Non-flammable preparative reversed-phase liquid chromatography of recombinant human insulin-like growth factor-I.

Acetonitrile is used as an eluent for reversed-phase chromatography. However, because it is a flammable solvent, using acetonitrile on a large scale requires expensive equipment and facilities specially designed for flammable solvents. Using a non-flammable solvent as an eluent eliminates this expense. A method was developed to purify recombinant human insulin-like growth factor I by reversed-phase high-performance liquid chromatography using gradient elution with hexylene glycol, a non-flammable replacement for acetonitrile. The separation produced equivalent yield, purity and throughput as reversed-phase chromatography using elution with acetonitrile.

Real-time control of purified product collection during chromatography of recombinant human insulin-like growth factor-I using an on-line assay.

During preparative reversed-phase chromatography of recombinant human insulin-like growth factor-I (IGF), the separation of IGF from IGF aggregates cannot be determined using UV absorbance. An on-line reversed-phase chromatographic assay was developed that provides a quantitative measurement of IGF and IGF aggregates every 4 min, allowing real-time control of purified IGF collection. Process control using the on-line assay is a reliable and accurate method to collect purified IGF.

Insulin-like growth factor binding protein-1 induces insulin release in the rat.

Injections of human insulin-like growth factor binding protein (hIGFBP-1) are reported to induce hyperglycemia in the rat, suggesting that IGFBP-1 acutely regulates glucose homeostasis. We now report the effects on glucose and insulin levels of administering recombinant (r) hIGFBP-1. In a series of studies, normal and streptozotocin (STZ) diabetic male Wistar rats (180-210 g), fasted for 6 or 16 h, were injected with rhIGFBP-1 (i.v., 80-500 microg/rat). rhIGFBP-1 did not affect blood glucose acutely but did stimulate insulin release in normal rats (5 min post injection; PBS, 103.5 +/- 8.5; rhIGFBP-1 (500 microg), 166.8 +/- 15.7; rhIGFBP-1 (100 microg); 151.4 +/- 14.1% initial). rhIGFBP-1 pretreatment, in normal and diabetic rats, reduced the hypoglycemic response to rhIGF-I (diabetic rats after 20 min: PBS, 103.4 +/- 11.4; BP-1 (500 microg) +/- rhIGF-I (50 microg), 97.6 +/- 3.6; rhIGF-I, 48.2 +/- 4.3% initial) but did not affect the hypoglycemic response to des(1-3)IGF-I or insulin (0.5 U/kg). These studies show that rhIGFBP-1 causes insulin release, has a minimal effect on blood glucose, and inhibits the hypoglycemic effect of rhIGF-I. These data suggest that endogenous IGF-I tonically suppresses insulin secretion and imply that aberrant IGFBP levels or reduced IGF-I bioactivity may lead to chronic hyperinsulinemia.

Purification of insulin-like growth factor-I and related proteins using underivatized silica.

Adsorption chromatography using underivatized porous glass can be an effective capture step for the purification of recombinant proteins. Classical desorption techniques using chaotropic agents or harsh chemical solvents often result in elution of inactive material and may not be economical at the process scale. More recently, elution schemes have used tetramethylammonium chloride (TMAC) to obtain biologically active material. A TMAC elution was shown to be effective in the initial purification steps for the recovery of recombinant human insulin-like growth factor-I (rhIGF-I) from an Escherichia coli fermentation broth. However, TMAC also elutes other, more hydrophobic, proteins that are difficult to remove in subsequent purification steps. This paper describes the capture of IGF-I from a crude fermentation broth and a more specific elution using a combination of ethanol and NaCl rather than TMAC. This elution also can be used with other proteins including an IGF-I binding protein (BP3) expressed in mammalian cell culture.

Large scale, in situ isolation of periplasmic IGF-I from E. coli.

Human insulin-like growth factor I (IGF-I) accumulates in both folded and aggregated forms in the fermentation medium and cellular periplasmic space when expressed in E. coli with an endogenous secretory signal sequence. Due to its heterogeneity in form and location, low yield of IGF-I was obtained using a typical refractile body recovery strategy. To enhance recovery yield, a new procedure was developed to solubilize and extract IGF-I from cells while in fermentation broth. This method, called in situ solubilization, involves addition of chaotrope and reductant to alkaline fermentation broth and provides recovery of about 90% of all IGF-I in an isolated supernatant. To further enhance recovery, a new aqueous two-phase extraction procedure was developed which partitions soluble non-native IGF-I and biomass solids into separate liquid phases. This two-phase extraction procedure involves addition of polymer and salt to the solubilization mixture and provides about 90% recovery of solubilized IGF-I in the light phase. The performance of the solubilization and aqueous extraction procedures is reproducible at scales ranging from 10 to 1000 liters and provides a 70% cumulative recovery yield of IGF-I in the isolated light phase. The procedure provides significant initial IGF-I purification since most host proteins remain cell associated during solubilization and are enriched in heavy phase. ELISA analysis for E. coli proteins indicates that 97% of the protein in the light phase is IGF-I. Together, the techniques of in situ solubilization and aqueous two-phase extraction provide a new, high yield approach for isolating recombinant protein which is accumulated in more than one form during fermentation.

Incorporation of hydrophobic selectivity in capillary electrophoresis: analysis of recombinant insulin-like growth factor I variants.

A highly selective electrophoretic system employing differential hydrophobic interaction was evaluated for the quantitative determination of recombinant insulin-like growth factor I (IGF-I) variants. The system consisted of mixed aqueous-organic buffers containing suitable amounts of a zwitterionic detergent. In addition, a neutral hydrophilic coating was attached to the wall of the capillary to minimize analyte adsorption and electroosmotic flow. The zwitterionic detergent acted as a hydrophobic selector, allowing independent optimization of the electrophoretic and hydrophobic selectivities in the separation system. The extent of hydrophobic interaction was conveniently adjusted by varying the type and amount of organic modifier. Complete resolution of a mixture of IGF-I variants with closely related mass-to-charge ratios was achieved. Quantitative analysis of IGF-I process samples agreed well with HPLC results. Finally, the approach was found to be compatible with on-line capillary electrophoresis-mass spectrometry.

Preparative isolation of recombinant human insulin-like growth factor 1 by reversed-phase high-performance liquid chromatography.

The isolation of recombinant human insulin-like growth factor 1 (rhIGF-1) is complicated by the presence of several rhIGF-1 variants which co-purify using conventional chromatographic media. These species consist primarily of a methionine-sulfoxide variant of the properly folded molecule and a misfolded form and its respective methionine-sulfoxide variant. An analytical reversed-phase high-performance liquid chromatography procedure using a 5-micron C18 column, an acetonitrile-trifluoroacetic acid (TFA) isocratic elution, and elevated temperature gives baseline resolution of the four species. Using this analytical method as a development tool, a process-scale chromatography step was established. The 5-micron analytical packing material was replaced with a larger-size particle to reduce back-pressure and cost. Since the TFA counter-ion binds tightly to proteins and is difficult to subsequently dissociate, a combination of acetic acid and NaCl was substituted. Isocratic separations are not good process options due to problems with reproducibility and control. A shallow gradient elution using premixed mobile phase buffers at the same linear velocity was found to give an equivalent separation at low load levels and minimized solvent degassing. However, at higher loading there was a loss of resolution. A matrix of various buffers was evaluated for their effects on separation. Elevated pH resulted in a significant shift in both the elution order and relative retention times of the principal rh-IGF-1 variants, resulting in a substantial increase in effective capacity. An increase in the ionic strength further improved resolution. Several different media were evaluated with regard to particle size, shape and pore diameter using the improved mobile phase. The new conditions were scaled up 1305-fold and resulted in superimposable chromatograms, 96% recovery and > 99% purity. Thus, by optimizing the pH, ionic strength and temperature, a high-capacity preparative separation of rhIGF-1 from its related fermentation variants was obtained.

Immunochemical characterization of the distinct monocyte cyclic AMP-phosphodiesterase from patients with atopic dermatitis.

BACKGROUND: Previous findings have suggested that the immunopathology of patients with atopic dermatitis (AD) results from altered cellular responses caused by cyclic nucleotide regulatory abnormalities. One such defect is the increased degradation of the second messenger, cyclic adenosine monophosphate (cAMP), by elevated cAMP-phosphodiesterase (PDE) activity in patients with AD. METHODS: We used two monoclonal antibodies to identify the major PDE isoform in AD blood monocytes. We have also characterized the abnormal PDE activity by means of chromatofocusing and sucrose gradient centrifugation. RESULTS: The chromatofocusing technique allowed the separation of a PDE-containing fraction (isoelectric point = 6.1) from AD monocytes but not from normal cells. This monocyte fraction accounted for most of the elevated leukocyte-PDE activity and was a cytosolic, cAMP-specific, low Michaelis constant, calcium-calmodulin-dependent enzyme, inhibited by the cAMP-PDE inhibitor, Ro 20-1724. The majority of the PDE activity in this chromatofocused fraction was immunoadsorbed by the solid-phase immobilized antibodies against calcium-calmodulin-dependent PDE. CONCLUSIONS: The increased degradation of cAMP by a unique form of PDE may cause defective regulation of intracellular functions of AD monocytes, leading to the characteristic hyperreactive immune and inflammatory events. Characterization of PDE isoenzymes from different leukocyte subpopulations may allow further expansion of cell-directed therapy for inflammatory disease.

Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I.

Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent.

Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors.

Primary sequence information has been reported for more than 15 different mammalian cyclic nucleotide phosphodiesterases. Moreover, recent observations suggest that many of these isozymes are selectively expressed in a limited number of cell types. The fact that nearly all these different phosphodiesterases have unique primary sequences in their catalytic or regulatory domains and that they are often selectively expressed implies that it may be possible to modulate individual isozymes using specific drugs. Joe Beavo and David Reifsnyder summarize much of the evidence that has led to our current understanding of multiple isozymes of phosphodiesterase, with emphasis on aspects that may be relevant to drug design. They also discuss why many previous attempts to isolate isozyme-selective inhibitors may have failed.

Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets.

Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.

Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs.

We have identified and highly purified a "low Km" cAMP phosphodiesterase from bovine cardiac muscle. This phosphodiesterase was inhibited by low concentrations of cGMP and has, therefore, been temporarily designated as cGMP-inhibited phosphodiesterase. After a 16,000-fold increase in specific activity, the highly purified enzyme had a specific activity of 6 mumol/min-mg and contained three major polypeptides. Initial data indicated that all of these polypeptides were derived from a single common precursor by proteolysis. We used this enzyme preparation to generate polyclonal antisera and monoclonal antibodies directed against the "low Km" phosphodiesterase. Immunoadsorption and immunoblot analysis allowed us to identify and isolate several molecular weight species of phosphodiesterase, including a larger form than previously reported for any purified low Km phosphodiesterase. This large form of the enzyme had a subunit molecular weight of approximately 110,000 and was the only one seen in fresh extracts of cardiac muscle. Full catalytic activity was recovered in the phosphodiesterase-antibody complex and enzyme prepared by immunoprecipitation exhibited Michaelis-Menten kinetics for cAMP hydrolysis and for inhibition by cGMP. The Km for cAMP hydrolysis was 0.15 microM and the Ki for cGMP inhibition of cAMP hydrolysis was 0.06 microM. This immunoprecipitation approach also allowed us to determine that the enzyme was phosphorylated on serine residues by cAMP-dependent protein kinase, and that the low Km, cGMP-inhibited phosphodiesterase was selectively inhibited by several new cardiotonic agents. Milrinone, amrinone, and fenoximone were highly selective inhibitors of this isozyme, and the relative affinities of these inhibitors were consistent with their order of potency as positive inotropic agents. These studies suggest that the cGMP-inhibited phosphodiesterase is a receptor for several new cardiotonic drugs.

Intact cell and cell-free phosphorylation and concomitant activation of a low Km, cAMP phosphodiesterase found in human platelets.

A monoclonal antibody (CGI-5) directed against the cGMP-inhibited phosphodiesterase isolated from bovine heart was used to examine the phosphorylation of this isozyme in human platelets. PGE1 promoted the phosphorylation of this isozyme, identified as a 110 kDa peptide following SDS-gel electrophoresis. Phosphorylation resulted in approximately a 40% increase in the cGMP-inhibited phosphodiesterase activity. Cell-free experiments demonstrated that cAMP-dependent protein kinase phosphorylated the cGMP-inhibited phosphodiesterase, and that this could be blocked by the heat stable inhibitor peptide (PKI). Phosphorylation of the cGMP-inhibited phosphodiesterase increases the Vmax for cAMP hydrolysis approximately 50%, but does not affect the Km for cAMP (0.12 microM).

The use of low protein liquid diets to determine the methionine requirement and the efficacy of methionine hydroxy analogue for the three-week-old pig.

A mechanical feeding device that dispenses liquid diets hourly was developed to feed 3-week-old pigs under carefully controlled and sanitary conditions. Pigs were weaned at 19-21 days of age, placed in individual cages of the automatic feeder, and trained to eat low protein (9%) milk diets, which were supplemented with essential amino acids, glutamic acid and monosodium glutamate so as to be equivalent to 14% protein nitrogen. The basal 9% protein diet contained 0.25% L-methionine and 0.08% L-cysteine and was supplemented with L- or DL-methionine or DL-methionine hydroxy analogue (MHA) at various levels for evaluation of the methionine requirement. Pigs fed the basal diet showed a significant decrease in gain, feed efficiency and plasma urea (P less than 0.05) relative to animals that received supplemental methionine or MHA. The plasma methionine concentration remained below 0.2 mumol/ml plasma when pigs were fed diets containing 0.25-0.51% methionine; however, a significant increase in plasma methionine (P less than 0.05) was seen when pigs were fed diets that contained greater than 0.51% methionine activity in the form of L- or DL-methionine or DL-MHA. The highest average daily gain (470 g) obtained with a diet containing 0.51% methionine was significantly better (P less than 0.05) than diets containing more or less L- or DL-methionine, and the feed efficiency of this diet (1.58 kg feed per kilogram gain) was also significantly better (P less than 0.05) than the feed efficiency obtained with other dietary methionine levels. MHA (0.17%) added to the basal diet significantly improved the average daily gain (P less than 0.05) and lead to a significant decrease in plasma urea (P less than 0.05) relative to pigs that received the basal diet. Supplemental MHA (greater than 0.51% methionine level) produced significant increases (P less than 0.05) in plasma methionine. These data show that the methionine requirement of the 3-week-old pig can be satisfied with L- or DL-methionine or DL-MHA at a level equal to 0.51% of the dietary solids and that these three are equivalent. These experiments also show that low protein diets supplemented with amino acids can be used for liquid feeding of pigs weaned at 3 weeks of age, and average daily gains of greater than 400 g can be realized.