Samir U. Sane

Spectroscopic evaluation of the stabilization of humanized monoclonal antibodies in amino acid formulations

The protective effects of amino acids on stabilizing protein secondary structure were evaluated using diffuse reflectance FTIR spectroscopy, and interactions between proteins and arginine were detected using solid-state NMR spectroscopy. Upon freeze-drying, excipient-free anti-CD11a and anti-IgE antibodies underwent significant changes in their secondary structures. For both antibodies, the amount of intermolecular beta-sheet substantially increased and the native conformation of intramolecular beta-sheet content decreased considerably. The addition of amino acids to the formulations reduced protein secondary structure alterations in a concentration-dependent manner. Histidine and arginine appeared to be the most protective excipients (of the amino acids studied) in inhibiting protein secondary structural changes. Solid-state NMR illustrated that non-covalent interactions (e.g., hydrogen bonding, ion-dipole interactions) were formed between the arginine side chain and the protein. Glycine is the least effective additive of those studied in preventing secondary structure changes upon freeze-drying. Despite secondary structural changes, freeze-dried protein in the presence and absence of amino acids refolded back into its native conformation upon reconstitution in water.

Sustained delivery of human growth hormone from a novel gel system: SABER.

PURPOSE The purpose of this study was to evaluate the release of recombinant human growth hormone (rhGH) from a novel non-polymeric sustained release system, SABER. METHODS The SABER system consists of sucrose acetate isobutryate, a solvent and a polymeric release modifier. Spray dried formulations of zinc complexed rhGH in sodium bicarbonate containing sucrose and polysorbate 20 were homogenized with various SABER systems (10% w/v rhGH) and assessed in vitro and in vivo (rat studies). The effect of protein to sucrose ratio in the spray dried formulation and a release modifier, poly-D,L-lactic acid (PLA), in the SABER system, on the initial release was investigated along with the effect of dose volume. RESULTS The in vitro release studies with rhGH SABER suspensions indicate that increasing the sucrose content from 2 to 5 mg/ml in the rhGH formulations increased the initial release (24 h) from 78.0% to 93.5%. When the protein formulation was held constant and 1.0% w/w PLA was added to the solvent phase, the initial release was reduced from 78.0% to less than 5.0%. The initial release in vivo after subcutaneous administration (SC) in rats (15 mg/kg rhGH) decreased with increasing PLA content (1.0% w/w PLA, Cmax = 342.8 ng/ml; 10% w/w PLA, Cmax = 35.4 ng/ml), while increased sucrose content increased both the initial release (AUC(0-2) days) and persistence (AUC(2-7) days) over the 7 days from 64.2 to 228.4 ng day/ml (total AUC). A linear dose response (rhGH serum levels) was observed after SC administration of different rhGH SABER volumes greater than 100 microl. Histological examination of the injection sites indicated a mild inflammatory response similar to that observed after injection of PLA microspheres. CONCLUSIONS The addition of PLA reduced the initial release rate of protein release from SABER, while increasing the sucrose content of the protein formulation yielded increased rhGH persistence. These results demonstrate that the SABER delivery system allows weight-based dosing at volumes greater than 100 microl to achieve sustained release of intact rhGH in vivo for at least 7 days.

A new roadmap for biopharmaceutical drug product development: Integrating development, validation, and quality by design

Quality by design (QbD) is a science- and risk-based approach to drug product development. Although pharmaceutical companies have historically used many of the same principles during development, this knowledge was not always formally captured or proactively submitted to regulators. In recent years, the US Food and Drug Administration has also recognized the need for more controls in the drug manufacturing processes, especially for biological therapeutics, and it has recently launched an initiative for Pharmaceutical Quality for the 21st Century to modernize pharmaceutical manufacturing and improve product quality. In the biopharmaceutical world, the QbD efforts have been mainly focused on active pharmaceutical ingredient processes with little emphasis on drug product development. We present a systematic approach to biopharmaceutical drug product development using a monoclonal antibody as an example. The approach presented herein leverages scientific understanding of products and processes, risk assessments, and rational experimental design to deliver processes that are consistent with QbD philosophy without excessive incremental effort. Data generated using these approaches will not only strengthen data packages to support specifications and manufacturing ranges but hopefully simplify implementation of postapproval changes. We anticipate that this approach will positively impact cost for companies, regulatory agencies, and patients, alike.

Drug Substance Frozen Storage and Thawing

Preservation through freezing is routinely used in the food industry. The same strategy can also be applied to biotechnology protein based drugs. However, in this case, careful considerations must be given to the process as the freeze and thaw operations put additional stresses on the protein, which may have negative impact on its function. In this chapter, we present a Quality by Design (QbD) approach for handling the risks associated with a commercial production freeze-thaw process for recombinant protein drug substances. A brief treatment on the physics of freezing and thawing is given, followed by a description of the commercial freeze-thaw unit operation and associated equipment. The QbD risk-based strategy is then discussed in detail. The overall approach is illustrated through a case study that shows how small-scale models are leveraged for characterization of freeze-thaw production process.

Lyophilization Process Design and Development Using QbD Principles

Lyophilization is a unit operation that is routinely used to stabilize an otherwise unstable molecule to achieve pharmaceutically acceptable shelf life. The lyophilization process is based on fundamental principles of heat and mass transfer, and the overall understanding of the impact of process parameters on product quality attributes has increased significantly over the past few decades. Quality by design (QbD) aims at building quality within the process rather than monitoring offline at the end of the process. This chapter describes how QbD elements—risk assessment, process characterization, PAT (Process Analytical Tool), and Design Space—can be applied to design, develop, and scale-up of the lyophilization process.

Near infrared spectroscopic characterisation of secondary structure content of proteins

Near infrared (NIR) spectroscopy was investigated as a non-destructive and rapid method to characterise the secondary structures of proteins in solid state. The absorption spectra of 11 reference proteins (0-46% α-helix, 6-73% β-sheet) were obtained using an Antaris NIR Analyser and quantitatively analysed using a chemometric software program to correlate the NIR spectral data to the secondary structure content obtained from X-ray crystallography and Raman spectroscopy. Simple linear regression analyses of the normalised second derivative NIR spectra indicated that 6289 cm−1 (R2 = 0.99, RMSEC = 2.6) and 4602 cm−1 (R2 = 0.96, RMSEC = 5.6) were highly sensitive wave number regions for β-sheet structure and the calibration models were highly predictive for three additional proteins which served as external validation standards (average error 2% and 3%, respectively). The second derivative NIR spectra at 4602 cm−1 was also found to be sensitive to α-helical content (R2 = 0.93, RMSEC = 5.8) and predictive of the validation standards (average error 6%). The results demonstrate the potential of NIR spectroscopy as a rapid and non-invasive tool for quantification of secondary structure content of proteins in solid state.