Studies on Stable Formulations for a Hydrophobic Cytokine

The goal of the thesis was to analyze the impact of the formulation conditions on the stabilization of a hydrophobic cytokine. A standard way to formulate the cytokine at physiological pH is using Human Serum Albumin (HSA) as excipient. Based on a lyophilized formulation with mannitol as bulking agent and HSA as stabilizer analogous to commercially available systems, the impact of HSA-stabilizers, NaCl and pH on particle formation and stability of the cytokine in the liquid state was elucidated. As NaCl can impact the freezing and lyophilization behavior, the impact of NaCl on the behavior of mannitol-HSA placebo mixtures based on the cytokine-HSA formulations was studied. The goal was to understand how freezing, lyophilization and a subsequent storage of the formulations are influenced by NaCl and the HSA-stabilizers. The use of HSA as excipient is related to the risk of blood born pathogens, enhanced immunogenicity, as well as to analytical difficulties. Therefore, approaches to replace HSA in the cytokine formulation and to achieve stable HSA-free formulations were developed. For the lyophilized formulations HSA was replaced by sucrose as amorphous stabilizer and the physico-chemical properties of the system mannitol–sucrose during freezing and lyophilization were analyzed. To achieve a stable HSA-free formulation, cytokine adsorption and its low solubility were the major issues that had to be overcome. Overall, it was feasible to stabilize the cytokine without the use of HSA at low pH as liquid and lyophilized formulation. By using glass type I+ vials, respectively adding polysorbate 20 it was possible to minimize protein adsorption. Thus, the studies provided an extensive characterization of the hydrophobic cytokine, its stabilization with HSA and the pH dependent instability of HSA-containing systems. As an alternative a stable HSA-free formulation for the cytokine could be presented. Especially for the lyophilized formulations it could be shown that subtle changes of excipients e.g. in the NaCl content can have a detrimental impact

A New Strategy to Stabilize Oxytocin in Aqueous Solutions: I. The Effects of Divalent Metal Ions and Citrate Buffer

In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. and divalent metal ions (Ca2+, Mg2+, and Zn2+) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl2, MgCl2, or ZnCl2 and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca2+, Mg2+, or Zn2+, while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions

Ongoing Challenges to Develop High Concentration Monoclonal Antibody-based Formulations for Subcutaneous Administration: Quo Vadis?

Although many subcutaneously (s.c.) delivered, high-concentration antibody formulations (HCAF) have received regulatory approval and are widely used commercially, formulation scientists are still presented with many ongoing challenges during HCAF development with new mAb and mAb-based candidates. Depending on the specific physicochemical and biological properties of a particular mAb-based molecule, such challenges vary from pharmaceutical attributes e.g., stability, viscosity, manufacturability, to clinical performance e.g., bioavailability, immunogenicity, and finally to patient experience e.g., preference for s.c. vs. intravenous delivery and/or preferred interactions with health-care professionals. This commentary focuses on one key formulation obstacle encountered during HCAF development: how to maximize the dose of the drug? We examine methodologies for increasing the protein concentration, increasing the volume delivered, or combining both approaches together. We discuss commonly encountered hurdles, i.e., physical protein instability and solution volume limitations, and we provide recommendations to formulation scientists to facilitate their development of s.c. administered HCAF with new mAb-based product candidates

Therapeutic Proteins and Advanced Therapy Medicinal Products

This chapter is divided in two parts: Therapeutic proteins and Advanced Therapy Medicinal Products (ATMPs). Therapeutic Proteins. Therapeutic proteins are large molecules that are different from low molecular weight medicines. They consist of amino acid chains, sometimes decorated with polysaccharides (glycoproteins) that fold in space in secondary and tertiary structures. The first section describes the production process which is divided into upstream and downstream processing. It encompasses cell line development and expression of biopharmaceutical products in Upstream Processing (USP), the purification of the products during Downstream Processing (DSP), as well as the subsequent formulation process (which may be part of DSP or done afterwards). Therapeutic proteins are sensitive to chemical and physical degradation. The formulation section describes the forces that stabilize a therapeutic protein and potential degradation pathways. It then discusses the challenges a formulator encounters when developing a protein medicinal product: (1) the development of analytical techniques for monitoring critical quality attributes, (2) the selection of the relevant tests to optimize stability and the excipients available, (3) the options for primary packaging materials and (4) the link with the preferred routes of administration. Ultimately, this should lead to achieving the predefined target product formulation profile for a biological. The predominant routes of administration for therapeutic proteins are the intravenous and subcutaneous (s.c.) route. In contrast to intravenous administration, absorption of the protein from the subcutaneous reservoir upon s.c. injection is an extra step, resulting in a longer time to reach the maximum plasma concentration (Cmax) as compared to intravenous administration. Therapeutic proteins are very important treatment options for a variety of diseases. An adverse event specifically linked to therapeutic proteins is immunogenicity. An immune response against a therapeutic protein can have a major impact on efficacy and/or safety and can be influenced by either patient-, disease-, and product-related factors. Especially, the product-related factors are important to take into consideration during product development. ATMPs. The second part of the chapter is devoted to the discussion of Advanced Therapy Medicinal Products (ATMPs). These are medicines made from, or consisting of, cells, genes or tissues. They have introduced a new area of specialism to the pharmacy workforce. The section about ATMPs will explore the role of the pharmacy team in the delivery of ATMPs recognising that operationalising ATMPs requires a collaborative multidisciplinary approach to ensure that the medicines are optimised for patients. This involves ensuring that whilst appropriate pharmacovigilance and pharmaceutical procedures are in place, handling is undertaken by a trained workforce that is competent in the handling of cellular products. The second part describes the legislation applicable to ATMPs and focuses on specific operational considerations including the logistics and product handling requirements related to ATMPs and the implications for pharmacy

Increasing community capacity to prevent childhood obesity: challenges, lessons learned and results from the Romp & Chomp intervention

BackgroundObesity is a major public health issue; however, only limited evidence is available about effective ways to prevent obesity, particularly in early childhood. Romp &amp; Chomp was a community-wide obesity prevention intervention conducted in Geelong Australia with a target group of 12,000 children aged 0-5 years. The intervention had an environmental and capacity building focus and we have recently demonstrated that the prevalence of overweight/obesity was lower in intervention children, post-intervention. Capacity building is defined as the development of knowledge, skills, commitment, structures, systems and leadership to enable effective health promotion and the aim of this study was to determine if the capacity of the Geelong community, represented by key stakeholder organisations, to support healthy eating and physical activity for young children was increased after Romp &amp; Chomp.MethodsA mixed methods evaluation with three data sources was utilised. 1) Document analysis comprised assessment of the documented formative and intervention activities against a capacity building framework (five domains: Partnerships, Leadership, Resource Allocation, Workforce Development, and Organisational Development); 2) Thematic analysis of key informant interviews (n = 16); and 3) the quantitative Community Capacity Index Survey.ResultsDocument analysis showed that the majority of the capacity building activities addressed the Partnerships, Resource Allocation and Organisational Development domains of capacity building, with a lack of activity in the Leadership and Workforce Development domains. The thematic analysis revealed the establishment of sustainable partnerships, use of specialist advice, and integration of activities into ongoing formal training for early childhood workers. Complex issues also emerged from the key informant interviews regarding the challenges of limited funding, high staff turnover, changing governance structures, lack of high level leadership and unclear communication strategies. The Community Capacity Index provided further evidence that the project implementation network achieved a moderate level of capacity.ConclusionsRomp &amp; Chomp increased the capacity of organisations, settings and services in the Geelong community to support healthy eating and physical activity for young children. Despite this success there are important learnings from this mixed methods evaluation that should inform current and future community-based public health and health promotion initiatives.Trial Registration Number: ANZCTRN12607000374460<br /

The effects of an area-based intervention on the uptake of maternal and child health assessments in Australia: A community trial

Background Recognition of the importance of the early years in determining health and educational attainment and promotion of the World Health Organization Health for All (HFA) principles has led to an international trend towards community-based initiatives to improve developmental outcomes among socio-economically disadvantaged children. In this study we examine whether, Best Start, an Australian area-based initiative to improve child health was effective in improving access to Maternal and Child Health (MCH) services. Methods The study compares access to information, parental confidence and annual 3.5 year Ages and Stages visiting rates before (2001/02) and after (2004/05) the introduction of Best Start. Access to information and parental confidence were measured in surveys of parents with 3 year old children. There were 1666 surveys in the first wave and 1838 surveys in the second wave. The analysis of visiting rates for the 3.5 year Ages and Stages visit included all eligible Victorian children. Best Start sites included 1,739 eligible children in 2001/02 and 1437 eligible children in 2004/05. The comparable figures in the rest of the state were and 45, 497 and 45, 953 respectively. Results There was a significant increase in attendance at the 3.5 year Ages and Stages visit in 2004/05 compared to 2001/02 in all areas. However the increase in attendance was significantly greater at Best Start sites than the rest of the state. Access to information and parental confidence improved over the course of the intervention in Best Start sites with MCH projects compared to other Best Start sites. Conclusion These results suggest that community-based initiatives in disadvantaged areas may improve parents' access to child health information, improve their confidence and increase MCH service use. These outcomes suggest such programmes could potentially contribute to strategies to reduce child health inequalities

Quality of Original and Biosimilar Epoetin Products

# The Author(s) 2010. This article is published with open access at Springerlink.com Purpose To compare the quality of therapeutic erythropoietin (EPO) products, including two biosimilars, with respect to content, aggregation, isoform profile and potency. Methods Two original products, Eprex (epoetin alfa) and Dynepo (epoetin delta), and two biosimilar products, Binocrit (epoetin alfa) and Retacrit (epoetin zeta), were compared using (1) high performance size exclusion chromatography, (2) ELISA, (3) SDS-PAGE, (4) capillary zone electrophoresis and (5) in-vivo potency. Results Tested EPO products differed in content, isoform composition, and potency. Conclusion Of the tested products, the biosimilars have the same or even better quality as the originals. Especially, the potency of originals may significantly differ from the value on the label

Towards Heat-stable Oxytocin Formulations: Analysis of Degradation Kinetics and Identification of Degradation Products

Purpose. To investigate degradation kinetics of oxytocin as a function of temperature and pH, and identify the degradation products. Materials and Methods. Accelerated degradation of oxytocin formulated at pH 2.0, 4.5, 7.0 and 9.0 was performed at 40, 55, 70 and 80°C. Degradation rate constants were determined from RP-HPLC data. Formulations were characterized by HP-SEC, UV absorption and fluorescence spectroscopy. Degradation products were identified by ESI-MS/MS. Results. The loss of intact oxytocin in RP-HPLC was pH- and temperature-dependent and followed (pseudo) first order kinetics. Degradation was fastest at pH 9.0, followed by pH 7.0, pH 2.0 and pH 4.5. The Arrhenius equation proved suitable to describe the kinetics, with the highest activation energy (116.3 kJ/mol) being found for pH 4.5 formulations. At pH 2.0 deamidation of Gln 4, Asn 5, and Gly 9-NH2, as well as combinations thereof were found. At pH 4.5, 7.0 and 9.0, the formation of tri- and tetrasulfidecontaining oxytocin as well as different types of disulfide and dityrosine-linked dimers were found to occur. Beta-elimination and larger aggregates were also observed. At pH 9.0, mono-deamidation of Gln 4, Asn 5, and Gly 9-NH2 additionally occurred. Conclusions. Multiple degradation products of oxytocin have been identified unequivocally, including various deamidated species, intramolecular oligosulfides and covalent aggregates. The strongly pH dependent degradation can be described by the Arrhenius equation. KEY WORDS: aggregation; Arrhenius kinetics; degradation; mass spectrometry; oxytocin