A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

PurposeThe feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied.Materials and MethodsA hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.5 and 10.5. The stability of the sCT formulations was analyzed over 1 week by the following assays: (1) protein concentration, (2) volume control by measuring pathlength, (3) turbidity (absorbance at 350 nm), (4) intrinsic tyrosine fluorescence, (5) 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence, (6) Nile Red fluorescence. Addition of the dyes (Nile Red and ANS) was used to study protein conformational changes.ResultsAfter 1 day, 27 out of the 100 formulations of salmon calcitonin were stable. After 7 days, 12 stable sCT formulations remained. The best salmon calcitonin formulation was in 10 mM sodium acetate buffer with pH values between 3.5 and 5.5.ConclusionsThe findings are in accordance with the sCT formulations that were patented and used commercially. This can be considered as a proof of concept for the high throughput protein formulation platform.

[1]  Manuel Muñoz-Torres,et al.  Calcitonin Therapy in Osteoporosis , 2004, Treatments in endocrinology.

[2]  George M. Nixon,et al.  Practical Considerations of , 1945 .

[3]  Gregory V. Kaler,et al.  Monitoring structural and ligand-loading status of serum albumin with an uncharged hydrophobic fluorescent probe, Nile red , 1996, Photonics West.

[4]  Robert Gurny,et al.  Detection and characterization of protein aggregates by fluorescence microscopy. , 2007, International journal of pharmaceutics.

[5]  A. Fink,et al.  Conformational states of beta-lactamase: molten-globule states at acidic and alkaline pH with high salt. , 1989, Biochemistry.

[6]  A. Cudd,et al.  Enhanced potency of human calcitonin when fibrillation is avoided. , 1995, Journal of pharmaceutical sciences.

[7]  Management of osteoporosis in postmenopausal women: 2006 position statement of The North American Menopause Society , 2006, Menopause.

[8]  D. Matulis,et al.  1-Anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation. , 1998, Biophysical journal.

[9]  Hanns-Christian Mahler,et al.  Induction and analysis of aggregates in a liquid IgG1-antibody formulation. , 2005, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[10]  Stephen A Charles,et al.  SuperGenerics: a better alternative for biogenerics. , 2005, Drug discovery today.

[11]  D L Sackett,et al.  Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces. , 1987, Analytical biochemistry.

[12]  E L McGown,et al.  Multichannel pipettor performance verified by measuring pathlength of reagent dispensed into a microplate. , 1998, Analytical biochemistry.

[13]  Nadine Ritter,et al.  Determining protein concentration. Part 1: Methodology , 2002 .

[14]  Connie M Weaver,et al.  The role of calcium in peri- and postmenopausal women: 2006 position statement of the North American Menopause Society. , 2006, Menopause.

[15]  V. Bhakuni,et al.  8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions. , 1999, Biochemistry.

[16]  Carole S Ben-Maimon.  Point: Why biogenerics make sense , 2006, Nature Biotechnology.

[17]  Ralf Thiericke,et al.  Evaluation of Liquid Handling Conditions in Microplates , 2001, Journal of biomolecular screening.

[18]  S. Wimalawansa,et al.  Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily. , 1997, Critical reviews in neurobiology.

[19]  Carole S Ben-Maimon,et al.  Biogenerics at the crossroads , 2006, Nature Biotechnology.

[20]  David G I Kingston,et al.  A fluorescence-based high-throughput assay for antimicrotubule drugs. , 2003, Analytical biochemistry.

[21]  A. Drake,et al.  Comparative study of human and salmon calcitonin secondary structure in solutions with low dielectric constants. , 1993, The Journal of biological chemistry.

[22]  Robert Gurny,et al.  High throughput screening of protein formulation stability: practical considerations. , 2007, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[23]  A. Drake,et al.  The structure and mechanism of formation of human calcitonin fibrils. , 1993, The Journal of biological chemistry.

[24]  P. Corran,et al.  Multicenter collaborative study to calibrate salmon calcitonin by bioassay and high-performance liquid chromatography: establishment of the third international standard. , 2001, Bone.

[25]  B M Eckhardt,et al.  A turbidimetric method to determine visual appearance of protein solutions. , 1994, Journal of pharmaceutical science and technology : the official journal of PDA.

[26]  L. Stryer,et al.  The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. , 1965, Journal of molecular biology.