Steven Shire

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

... Auteur(s) / Author(s). CIPOLLA DC ; CLARK AR ; CHAN H.-K. ; GONDA I. ; SHIRE SJ ; Affiliation(s) du ou des auteurs / Author(s) Affiliation(s). Genentech Inc., South San Francisco CA 94080, ETATS-UNIS ... Langue / Language. Anglais Revue : Français Editeur / Publisher. ...

A filter binding assay was developed to study interactions between purified TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, and trp specific transcripts. TRAP formed stable complexes with trpEDCFBA leader RNA; binding was L-tryptophan-dependent and was complete within 60 s. TRAP binds to a segment of the trp leader transcript that includes part of an RNA antiterminator structure. Binding to this segment allows formation of an RNA terminator structure, thereby promoting transcription termination. Using several trpEDCFBA leader deletion transcripts, we identified several closely spaced trinucleotide repeats (seven GAG and four UAG repeats) in the trp leader transcript that appeared to be required for TRAP binding. We also showed that TRAP binds to a segment of the trpG transcript that includes the trpG ribosome binding site; the nucleotide sequence of this segment contains several appropriately spaced trinucleotide repeats (seven GAG, one UAG, and one AAG). TRAP binding to the trpG transcript would block translation initiation. RNA footprint analysis confirmed interaction between TRAP and the trinucleotide repeats in the various transcripts. TRAP, in the presence or absence of L-tryptophan, appears to consist of 11 or 12 identical 8-kDa subunits. Our findings suggest that each tryptophan-activated TRAP subunit can bind one G/UAG repeat in a target transcript. Multiple protein-RNA interactions are required for stable association.

ABSTRACT The use of analytical ultracentrifugation for probing macromolecular interactions in recombinant deoxyribonucleic acid (DNA) technology was discussed. Analytical ultracentrifugation was used to determine absolute molecular weights without the use of molecular weight standards or interference from the sieving matrix used for separation. The instrument used for analytical ultracentrifugation comprised of an ultracentrifuge, designed to centrifuge components in a well controlled environment with good temperature control and a stable centrifugal field.

Monoclonal antibodies (MAb) have become a crucial therapeutic agent. However, some applications such as a subcutaneous injection need highly concentrated protein solutions that can have undesired large viscosity. Therefore, it is of great importance to examine the relation between MAb self-associated nanostructure and its viscosity under various concentration and excipient conditions with techniques which can probe both structure and dynamics that span the length scale of an individual protein to a larger aggregate. We have investigated the protein-protein interactions and diffusive properties of highly concentrated MAbs using small angle neutron scattering (SANS) and neutron spin echo (NSE). Our results indicate that concentration, temperature, pH and surfactant do not have a strong effect on the individual MAb conformation for our MAb samples. The short-range attraction of some MAb proteins is found to be highly anisotropic in contrast to many other protein solutions at low ionic ...

Light scattering intensity measurements of solutions of two purified monoclonal antibodies were performed over a wide range of concentrations (0.5-275 mg/mL) and ionic strengths (0.02 to 0.6 M). Despite extensive sequence homology between these mAbs, alteration of ∼20 amino acids in the complementarity determining regions resulted in different net intermolecular interactions and responses to solution ionic strength. The concentration dependence of scattering was analyzed by comparison with the predictions of three models, allowing for intermolecular interaction of various types. In order of increasing complexity, the three models account for: (1) steric repulsions (simple hard-sphere model), (2) steric repulsion with short-ranged attractive interactions of varying magnitude (adhesive hard-sphere model), and (3) steric and nonsteric repulsive interactions between several species whose relative concentrations may change as a function of total protein concentration as dictated by equil...

To determine the effect of moisture and the role of the glass transition temperature (Tg) on the stability of a high concentration, lyophilized, monoclonal antibody. A humanized monoclonal antibody was lyophilized in a sucrose/histidine/polysorbate 20 formulation. Residual moistures were from 1 to 8%. Tg values were measured by modulated DSC. Vials were stored at temperatures from 5 to 50 degrees C for 6 or 12 months. Aggregation was monitored by size exclusion chromatography and Asp isomerization by hydrophobic interaction chromatography. Changes in secondary structure were monitored by Fourier transform infrared (FTIR). T. values varied from 80 degrees C at 1% moisture to 25 degrees C at 8% moisture, there was no cake collapse and were no differences in the secondary structure by FTIR. All formulations were stable at 5 degrees C. High moisture cakes had higher aggregation rates than drier samples if stored above their Tg values. Intermediate moisture vials were more stable to aggr...

The purpose of this work is to utilize electron paramagnetic resonance (EPR) spectroscopy in conjunction with analytical ultracentrifugation (AUC) to investigate the binding of surfactants to proteins with a transmembrance domain. As an example these methods have been used to study the interaction of a nonionic surfactant, C12E8, to recombinant human tissue factor (rhTF) in liquid formulations. The complementary nature of the two techniques aids in data interpretation when there is ambiguity using a single technique. In addition to binding stoichiometries, the possibility of identifying the interacting domains by using two forms of rhTF is explored. Two recombinant, truncated forms of human tissue factor were formulated in the absence of phospholipids. Neither of the recombinant proteins, produced in E. coli, contains the cytoplasmic domain. Recombinant human tissue factor 243 (rhTF 243) consists of 243 amino acids and includes the transmembrane sequences. Recombinant human tissue f...

Interaction of human IgE with its high affinity receptor (Fc epsilon RI) on mast cells and basophils is an important step for initiating IgE mediated immune responses. To characterize the IgE and Fc epsilon RI interaction, we investigated this interaction in terms of stoichiometry and binding affinity in solution. The binding of IgE and IgE Fc epsilon RI alpha chain, the extracellular portion of IgE high affinity receptor (sFc epsilon RI alpha) was compared with the binding of IgE and IgE immunoadhesin (Fc epsilon RI alpha-IgG). The interaction was characterized by analytical ultracentrifugation, size exclusion chromatography, light scattering and ELISA. We show that the sFc epsilon RI alpha is only able to bind to one IgE, while the immunoadhesin can bind to two IgE. The interaction between IgE and Fc epsilon RI is very strong. Both forms of soluble receptors have similar intrinsic binding affinity with IgE. Both soluble receptors (Fc epsilon RI alpha-IgG and sFc epsilon RI alpha) ...

Plasminogen activators (PA) are endogenous serine proteases involved in a cascade of events leading to the dissolution of a blood clot. These proteins are classified in two distinct groups: urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen ...

... techniques, such as lyophilization and spray drying [6]. Other techniques, such as crystallization [11] and microparticle-based technology [12 ... First, the rapid pumping and continuous circulation through narrow pathways may generate sufficient shear and cavitation stresses on a ...

ABSTRACT Since the emergence of recombinant DNA technology from the lab to commercialization, biotechnology protein drugs have garnered an increased share of new molecular entities (NMEs) in development pipelines (Mullin 2004). Monoclonal antibodies have rapidly become one of the major shares of this market with about 25% of biotech products in development. As with any other protein pharmaceutical monoclonal antibodies can degrade both by chemical and physical pathways. One of the most important pathways for protein physical degradation is the generation of protein aggregates. The ability of proteins to aggregate has been recognized from the early beginnings in protein biochemistry, and has become an important degradation pathway that can have a major impact on the safety and efficacy of protein drugs.

The purpose of this study was to evaluate the mechanisms of aggregate formation and excipient stabilization in freeze-dried formulations of a recombinant humanized monoclonal antibody. Protein degradation was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native size exclusion chromatography, and protein structure was studied using Fourier transform-infrared spectrometry and circular dichroism. The results showed that protein aggregates present following reconstitution were composed of native antibody structure and a reduced amount of free thiol when compared to protein monomer, which implied that intermolecular disulfides were involved in the aggregation mechanism. An excipient-free formulation resulted in reversible solid-state protein structural alteration and increased aggregation during storage. This correlated with dehydration to an extent that the amount of water was less than the estimated number of surface-accessible hydrogen-bonding sites on the protein. Improved native-like solid-state protein structure and reduced aggregation were obtained by formulation with enough carbohydrate to fulfill the hydrogen-bonding sites on the surface of the protein. Carbohydrate in excess of this concentration has less of an influence on protein aggregation. Reduced aggregation during storage was obtained by the addition of sufficient excipient to both stabilize solid-state protein structure and provide an environment that consisted of an amorphous glassy state matrix.

Relaxin was discovered more than 50 years ago. However, only with the advent of recombinant DNA technology have sufficient amounts of human relaxin been produced and made available for in-depth biochemical characterization and pharmacological studies. ...

ABSTRACT Development of proteins as pharmaceuticals is more challenging compared to traditional small molecule drugs because of the increased complexity of the chemical and physical stability of protein pharmaceuticals. This necessitates the use of both analytical and biophysical methods to investigate the stability of proteins. The choice of which biophysical methods to use is dictated by the types of studies and specific requirements that support protein drug development. This chapter discusses the various types of biophysical studies that are often performed during protein formulation development including (1) early screening assessments, (2) intense characterization, and (3) confirmatory studies. Case studies with representative examples from Genentech are presented for each of the above topics.

Multiple molecular driving forces mediate protein stability, association, and recognition in concentrated solutions. Here we investigate the interactions that modulate the nonideal solution behavior of two immunoglobulins (IgG1s) in highly concentrated solutions using two-dimensional vibrational correlation spectroscopy (2D-COS) and principal components analysis (PCA). A specific sequence of changes is observed in the concentration-dependent vibrational spectra of the highly viscous IgG solution that deviates from ideality, whereas that sequence is reversed for all other conditions examined. The asynchronous spectra reveal variation in beta-sheet and turn regions occur before intensity variations in disordered and alpha-helical regions as the concentration is increased for the highly viscous regime. This is in contrast to the sequence observed for all other conditions studied and to the idea that beta-sheet regions are resistant to concentration-dependent affects. Finally, we show that increased hydrogen bonding and electrostatics primarily modulate the intermolecular association and nonideal behavior. Specifically, 2D-COS and PCA analysis of the amide II region suggests that Glu and Asp residues trigger the change resulting in increased viscosity and association of one IgG.

The ends of chromosomes contain repeats of guanine-rich sequences that can assume highly compact conformations and are presumed necessary for their biological role in chromosomal stabilization and association. We have investigated the conformational behavior of d(G3T4G3) as a function of the addition of either KCl or NaCl, in the concentration range of 50-200 mM, by using a spectrum of physical techniques and conclude that these salts induce a quadruplex species composed of two strands, each in a hairpin conformation. When salt is added, a large positive signal appears near 290 nm in the CD spectra. UV thermal denaturation curves show a single concentration-dependent transition and provide data for quantitating the thermodynamics of quadruplex formation. In electrophoresis experiments, the quadruplex structure migrates as a single species and more rapidly than the unstructured single strand. NMR spectra in the presence of KCl or NaCl indicate that the structure formed is asymmetric. Equilibrium ultracentrifugation studies confirm that these quadruplexes are composed of two strands of d(G3T4G3). Possible models for this structure are discussed.

The mechanism of assembly of tobacco mosaic virus has been investigated under conditions in which the rates of incorporation of the 4S and 20S proteins can each be directly measured by analytical centfrifugation. Under these conditions, pH 6.5, 6.5 degrees C, 0.10 M ionic strength potassium orthophosphate, the protein can be made to exist as a metastable 20S aggregate that is necessary for efficient reconstitution. The overall assembly process consists of an initiation (nucleation) reaction that requires two to three 20S disk aggregates per RNA molecule and is followed by an elongation (growth) reaction. In the elongation phase of assembly the 4S protein is incorporated 50 to 70 times faster than the 20S disk, calculated on the basis of a steady-state kinetic analysis. Therefore, under these conditions, in which the rate of assembly is about 0.06 of that at pH 7, 20 degrees C, 0.10 M ionic strength orthophosphate, the 4S protein preferentially participates in the elongation phase. At this slow reconstitution rate intermediate assembly states (about 70-168 S) can be observed. The kinetics of both protein incorporation and nucleoprotein formation suggest that the elongation process is composed of at least two different, possibly sequential, rate-limiting reactions.

The mechanism of assembly of tobacco mosaic virus (TMV) has been investigated at pH 7.0 and 20 degrees C by analytical ultracentrifugation. Under these conditions the overall rates of interconversion of 4S and 20S TMV coat protein are sufficiently slow to make possible measurements of the concentrations of remaining 4S and 20S TMV coat protein after addition of homologous RNA to solutions containing, initially, various mass ratios of 20S protein to 4S protein. It has been possible to measure, by schlieren boundary analysis, the relative rates of incorporation of 4S and 20S TMV protein into the growing nucleoprotein rod over the range of initial 20S:4S protein mass ratios from 93:7 to 18:82. The results show that the amount of incorporation of 20S TMV protein depends on the initial 20S:4S mass ratio between approximately 100% and 60% 20S protein but that reconstitution can proceed with approximately 100% 20S TMV protein to form full virus-size rods. However, when the initial protein solutions have less than 60% 20S protein, approximately 80% of the reconstituted nucleoprotein is preferentially formed from 4S coat protein. The remaining approximately 20% appears to require preformed 20S coat protein. These results suggest that a larger region of RNA than previously estimated is involved in the rate-limiting nucleation step in assembly and may explain previously conflicting results concerning the elongation phase of assembly when starting with partially assembled rods.

... Ming-Yu R. Wang,Ia Brian M. Hoffman,*la Steven J. Shire,lb and Frank RN Gurd*Ib Contribution from the Department of Chemistry, Northwestern University, Ecanston, Illinois 60201, and the Department of Chemistry, Indiana University, Bloomington ... 25 -40 -50 -60 -70 ASo (e u ...

The viscosity profiles of four different IgG(1) molecules were studied as a function of concentration at pH 6.0. At high concentrations, MAb-H and -A showed significantly higher viscosities as compared to MAb-G and -E. Zeta Potential (ξ) measurements showed that all the IgG(1) molecules carried a net positive charge at this pH. MAb-G showed the highest positive zeta potential followed by MAb-E, -H, and -A. A consistent interpretation of the impact of net charge on viscosity for these MAbs is not possible, suggesting that electroviscous effects cannot explain the differences in viscosity. Values of k(D) (dynamic light scattering) indicated that the intermolecular interactions were repulsive for MAb-E and -G; and attractive for MAb-H and -A. Solution storage modulus (G') in high concentration solutions was consistent with attractive intermolecular interactions for MAb-H and -A, and repulsive interactions for MAb-G and -E. Effect of salt addition on solution G' and k(D) indicated that the interactions were primarily electrostatic in nature. The concentration dependent viscosity data were analyzed using a modified Ross and Minton equation. The analysis explicitly differentiates between the effect of molecular shape, size, self-crowding, and electrostatic intermolecular interactions in governing high concentration viscosity behavior.

The effect of calcium ions on the structure and stability of recombinant human DNase I (rhDNase) in the aqueous and solid (lyophilized) states was investigated. Fourier transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure, while chemical stability was monitored in terms of deamidation and soluble aggregate formation at 40 degrees C. The exogenous calcium was removed by EGTA. This process can remove all but approximately one calcium ion per protein molecule. Analysis of the FTIR spectra in the amide III region in either the aqueous or lyophilized state demonstrated that removal of exogenous Ca2+ by EGTA-treatment had little effect on the secondary structure (and lyophilization-induced rearrangement thereof). For the aqueous solution, circular dichroism was used as an independent technique and confirmed that there was no large overall change in the secondary or tertiary structure upon the removal of calcium. The primary degradation route for the aqueous protein was deamidation. For the EGTA-treated protein, there was also severe covalent aggregation, e.g., formation of intermolecular disulfides facilitated by the cleavage of Cys173-Cys209. The aggregates exhibited a markedly different secondary structure compared to the native protein. For instance, the beta-sheet band observed at ca. 1620 cm-1 wavenumber in the amide I second derivative spectra was increased. Enzymatic activity was completely lost upon aggregation, consistent with the cleavage of the aforementioned native disulfide. For the protein lyophilized in the presence of Ca2+, there was no increase in deamidated species during solid-state storage; however, some aggregation was observed. For the lyophilized EGTA-treated protein, aggregation was even more pronounced, and there was some loss in enzymatic activity upon reconstitution. Thus, the removal of calcium ions by EGTA-treatment decreased the stability of rhDNase in both the aqueous and solid states even though no large overall calcium-induced structural changes could be observed by the techniques used in this study.