Protein isolated from biopharmaceutical formulations cannot be used for comparative studies: Follow-up to "a case study using Epoetin Alfa from Epogen and EPREX".

In the biotechnology area, the issue of comparability with an innovator product is complex. Ideally, a side-by-side comparison of physical properties would be part of the demonstration of comparability. However, biogeneric companies do not have access to the bulk drug substance from the innovator company for biophysical comparison, and isolation of protein from marketed product cannot be guaranteed to produce material that is identical to the bulk drug substance from which it was prepared. In a recently published study, protein was isolated from marketed product and comparative studies performed. In a follow-up investigation of the published work, we demonstrate here that even a simple isolation procedure can significantly compromise the protein, which raises serious questions about the interpretation of that study, and in a broader context the value of any studies done with such "out-of-process" protein.

Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.

Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein conjugate. Baseline resolution of the chromatographic peaks is not required; peaks need only be sufficiently separated to represent relatively pure fractions. An improved technique for determining the polypeptide-only mass of protein conjugates is also described. These techniques are applied to determining the degree of erythropoietin glycosylation, the degree of polyethylene glycol conjugation to RNase A and brain-derived neurotrophic factor, and the solution association states of these molecules. Calibration methods for the RI, UV, and LS detectors will also be addressed, as well as online methods to determine protein extinction coefficients and dn/dc values both unconjugated and conjugated protein molecules.

Copper staining method for extracting biologically active proteins from native gels.

An attempt was made to make protein bands visible on native gel using copper staining, since such a mild staining procedure would make the entire native gel electrophoresis process non-denaturing. Copper staining not only was able to detect various proteins on native gel with reasonable sensitivity, but also made extraction and recovery of active proteins possible from the gel using a gentle procedure.

Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin.

Thermal stress was used to assess the stability of recombinant human erythropoietin (EPO) derived from Chinese hamster ovary cells. In 20 mm phosphate at pH 7.0, this protein had a highly reversible thermal unfolding as observed by far UV circular dichroism (CD) and native gel analysis, with no indication of protein aggregation. It had a relatively low melting temperature at 53 degrees C. Assuming a two-state transition, the observed reversibility permits thermodynamic analysis of the unfolding of EPO, which shows that the free energy of unfolding at 25 degrees C is only 6-7 kcal/mol. Upon heating to 79 degrees C over 30 min, however, this protein does undergo aggregation as assessed by native gel. In 20 mm phosphate and citrate at pH 7.0, the results are similar, i.e., EPO suffered a substantial aggregation, while it showed little aggregation in 20 mm Tris or histidine at pH 7.0 and 20 mm glycine at pH 6.3 under identical heat treatment.

Molecular cloning of groESL locus, and purification and characterization of chaperonins, GroEL and GroES, from Bacillus brevis.

The groESL locus of a protein-hypersecreting bacterium, Bacillus brevis, was cloned by PCR using primers designed based on the DNA sequence of a B. subtilis homolog. GroEL protein was purified to apparent homogeneity and its ATPase activity was characterized: it hydrolyzed ATP, CTP, and TTP in this order of reaction rate, and its specific activity for ATP was 0.1 micromole/min/mg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% alpha-helix, 24% beta-sheet, and 19% turn structures, by CD measurement. GroES protein was also highly purified to examine its chaperonin activity. GroEL protected from thermal inactivation of and showed refolding-promoting activity for malate dehydrogenase, strictly depending on the presence of ATP and GroES.

Overview of the quantitation of protein interactions.

The biological function of many proteins involves reversible interactions with other proteins, nucleic acids, or other non-protein ligands. Such interactions play many different roles in a wide range of cellular processes. A few examples are: (1) storing or transporting key metabolites (e.g., O(2) storage by myoglobin); (2) forming and maintaining the quaternary structure of multi-subunit enzymes; (3) specific binding and recognition events (antigen-antibody, hormone-receptor, transcription factor-promoter); and (4) self-assembly of large structures (microtubules, chromatin). Thus, the quantitative characterization of such interactions represents an important part of understanding the function of such proteins and their role in these cellular events. This unit sets the tone for the rest of the chapter, and gives important information necessary to understand some of the topics that will be covered in future supplements, such as sedimentation equilibrium (analytical and micro-preparative), surface plasmon resonance (SPR), size-exclusion chromatography (SEC) with on-line light scattering, and chemical cross-linking.

A chimera-like alpha-amylase inhibitor suggesting the evolution of Phaseolus vulgaris alpha-amylase inhibitor.

White kidney bean (Phaseolus vulgaris) contains two kinds of alpha-amylase inhibitors, one heat-stable (alpha AI-s) and one heat-labile (alpha AI-u). alpha AI-s has recently been revealed to be a tetrameric complex, alpha(2)beta(2), with two active sites [Kasahara et al. (1996) J. Biochem. 120, 177-183]. The present study was undertaken to reveal the molecular features of alpha AI-u, which is composed of three kinds of subunits, alpha, beta, and gamma. The gamma-subunit, in contrast to the alpha- and beta-subunits that are indistinguishable from the alpha- and beta-subunits of alpha AI-s, was found to correspond to a subunit of an alpha-amylase inhibitor-like protein, which has been identified as an inactive, evolutionary intermediate between arcelin and the alpha-amylase inhibitor in a P. vulgaris defense protein family. The polypeptide molecular weight of alpha AI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that alpha AI-u is a trimeric complex, alpha beta gamma. The inhibition of alpha AI-u by increasing amounts of porcine pancreatic alpha-amylase (PPA) indicates that an inactive 1:1 complex is formed between alpha AI-u and PPA. Molecular weight estimation of the complex by the light-scattering technique confirmed that it is a complex of alpha AI-u with one PPA molecule. Thus it seems probable that alpha AI-u is an evolutionary intermediate of the P. vulgaris alpha-amylase inhibitor.

A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions.

The time-derivative method for deriving the sedimentation coefficient distribution, g(s*), from sedimentation velocity data that was developed by Walter Stafford has many advantages and is now widely used. By fitting Gaussian functions to the g(s*) distribution both sedimentation and diffusion coefficients (and therefore molecular masses) for individual species can be obtained. However, some of the approximations used in these procedures limit the accuracy of the results. An alternative approach is proposed in which the dc/dt data are fitted rather than g(s*). This new approach gives improved accuracy, extends the range to sedimentation coefficients below 1 S, and enhances resolution of multiple species. For both approaches the peaks from individual species are broadened when the data cover too wide a time span, and this effect is explored and quantified. An alternative algorithm for calculating g(s*) from the dc/dt curves is presented and discussed. Rather than first averaging the dc/dt data for individual scan pairs and then calculating g(s*) from that average, the g(s*) distributions are calculated for every scan pair and then subsequently averaged. This alternative procedure yields smaller error bars for g(s*) and somewhat greater accuracy for fitted hydrodynamic properties when the time span becomes large.

[Applications of analytical ultracentrifuge to molecular biology and pharmaceutical science].

Analytical ultracentrifuges, XL-A and XL-I, developed by Beckman Company now find a broad application not only in universities but also in industries. Especially they are utilized conveniently in industries aiming at the development of proteins as a therapeutic drug or in those targeting drugs composed of small molecules developed on the basis of the structures of proteins. Sedimentation techniques can be used 1) to determine the molecular weight of proteins in solution, 2) to examine protein aggregation, 3) to evaluate the molecular shape of proteins, 4) to study the interaction of proteins, e.g. between ligands and receptors, and 5) to obtain insight into biological functions of homologous proteins. Application of this technique to molecular biology and pharmaceutical science will be reviewed with ample examples.

Reversibility of heat-induced denaturation of the recombinant human megakaryocyte growth and development factor.

PURPOSE: The present study was performed to examine the effect of solution conditions on the reversibility of the thermal denaturation of megakaryocyte growth and development factor (rHuMGDF). METHODS: Changes in the far UV CD spectra of rHuMGDF with temperature were used to monitor the thermal denaturation of the protein, and the recovery of folded protein following a return to room temperature. The effect of protein concentration, scan rate, and buffer composition on thermal denaturation and on the reversibility were determined. Surface tension measurements were used to determine the effect of this unfolding reaction on the surface adsorption of the protein. Sedimentation velocity was used to assess recovery of native monomer and the size of soluble aggregates. In addition, monomeric protein remaining in solution after incubation at 37 degrees C for 2 weeks in either 10 mM imidazole of 10 mM phosphate was determined. RESULTS: In phosphate buffer the rHuMGDF irreversibly precipitates upon unfolding under all the conditions examined. In imidazole the unfolding is at least partially reversible, with no visible precipitate seen; the degree of reversibility increased by lowering both protein and salt concentrations, and the amount of time spent at elevated temperature. In order to compare thermal unfolding occuring with different degrees of reversibility, the melting temperature was defined as the temperature at which melting begins. The melting temperature itself is relatively independent of the buffer composition, or experimental conditions. At low protein concentrations the protein stabilizer sucrose had a marginal effect on the thermal transition of rHuMGDF, while at protein concentrations of about 2 mg/ml the inclusion of sucrose increased the apparent melting temperature by about 4 degrees C, to that seen at low protein concentrations, but had little effect on the reversibility of denaturation. Inclusion of 1 or 2 M urea did not affect the reaction. Surface tension measurements of rHuMGDF solutions showed little difference before and after melting, and in the presence or absence of sucrose. When unfolding is irreversible, the MGDF appears to form soluble aggregates of tetramers to 14-mers, while under reversible conditions native monomer is recovered. More monomeric MGDF remained in solution following storage for 2 weeks at 37 degrees C in imidazole than in phosphate, in both the presence and absence of sucrose. CONCLUSIONS: These results can be explained by assuming that thermal denaturation proceeds as a two-step reaction, the first step being the equilibrium between folded and unfolded states, while the second step is a slow irreversible aggregation. The different buffer systems affect the rate of the aggregation step, but not the intrinsic thermal stability nor the rate of the unfolding step.

Heparin is essential for a single keratinocyte growth factor molecule to bind and form a complex with two molecules of the extracellular domain of its receptor.

Keratinocyte growth factor (KGF or FGF-7) is a member of the heparin binding fibroblast growth factor (FGF) family and is a paracrine mediator of proliferation and differentiation of a wide variety of epithelial cells. To examine the stoichiometry of complexes formed between KGF and its receptor, we have utilized a soluble variant of the extracellular region of the KGF receptor containing two tandem immunoglobulin-like loops, loops II and III (sKGFR). Ligand-receptor complexes were examined by size exclusion chromatography, light scattering, N-terminal protein sequencing, and sedimentation velocity. In the presence of low-molecular mass heparin ( approximately 3 kDa), we demonstrate the formation of complexes containing two molecules of sKGFR and one molecule of KGF. In the absence of heparin, we were unable to detect any KGF-sKGFR complexes using the above techniques, and additional studies in which sedimentation equilibrium was used show that the binding is very weak (Kd >/= 70 microM). Furthermore, using heparin fragments of defined size, we demonstrate that a heparin octamer or decamer can promote formation of a 2:1 complex, while a hexamer does not. Utilizing the highly purified proteins and defined conditions described in this study, we find that heparin is obligatory for formation of a KGF-sKGFR complex. Finally, 32D cells, which appear to lack low-affinity FGF binding sites, were transfected with a KGFR-erythropoeitin receptor chimera and were found to require heparin to achieve maximal KGF stimulation. Our data are consistent with the previously described concept that cell- or matrix-associated heparan sulfate proteoglycans (HSPGs) and FGF ligands participate in a concerted mechanism that facilitates FGFR dimerization and signal transduction in vivo.

Recombinant rat fibroblast growth factor-16: structure and biological activity.

Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties.

Biochemical, biophysical, and pharmacological characterization of bacterially expressed human agouti-related protein.

The agouti-related protein gene (Agrp) is a novel gene implicated in the control of feeding behavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp in transgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, these data suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in the central nervous system. The availability of recombinant, highly purified protein is required to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fraction of Escherichia coli and appeared as large intermolecular disulfide-bonded aggregates. Following oxidation, refolding, and purification, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily random coil and beta-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated that MKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressed AGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both forms act as competitive antagonists of alpha-melanocyte stimulating hormone (alpha-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstration that AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within the central nervous system, and has important implications in the control of feeding.

Interactions between NFkappaB and its inhibitor ikappaB: biophysical characterization of a NFkappaB/ikappaB-alpha complex.

The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E. coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography. The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis. The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA. The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops. By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops. Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results. The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.

Granulocyte-colony stimulating factor maintains a thermally stable, compact, partially folded structure at pH2.

At acidic pH many proteins exist in a partially unfolded form, called the "A" state. This is defined as a flexible, expanded structure with well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induced denaturation. Granulocyte-colony stimulating factor (G-CSF), a four-helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.0. We therefore examined the conformation and thermal unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stoke's radius even at pH 2.0. The thermal-induced denaturation of G-CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some alpha-helical structure persists even at 86 degrees C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G-CSF at pH 2.0 is thus revealed as compact, with a unique, three-dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the "A" state.

Changes in conformation and stability upon formation of complexes of erythropoietin (EPO) and soluble EPO receptor.

Erythropoietin (EPO) is a glycoprotein hormone which belongs to the four-helical-bundle cytokine family and regulates the level of circulating red blood cells. The EPO receptor (EPOR) belongs to the cytokine-receptor family of proteins. While many of the downstream events following receptor/ligand interaction have been defined, both ligand-induced receptor dimerization and conformational changes induced by binding have been implicated as the initial step in signal transduction. In a recent paper [Philo et al. (1996), Biochemistry 38, 1681-1691] we described the formation of both 1:1 and 2:1 EPOR/EPO complexes. In this paper, we examine changes in protein conformation and stability resulting from the formation of both 1:1 and 2:1 complexes of the soluble extracellular domain of EPOR and the recombinant EPO derived from either Chinese hamster ovary cells or from Escherichia coli cells. Occupation of the first binding site results in a slight conformational change that is apparent in both the far- and near-UV circular dichroism spectra. Formation of the 2:1 complex results in an even greater change in conformation which involves the local environment of one or more aromatic amino acids, accompanied perhaps by a small increase in helical content of the complex. This change in local conformation could occur in the EPO molecule, in the EPOR, in both EPOR molecules due to dimerization, or in all molecules in the trimer. The 1:1 complex exhibits increased stability to thermal-induced denaturation relative to the individual protein component; indeed, the E. coli-derived (nonglycosylated) EPO stays folded in the complex at temperatures where the EPO alone would have unfolded and precipitated. Glycosylation of the receptor increases the reversibility of thermal denaturation, but does not affect the temperature at which this unfolding reaction occurs.

Structural characterization of an alpha-amylase inhibitor from a wild common bean (Phaseolus vulgaris): insight into the common structural features of leguminous alpha-amylase inhibitors.

The primary structures of two subunits of an alpha-amylase inhibitor (alpha AI-2) from a wild common bean (Phaseolus vulgaris) were revealed by a comparison of the amino acid sequence previously deduced from the nucleotide sequence with the amino- and carboxyl-terminal amino acid sequences determined by conventional methods. The polypeptide molecular weight of alpha AI-2 obtained by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that alpha AI-2 has the subunit stoichiometry of an alpha 2 beta 2 complex. These structural features were closely similar to those recently elucidated for a white kidney bean (P. vulgaris) alpha-amylase inhibitor, which is quite different in the inhibitory specificity from alpha AI-2. The post-translational processing of the precursor glycoproteins to form the tetrameric structure appeared to require an Arg residue close to the processing site. Further, the proper associations of the subunits into the tetrameric structures seemed to be strictly controlled by a few amino acids on the subunit interfaces.

Dimerization of granulocyte-colony stimulating factor receptor: the Ig plus CRH construct of granulocyte-colony stimulating factor receptor forms a 2:2 complex with a ligand.

We have previously shown that the extracellular domain of granulocyte-colony stimulating factor receptor (soluble G-CSFR), prepared from CHO cell conditioned media, dimerizes upon binding its ligand, G-CSF. The most stable ligand-receptor complex occurs at a 2:2 stoichiometry, unlike the growth hormone and erythropoietin systems. In the latter cases, each ligand uses two sites to bring two receptors together. In this study, we have generated a truncated G-CSF receptor, known to be sufficient for high affinity ligand binding, which consists of an Ig-like domain and a cytokine receptor homology module. With an affinity purified receptor, sedimentation equilibrium experiments clearly demonstrated that this truncated form of the receptor behaves very similarly to the entire extracellular domain. The sedimentation equilibrium data are consistent with the model that the truncated receptor has a weak tendency to self-associate into a dimer in the absence of a ligand, this receptor-receptor interaction is enhanced by ligand binding, and the most stable complex occurs at a 2:2 stoichiometry. These results are very different from those described by others for various murine G-CSF receptor constructs from either Escherichia coli or insect expression systems.

An improved function for fitting sedimentation velocity data for low-molecular-weight solutes.

Many traditional approaches to the analysis of sedimentation velocity data work poorly with data for low-molecular-weight solutes, which have sedimentation boundaries that are severely broadened by diffusion. An approach that has previously had some success is to directly fit these broad boundaries to approximate solutions of the Lamm equation that directly account for the high diffusion. However, none of the available approximate solutions work well at times both early and late in the run, or give boundary shapes that are highly accurate, especially for species of molecular weight < 10,000. An improved fitting function has been developed to overcome some of these limitations. The new function adds two correction terms to the Fujita-MacCosham solution. The optimum coefficients for these new correction terms were determined by a least-squares approach. The accuracy and limitations of fitting with this new function were tested against synthetic data sets obtained by finite-element methods, for analysis of samples containing either single species or several noninteracting species. We also compare the strengths and weaknesses of this method of analysis, and its ability to work with noisy data, relative to recently developed time-derivative methodologies.