Characterization of the aggregation propensity of charge variants of recombinant human growth hormone.

Biopharmaceutical products are subject to in depth analysis to ensure and improve their safety and efficacy. As part of this effort the stability and aggregation mechanisms of the therapeutic protein is characterized over the whole life cycle. The stability and aggregation behavior of single charge variants present in biopharmaceuticals were hardly investigated. In this study we applied a previously established methodology to assess the charge variants of the drug substance (DS) of human growth hormone (hGH). We assessed the stability and aggregation propensity of an acidic variant which forms in DS at a larger extent during short time storage at elevated temperatures. We developed a semi-preparative method to separate and analyze the charge species. Thermal and colloidal stability of this variant was analyzed by light scattering methods and a stability testing in different buffer formulations. The acidic variant showed slightly attractive self-interaction at lower pH. Thermal stress did not result in increased aggregation propensity or decreased stability compared to the DS. Thus, the methodology enabled to assess the risk of a single protein variant within the DS of hGH. The approach can also be utilized for other protein drugs as previously shown for a monoclonal antibody.

Identification of monoclonal antibody variants involved in aggregate formation - Part 2: Hydrophobicity variants.

Monoclonal antibodies (mAbs) are valuable tools both in therapy and in diagnostic. Their tendency to aggregate is a serious concern. Since a mAb drug substance (DS) is composed of different variants, it is important for manufacturers to know the behavior and stability not only of the mAb as a whole, but also of the variants contained in the product. We present a method to separate hydrophobicity variants of a mAb and subsequently analyzed these variants for stability and aggregation propensity. We identified a potentially aggregation prone hydrophilic variant which is interrelated with another previously identified aggregation prone acidic charge variant. Additionally, we assessed the risk posed by the aggregation prone variant to the DS by spiking hydrophobicity variants into DS and did not observe an enhanced aggregation propensity. Thus we present an approach to separate, characterize and analyze the criticality of aggregation prone variants in protein DS which is a step forward to further assure drug safety.

Identification of monoclonal antibody variants involved in aggregate formation - Part 1: Charge variants.

Biopharmaceutical products contain conformational and chemical variants, that are typically well characterized regarding identity and activity. However, little is known about their self-interaction propensity and tendency to unfold, which are critical characteristics for drug stability and safety. This study aimed to separate and compare charge variants of a monoclonal antibody (mAb) and to identify aggregation prone species. We show a semi-preparative cation exchange method, that we developed to separate the individual acidic and basic variants from the naïve mAb. Additionally, we demonstrate, that the yield and purity of the fractionated charge species, extracted by that method, were sufficient for subsequent analysis of aggregate content, conformation stability and self-interaction. Our analysis revealed a differently behaving acidic variant and confirmed its increased aggregation propensity by molecular modeling. During a stability study, the potentially aggregation prone charge variant posed a limited risk to the drug substance (DS). We are the first to look at the stability of single charge variants of biopharmaceuticals, and thus present manufacturers and regulatory authorities with a method to enhance drug safety.