Biomedical subjects

R L Remmele

Publications and source records attributed to R L Remmele.

7 recordsLinked to original sources

PubMed2002

Designing proteins that work using recombinant technologies.

Therapeutic proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Fusion proteins bring together functions from two different molecules creating therapeutics with completely novel activities. Protein engineering technologies have relied on rational design, directed evolution, DNA shuffling, RNA-peptide fusion, phage and ribosomal display methods to select out candidate protein forms with the desired therapeutic properties. Engineered site-specific pegylation and glycosylation strategies have improved circulation half-life, reduced immunogenicity and increased protein therapeutic stability. In this review we describe how protein engineering techniques have been used to select out, improve stability and clinical efficacy of protein therapeutics.

Animals↗

PubMed2001

Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule.

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region.

Animals↗

PubMed1999

Minimization of recombinant human Flt3 ligand aggregation at the Tm plateau: a matter of thermal reversibility.

This study elucidates the importance of thermal reversibility as it pertains to the minimization of recombinant human Flt3 ligand aggregation and its potential role for determining solution conditions that can achieve the greatest long-term storage stability. Both thermal reversibility and Tm were evaluated as microcalorimetric parameters of stability within the range extending from pH 6 to 9, where the Tm was shown to plateau near 80 degrees C. Within this region, the reversibility was shown to decrease from 96. 6% to 15.2% while the pH was increased from 6 to 9, respectively. Accelerated stability studies conducted at 50 degrees C exhibited rates of aggregation augmented by pH that inversely correlated with the thermal reversibility data. Namely, high thermal reversibility at the Tm plateau correlated with slower rates of aggregation. Enthalpic calorimetric to van't Hoff ratios (DeltaH1/DeltaHv) yielded results close to unity within the plateau region, suggesting that the unfolding of rhFlt3 ligand was approximately two-state. Evidence that unfolding preceded the formation of the aggregate was provided by far-UV CD data of a soluble islolate of the aggregated product exhibiting a 28% loss of alpha-helix offset by a 31% gain in beta-sheet. This information combined with the thermal reversibility data provided compelling evidence that unfolding was a key event in the aggregation pathway at 50 degrees C. Minimization of aggregation was achieved at pH 6 and corroborated by evidence acquired from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion data. Correspondingly, the bioactivity was found to be optimal at pH 6. The findings link thermal reversibility to the propensity of Flt3 ligand to aggregate once unfolded in the Tm plateau region and provide a basis for relating the reversibility of thermal denaturation to the prediction of long-term storage stability in aqueous solution.

Animals↗

PubMed1999

Incorporation of an isoleucine zipper motif enhances the biological activity of soluble CD40L (CD154).

Recent progress in the understanding of immune function indicates that the interaction of CD40L with its receptor, CD40, plays a pivotal role in both humoral immunity and cell-mediated defense against pathogens. Functional studies of this interaction on both dendritic cells and malignant cells have demonstrated that CD40L also plays an important role in immune surveillance and anti-tumor immunity. CD40L exists in nature predominantly as a membrane-anchored molecule. To develop CD40L as a potential therapeutic, it is important to optimize soluble forms of this molecule that could be used in a clinical setting. Several reports have shown that soluble forms of CD40L, like CD40 antibodies, are biologically active. In the present report we demonstrate that the incorporation of an isoleucine zipper trimerization motif significantly enhances the biological activity of soluble CD40L.

Animals↗

PubMed1998

Interleukin-1 receptor (IL-1R) liquid formulation development using differential scanning calorimetry.

PURPOSE: To elucidate the solution conditions that confer stability of aqueous IL-1R using differential scanning calorimetry (DSC). METHODS: Optimal pH conditions were determined by monitoring degradation products encountered during accelerated studies (at elevated temperatures) using SDS-PAGE. At the pH optimum, DSC screened for excipients that enhanced thermal stability by shifting the Tm to higher values. Using SEC the relationship between thermal unfolding and stability was investigated by considering if lower Tm's in the presence of preservatives correlated with degradation products at 37 degrees C over time. The degree of aggregation relative to that of a control determined the level of stability achieved. RESULTS: Circular dichroism (CD) measurements confirmed molecular modeling studies showing IL-1R to be about 39% beta-sheet. Two major transitions characterized the DSC data with Tm's observed near 47 degrees C and 66 degrees C. Among 21 excipients screened, NaCl exhibited the greatest stabilizing influences based on shifting the low temperature transition to 53 degrees C. The low temperature transition was later found to comprise two transitions, yielding a total of three melting transitions for IL-1R. High Tm's arising from the presence of preservatives correlated with the order of stability (i.e., 0.065% phenol > 0.1% m-Cresol > 0.9% benzyl alcohol). CONCLUSIONS: The three melting transitions are consistent in origin with the cooperative unfolding of three unique immunoglobulin-like domains of IL-1R. Optimal stability was achieved in 20 mM sodium citrate at pH 6 with sufficient NaCl to attain the tonicity of human serum. A correlation between the predicted ranking of stability and the extent of aggregation was demonstrated using DSC.

Calorimetry, Differential Scanning↗

PubMed1997

Real-time in situ monitoring of lysozyme during lyophilization using infrared spectroscopy: dehydration stress in the presence of sucrose.

PURPOSE: First, to investigate the role of sucrose in stabilizing protein structure (as measured by changes in the amide I band of lysozyme) caused by dehydration encountered during lyophilization. Second, to demonstrate the utility of internal reflection spectroscopy as a tool for conducting controlled lyophilization experiments. METHODS: A custom-built internal reflection FTIR accessory was used to follow the entire freeze-drying process of solutions consisting of 49.4 mg/mL lysozyme in the presence and absence of 10% sucrose in real-time. Studies were carried out using D2O as a transparent medium in the infrared region of the protein amide bands. Potential self-association of the protein in the presence of sucrose was investigated using dynamic light scattering. Hydration levels were determined using a multiple regression equation. Differential scanning calorimetry (DSC) permitted characterization of the final lyophilized product. Moisture content was determined using Karl Fischer titration. RESULTS: Throughout freezing and drying, minimal changes were observed both in frequency (1647 +/- 1 cm-1) and bandwidth (46 +/- 1 cm-1) of the amide I band in the presence of sucrose. In contrast, greater changes in frequency and band width were seen in the absence of sucrose. A successfully lyophilized cake was obtained which had properties of a glass as measured by DSC, with a Tg of 50 degree C. The lyophilized product containing sucrose had 4% moisture by weight. Three distinct rates of water desorption were discovered during drying under vacuum (50 mg/hr within the sample temperature range from -35 degrees to -25 degrees C; 30 mg/hr from -10 degrees to 25 degrees C; 1.2 mg/hr from 27 degrees to 38 degrees C). CONCLUSIONS: The inclusion of sucrose served to minimize perturbations of protein structure caused by freezing and dehydration stresses encountered during lyophilization (compared to studies conducted in absence of sucrose). The results support the water replacement hypothesis and underscore the role of the sugar in preserving a native structure in the dried state. This investigation demonstrates the usefulness of infrared spectroscopy in evaluating lyophilization process parameters and formulation design.

Freeze Drying↗

PubMed1990

Raman spectroscopic studies of hen egg-white lysozyme at high temperatures and pressures.

In situ high-temperature, high-pressure Raman experiments on 3 mM (pH 5) aqueous solutions of hen egg-white (HEW) lysozyme show a decrease in the relative height of the 505 cm-1 band associated with S-S stretching vibrations at 72 degrees C (1 bar). The peak height changes are accompanied by significant band broadening, and the integrated band intensity does not change within experimental error. The effect of increased pressure at 72 degrees C was to hinder broadening of the 505 cm-1 band. HEW lysozyme (2.4 mM, pH 5) was also heated at 76 degrees C, 80 degrees C, and 95 degrees C for different periods of time, and aliquots were quenched to room temperature for Raman and enzymatic activity measurements. After 9 hr at 76 degrees C, the protein exhibits enzyme activity less than 50% of the initial value, and approximately 50% reduction in activity is achieved after 3 hr at 80 degrees C or 1 hr at 95 degrees C. The Raman results suggest that different irreversibly denatured conformations are attained during prolonged exposures at these different temperatures. It is apparent from these studies that the S-S stretch intensity is decreased irreversibly.

Amino Acid Sequence↗