Considerations for the Use of Polysorbates in Biopharmaceuticals.

PURPOSE: Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations. METHODS: A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations. RESULTS: Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients. CONCLUSIONS: An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

Application of a high-throughput screening procedure with PEG-induced precipitation to compare relative protein solubility during formulation development with IgG1 monoclonal antibodies.

Protein solubility is a critical attribute in monoclonal antibody (mAb) formulation development as insolubility issues can negatively impact drug stability, activity, bioavailability, and immunogenicity. A high-throughput adaptation of an experimental method previously established in the literature to determine apparent protein solubility is described, where polyethylene glycol (PEG) is used to reduce protein solubility in a quantitatively definable manner. Utilizing an automated, high-throughput system, an immunoglobulin G (IgG)1 mAb in a variety of buffer conditions was exposed to increasing concentrations of PEG and the amount of protein remaining in solution was determined. Comparisons of PEG(midpt) values (the weight% PEG in solution required to decrease the protein concentration by 50%) to extrapolated values of apparent protein solubility (in the absence of PEG) were performed. The determination of PEG(midpt) by using sigmoidal curve fitting of the entire data set was shown to be the most precise and reproducible approach for use during high-throughput screening experiments. The high-throughput PEG methodology was then applied to the screening of different formulations to optimize relative protein solubility profiles (weight% PEG vs. protein concentration and their corresponding PEG(midpt) values) in terms of solution pH and buffer ions for both human and chimeric IgG1 mAbs. Other comparisons included evaluating relative solubility profiles of an IgG1 mAb produced from different cell lines (Chinese hamster ovary vs. murine) as well as for different IgG1 mAbs (produced from the same cell line) in a series of formulation buffers. Based on these comparisons, it was concluded that rapid, high-throughput determinations of relative protein solubility profiles can be used as a practical, experimental tool to compare mAb preparations and to rank order buffer and pH conditions during formulation development.

PEGylated bioactive molecules in biodegradable polymer microparticles.

Injectable peptide and oligonucleotide biotherapeutics offer great promise for treatment of serious chronic diseases but almost always need further formulation work to increase stability and circulation lifetimes. Covalent attachment of poly(ethylene glycol) (PEG) will increase circulation lifetimes up to a week or so and decrease degradation in favorable cases. Encapsulation in biodegradable polymer microparticles has been highly successful, mostly for peptides to provide sustained release up to several months after injection. Although products are on the market using these technologies, PEGylation and microparticle encapsulation each have drawbacks that prevent more widespread use. When they are combined, the limitations of one technology may be resolved by the other. Work in several laboratories on encapsulation of PEGylated bioactive molecules has revealed a synergy. Activity reduction and restricted circulation lifetimes for PEGylated bioactive agents is addressed by microencapsulation and using a lower PEG molecular weight. Chemical degradation, excessive burst release and limited drug content are typical problems for microparticles that are ameliorated by using PEGylated actives. The case for synergy between PEGylation and microencapsulation is illustrated in this review by work with several proteins and peptides including insulin, and the oligonucleotide therapeutic, pegaptanib.

PEGylated insulin in PLGA microparticles. In vivo and in vitro analysis.

A novel controlled release formulation has been developed with PEGylated human insulin encapsulated in PLGA microspheres that produces multi-day release in vivo. The insulin is specifically PEGylated at the amino terminus of the B chain with a relatively low molecular weight PEG (5000 Da). Insulin with this modification retains full biological activity, but has a limited serum half-life, making encapsulation necessary for sustained release beyond a few hours. PEGylated insulin can be co-dissolved with PLGA in methylene chloride and microspheres made by a single o/w emulsion process. Insulin conformation and biological activity are preserved after PEGylation and PLGA encapsulation. The monolithic microspheres have inherently low burst release, an important safety feature for an extended release injectable insulin product. In PBS at 37 degrees C, formulations with a drug content of approximately 14% show very low (< 1%) initial release of insulin over one day and near zero order drug release after a lag of 3-4 days. In animal studies, PEG-insulin microspheres administered subcutaneously as a single injection produced < 1% release of insulin in the first day but then lowered the serum glucose levels of diabetic rats to values < 200 mg/dL for approximately 9 days. When doses were given at 7-day intervals, steady state drug levels were achieved after only 2 doses. PEG-insulin PLGA microparticles show promise as a once-weekly dosed, sustained release basal insulin formulation.

Effects of PEG conjugation on insulin properties.

The goal of this research was to determine whether the site-specific attachment of poly(ethylene glycol) to insulin could enhance the physical and pharmacological properties of insulin without negatively affecting its biological activity or immunological properties. Electrophilically activated derivatives of low-molecular-weight monomethoxypoly(ethylene glycol) (mPEG) were chemically coupled to insulin via its amino groups at positions phenylalanine-B1 or lysine-B29, with an amide bond being formed between the polymer and protein. The site-specific attachment of mPEG to insulin did not substantially alter insulin's secondary/tertiary structure, self-association behavior, or potency in vivo. However, mPEG attachment did significantly enhance insulin's resistance to aggregation. In addition, the pegylation of insulin almost completely eliminates the resultant conjugate's immunogenicity, allergenicity, and antigenicity. Finally, the conjugates were observed to remain in the systemic circulation for longer periods of time than unmodified insulin after subcutaneous administration.