjOH ratio on the mechanical properties and swelling behaviour of the derived polyurethane films were investigated. At low lignin content, flexible but weak polyurethanes were produced regardless of the NCO/OH ratio. Rather tough polyurethanes were prepared at intermediate NCOjOH ratios and lignin contents of 15-25 wt%. At lignin contents of 30 wt% or above, hard and brittle films were obtained irrespective of the NCO/OH ratio employed. Hard and glassy polyurethanes were produced at NCOjOH ratios of I.8 or greater which were not testable. 0 1997 Published by Elsevier Science Ltd.

The American Journal of Pathology, 1998

Journal of Agricultural and Food Chemistry, 1993

A series of monoclonal antibodies were generated that can bind dimetridazole, a nitroimidazole dr... more A series of monoclonal antibodies were generated that can bind dimetridazole, a nitroimidazole drug used in veterinary medicine. A competition enzyme-linked immunosorption assay (cELISA) is described and is used to characterize the binding of these antibodies to a number of nitroimidazole drugs. The 50% inhibition of control occurred for the most sensitive of these antibodies at 12 ng/mL for dimetridazole, 75 ng/mL for hydroxydimetridazole, 25 ng/mL for ipronidazole, 2000 ng/mL for hydroxyipronidazole, and 20 000 ng/mL for metronidazole. An extraction method for these nitroimidazoles that is compatible with the immunoassay is described. Using this method, as little as 1 ng of dimetridazole could be detected in turkey muscle. Eds.; ACS Symposium Series 451; American Chemical Society: Washington, DC, 1991; pp 108-123. Stone, L. R.; Lawrence, J. W.; Hobson, D. L. GLC Determination of l-methyl-2-hydroxymethyl-5-nitroimidazole in Swine Tissue.