Analysis of recombinant human platelet-derived growth factor by reversed-charge capillary zone electrophoresis.

Reversed-charge capillary zone electrophoresis (RC-CZE) has been developed as a clipping (proteolysis) assay for homodimeric protein recombinant human platelet-derived growth factor (rhPDGF-BB), a major serum mitogenic factor involved in subcutaneous wound healing. When expressed in yeast, the protein is excreted as a fully folded homodimeric protein consisting of two antiparallel B chains held together by two interchain disulfide bonds. During fermentation, internal proteolysis (clipping between residues Arg32 and Thr33) and C-terminal truncation (Arg32 and Thr109) may occur. Internal proteolysis yields three potential forms of rhPDGF-BB: intact (both B chains are intact), single-clipped (one B chain is clipped), and double-clipped (both B chains are clipped). Clipping also creates new C-terminal sites for further C-terminal truncations and leads to a very complex mixture of isoforms. Routine baseline resolution of these three forms by various modes of HPLC proved unsuccessful. When the disulfide bonds of antiparallel chains are reduced, the complex peptide mixture can be analyzed by RP-HPLC; however, only the level of total clipping is identified. Since RC-CZE separation relies upon differences in molecular charge/size ratio, it can resolve the three rhPDGF-BB forms differing in the additional exposed residues. The choice of reversed-charge CZE columns (amine-coated column) allows proteins of high pI such as rhPDGF-BB (pI > 10) to be readily analyzed while minimizing protein loss from column adsorption. To simplify the electropherogram of clipped forms, the sample is treated first with carboxypeptidase B to reduce the charge microheterogeneity of partial Arg32 truncation. Analysis of rhPDGF-BB by RC-CZE yields a baseline separation between the three forms, intact and single- and double-clipped rhPDGF-BB.

Classical light scattering quantitation of protein aggregates: off-line spectroscopy versus HPLC detection.

This paper describes the development and validation of a new off-line approach to quantitate both covalent and noncovalent, in-solution aggregates present in protein formulations and compares the new assay to established HPLC methods. This off-line analysis is well suited for use in QC release testing, formulation development and stability indicating applications. An inexpensive, continuous source HPLC fluorometer has been adapted with the addition of second order filters for use as a sensitive right-angle scatterometer which can determine the molecular weight of protein aggregates in solution. When used as an HPLC detector, right-angle light scattering is a sensitive method which can determine the molecular weight of peaks separable by HPLC, thus discriminating between monomers of different conformations and aggregates. The weight-averaged molecular weight of aggregate peaks can be calculated with system calibration, yielding the average number of monomers per aggregate. If the protein concentration is high enough for an adequate signal, the off-line technique of right-angle light scattering of protein formulations has advantages of convenience and speed over the HPLC approach. Samples are placed in standard fluorometer cuvettes and toluene is used as a calibrator. Data are presented which show the off-line (static) method to be extremely rapid, rugged and precise. The accuracy of this approach is demonstrated through cross-validation to traditional GPC analysis of protein aggregate distributions. This non-invasive light scattering approach is particularly useful when non-covalent protein aggregation is reversible and readily altered by chromatographic separations typically used for characterizing aggregates.

Evaluation of product equivalence during process optimization for manufacture of a human IgM monoclonal antibody.

We have developed a battery of tests to characterize monoclonal antibodies and assess the effect of potential manufacturing process changes. Tryptic peptide mapping, molecular weight determination by HPLC and classical light scattering, isoelectric focussing, oligosaccharide mapping by HPAE-PAD chromatography, circular dichroism spectra and differential scanning calorimetry were applied as sensitive assays of antibody structure. Biological activity was assessed by measurement of specific antigen binding activity, binding spectrum and opsonic activity. Pharmacokinetics was assessed by clearance rate studies in rats. The sensitivity of this battery of assays was demonstrated by the ability to readily detect differences between a human monoclonal antibody (IgM-2) produced by in vitro cell culture versus ascites culture. These same tests support equivalence of a second monoclonal antibody (IgM-1) produced before and after in vitro cell culture process improvements which resulted in a twofold increase in product titer.

Analysis of alkyl sulfates in protein solutions by isocratic and gradient ion chromatography.

An ion-chromatographic analysis for separation and quantitation of long-chain alkyl sulfates in both commercial samples of sodium dodecyl sulfate (SDS) (lauryl sodium sulfate) and protein solutions was developed. The separation was performed on a hydrophobic resin-based column utilizing tetrabutylammonium hydroxide as an ion-pair reagent and acetonitrile as an organic modifier. Sensitive and selective detection of alkyl sulfates was achieved with an anion suppressor and a conductivity detector. Gradient elution with acetonitrile was developed for the detection of a broad range of alkyl chain lengths (C-10--C-20) at high sensitivity. Because of the wide linear range of this method (0.2-700 micrograms/ml), trace levels of C-10, C-14, C-16, C-18, and C-20 alkyl sulfates can be accurately measured in the presence of high concentrations of C-12 alkyl sulfate (SDS). Thus the alkyl sulfate purity of commercial SDS solutions can be accurately and precisely determined without any sample treatment. For analysis of alkyl sulfates from protein solutions, sample treatment consisted of a one-step ion-pair extraction prior to chromatographic resolution and quantitation. Alkyl sulfates from 2-150 micrograms/ml were recovered in high yield from wide variety of protein solutions.

Reversible subunit dissociation of tumor necrosis factor during hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) of recombinant tumor necrosis factor (TNF) results in reversible dissociation of the quaternary protein structure yielding separation of trimer and monomer peaks. Gel electrophoresis, size exclusion, fluorescence polarization and rechromatography were used to identify the trimeric and monomeric species. Relative amounts of these peaks varied as a function of temperature and column contact time. When TNF was chromatographed in the presence of partially proteolyzed [14 kilodalton (kD)] TNF, two additional peaks, identified as the 14-kD monomer and heterotrimer of TNF and the 14-kD fragment, appeared. Rechromatography of this heterotrimer produced TNF monomer and 14-kD peaks establishing that the multiple peak pattern in HIC was due to quaternary dissociation. Incubating TNF in denaturants prior to non-denaturing size-exclusion chromatography resulted in apparent protein unfolding. However, free, undenatured monomer was not observed. We conclude that TNF is most likely a trimer, which is tightly held together by hydrophobic forces, and that the tertiary structure of the monomer is stabilized through this subunit association. The hydrophobicity of the sorbent surface mediates reversible dissociation of the trimers to monomers through hydrophobic stabilization of the monomeric tertiary structure. After elution, the TNF monomers reassociate to form the native TNF trimer.

Model of protein conformation in the reversed-phase separation of interleukin-2 muteins.

Thirty muteins* of interleukin-2 were studied by reversed-phase high-performance liquid chromatography in a gradient mode. Values of the stoichiometry-factor Z [from Geng and Regnier, J. Chromatogr., 296 (1984) 15] varied over a 2.5-fold range for these proteins of similar molecular weight and composition. It is proposed that the more hydrophobic and/or more stable proteins have smaller values of Z while the larger Z-values correspond to a higher degree of protein unfolding during reversed-phase retention. The practical utility of this approach was demonstrated when these Z values were used to predict correctly a reversal in elution order for two closely related interleukin-2 muteins, when shallower gradients were used.

Reversed-phase chromatography of interleukin-2 muteins.

Human recombinant interleukin-2 (IL-2) and related species have been characterized by chemical modifications, tryptic digestion, and cyanogen bromide digestion. The oxidation states of the cysteines and methionines in several IL-2 muteins have been determined. Reversed-phase high-performance liquid chromatography allowed us to distinguish the modifications in these muteins and to correlate retention behavior with their structure.

Improved liquid-chromatographic assay of quinidine and its metabolites in biological fluids.

We describe a simple, rapid, and specific assay for quinidine and its known metabolites in plasma, urine, and bile. Plasma proteins are precipitated by adding acetonitrile, which also contains the internal standard. The supernatant fluid is evaporated and the reconstituted residue is separated on a reversed-phase column, with fluorescence detection. The standard curve is linear and results are reproducible over the clinical concentration ranges: quinidine 0.4 to 8.0 mg/L and the three metabolites (quinidine 10,11-diol, 3-hydroxyquinidine, and quinidine-N-oxide) 0.05 to 1.5 mg/L. As little as 10 micrograms of the N-oxide metabolite per liter and 1 microgram of the other analytes per liter can be quantitated in 0.5 mL of plasma, urine, or bile. With the use of an automated chromatographic system, many samples can be analyzed in a continuous run.

Simultaneous determinations by theophylline and its major metabolites in urine by reversed-phase ion-pair high-performance liquid chromatography.

A new, highly slective high-performance liquid-chromatographic (HPLC) assay for theophylline and its major metabolites in urine is described. The method utilizes an ion-pair extraction followed by separation and quantitation by reversed-phase ion-pair gradient-elution HPLC. Comparison with several other methods showed that interferences were present in too many blank urine samples to allow for the accurate quantitation of the metabolites of theophylline by direct injection--isocratic HPLC assays. Sample processing involving ion-pair complexing and extraction together with gradient-elution systems is recommended for accurate pharmacokinetic studies.

On the mechanism of 2'-deoxyuridylate hydroxymethylase.

dUMP hydroxymethylase from SP01-infected Bacillus subtilis has been purified 160-fold by chromatography on DEAE-cellulose and ethylagarose. The enzyme catalyzes exchange of the 5-hydrogen of dUMP for protons of water in the presence or absence of the cofactor CH2-H4folate. Upon treatment with FdUMP and CH2-H4folate, an isolable covalent complex is formed which is believed to be structurally similar to a steady-state intermediate of the normal reaction. The FdUMP-CH2-H4folate-dUMP hydroxymethylase complex is stable toward denaturation with sodium dodecyl sulfate and shows a subunit molecular weight of 46 000. By analogy with chemical models and studies of dTMP synthetase, a mechanism is proposed for the reaction catalyzed by dUMP hydroxymethylase.