Kevin E. Van Cott

Tobacco protein separation by aqueous two-phase extraction

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.

High Throughput Quantification of N-Glycans Using One-Pot Sialic Acid Modification and Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then be released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method.

Layer-by-layer deposition and ordering of low-molecular-weight dye molecules for second-order nonlinear optics.

A combination of electrostatic interactions and covalent bonding is used to form films with low-molecular-weight chromophores by a layer-by-layer deposition process. Using a common, commercially available red dye, this deposition process results in noncentrosymmetric films (see scheme) that exhibit secondharmonic generation (red green), with (2) values as large as 11.3 10 9 esu, that is, six times that of quartz. K. E. Van Cott,* M. Guzy, P. Neyman, C. Brands, J. R. Heflin, H. W. Gibson, R. M. Davis . . . . . . . . . . . . . . . . 3236 ± 3238

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

N-glycosylation microheterogeneity and site occupancy of an Asn-X-Cys sequon in plasma-derived and recombinant protein C

Human protein C (hPC) is glycosylated at three Asn‐X‐Ser/Thr and one atypical Asn‐X‐Cys sequons. We have characterized the micro‐ and macro‐heterogeneity of plasma‐derived hPC and compared the glycosylation features with recombinant protein C (tg‐PC) produced in a transgenic pig bioreactor from two animals having approximately tenfold different expression levels. The N‐glycans of hPC are complex di‐ and tri‐sialylated structures, and we measured 78% site occupancy at Asn‐329 (the Asn‐X‐Cys sequon). The N‐glycans of tg‐PC are complex sialylated structures, but less branched and partially sialylated. The porcine mammary epithelial cells glycosylate the Asn‐X‐Cys sequon with a similar efficiency as human hepatocytes even at these high expression levels, and site occupancy at this sequon was not affected by expression level. A distinct bias for particular structures was present at each of the four glycosylation sites for both hPC and tg‐PC. Interestingly, glycans with GalNAc in the antennae were predominant at the Asn‐329 site. The N‐glycan structures found for tg‐PC are very similar to those reported for a recombinant Factor IX produced in transgenic pig milk, and similar to the endogenous milk protein lactoferrin, which may indicate that N‐glycan processing in the porcine mammary epithelial cells is more uniform than in other tissues.

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100--1800 μg ml−1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100--400 μg ml−1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein

Affinity purification of biologically active and inactive forms of recombinant human protein C produced in porcine mammary gland

Recombinant human protein C (rhPC) secreted in the milk of transgenic pigs was studied. Transgenes having different regulatory elements of the murine milk protein, whey acidic protein, were used with cDNA and genomic human protein C (hPC) DNA sequences to obtain lower and higher expressing animals. The cDNA pigs had a range of expression of about 0.1–0.5 g/l milk. Two different genomic hPC pig lines have expressed 0.3 and 1–2 g/l, respectively. The rhPC was first purified at yields greater than 60 per cent using a monoclonal antibody (mAb) to the activation site on the heavy chain of hPC. Subsequent immunopurification with a calcium‐dependent mAb directed to the γ‐carboxyglutamic acid domain of the light chain of hPC was used to fractionate a population having a higher specific anticoagulant activity in vitro. The higher percentages of Ca2+‐dependent conformers isolated from the total rhPC by immunopurification correlated well with higher specific activity and lower expression. A rate limitation in γ‐carboxylation of rhPC was clearly identified for the higher expressing animals. Thus, transgenic animals with high expression levels of complex recombinant proteins produced a lower percentage of biologically active protein.

Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels.

Colostrum and milk are natural vehicles for acquiring passive immunity and are valuable tools for decreasing neonatant mortality from diarrheal disease. The effects of recombinant human protein C (rhPC) expression levels on endogenous immunoglobulin and transferrin content of the milk of different lineages of transgenic pigs were studied. The levels of rhPC in the milk ranged from 40 to 1200 μg/ml. Transgenic pigs with rhPC expression levels less than 500 μg/ml had no significant differences in milk protein composition with respect to nontransgenic pigs. A line of transgenic pigs having rhPC expression levels of 960–1200 μg/ml had two- to three-fold higher IgG, IgM, and secretory IgA concentrations compared to other transgenic and nontransgenic pig groups (P < 0.05), and four- to five-fold higher transferrin levels than nontransgenic pigs (P < 0.05). Changes in milk protein composition were not associated with mastitis or other pathologic disruption of epithelial cell junctions as indicated by normal casein and albumin levels in milk. Since IgG, IgM, secretory IgA, and transferrin are transported into the milk by transcytosis, higher levels of these proteins indicate that transcyctosis in the mammary epithelial cell was likely upregulated in pigs having high rhPC expression levels. This study is the first that shows a statistically significant example that mammary tissue specific expression of a heterologous protein can enhance endogenous phenotypic characteristics of milk.

Role of local antibody density effects on immunosorbent efficiency

Abstract This study evaluates the effect of immobilized antibody density on the performance of an immunosorbent. In contrast to previous studies that emphasize the correlation of high volume averaged antibody density with immunosorbent performance, we have studied the effects of locally high antibody density and spatial distribution on the antigen binding efficiency under conditions of dynamic loading and elution. The distribution of an anti-human Protein C monoclonal antibody immobilized on 3M Emphaze AB1 Biosupport Medium was evaluated. The distribution of immobilized antibody was controlled by a two-step sequence of permeation and reaction. Labeled antibody was visualized by immunofluorescence. Conditions of low pH, low temperature, and the presence of a competitor nucleophile sufficiently depressed the Thiele modulus for coupling to enable permeation of the antibody. The adsorption of the permeated antibody was enhanced by the presence of 0.75 M Na2SO4, and then the pH was raised to achieve rapid covalent coupling. Bead-averaged antibody densities of 1–11 mg/ml of hydrogel support were studied. Immunosorbents containing more evenly distributed antibody gave a two- to three-fold greater antigen binding efficiency than those with locally high antibody densities. No appreciable changes in mass transfer characteristics were observed using breakthrough analysis for immunosorbents with distributed versus locally high antibody density.