Glycoproteins represent the largest group of the growing number of biologically-derived medicines. The associated glycan structures and their distribution are known to have a large impact on pharmacokinetics. A modelling framework was developed to provide a link from the extracellular environment and its effect on intracellular metabolites to the distribution of glycans on the constant region of an antibody product. The main focus of this work is the mechanistic in silico reconstruction of the nucleotide sugar donor (NSD) metabolic network by means of 34 species mass balances and the saturation kinetics rates of the 60 metabolic reactions involved. NSDs are the co-substrates of the glycosylation process in the Golgi apparatus and their simulated dynamic intracellular concentration profiles were linked to an existing model describing the distribution of N-linked glycan structures of the antibody constant region. The modelling framework also describes the growth dynamics of the cell population by means of modified Monod kinetics. Simulation results match well to experimental data from a murine hybridoma cell line. The result is a modelling platform which is able to describe the product glycoform based on extracellular conditions. It represents a first step towards the in silico prediction of the glycoform of a biotherapeutic and provides a platform for the optimisation of bioprocess conditions with respect to product quality.

Protein pharmacokinetic modulation is becoming an important tool in the development of biotherapeutics. Proteins can be chemically or recombinantly modified to alter their half-lives and bioavailability to suit particular applications as well as improve side effect profiles. The most successful and clinically used approach to date is chemical conjugation with poly(ethylene glycol) polymers (PEGylation). Here, therapeutic protein half-life can be increased significantly while retaining biological function, reducing immunogenicity and cross-reaction. Naturally occurring alternatives to such synthetic polymers could have major advantages such as lower side effects due to biodegradability and metabolism. Polysialic acid (PSA) has been investigated as a pharmacokinetic modulatory biopolymer with many successful examples in preclinical and clinical development. Single-chain Fvs (scFvs) are a choice antibody format for human therapeutic antibody discovery. Because of their small size, they are rapidly eliminated from the circulation and often are rebuilt into larger proteins for drug development and a longer half-life. Here we show that chemical polysialylation can increase the half-life of an antiplacental alkaline (PLAP) and anticarcinoembryonic antigen (CEA) scFv (F1 and MFE-23, respectively) 3.4-4.9-fold, resulting in a 10.6-15.2-fold increase in blood exposure. Amine-directed coupling of the MFE-23 scFv reduced its immunoreactivity 20-fold which was resolved by site-specific polysialylation through an engineered C-terminal thiol residue. The site-specifically polysialylated MFE-23 scFv demonstrated up to 30-fold improved tumor uptake while displaying favorable tumor:normal tissue specificity. This suggests that engineering antibody fragments for site-specific polysialylation could be a useful approach to increase the half-life for a variety of therapeutic applications.

Chemical coupling of a variety of polymers to therapeutic proteins has been studied as a way of improving their pharmacokinetics and pharmacodynamics in vivo. Conjugates have been shown to possess greater stability, lower immunogenicity, and a longer blood circulation time due to the chemicophysical properties of these hydrophilic long chain molecules. Naturally occurring colominic acid (polysialic acid, PSA) has been investigated as an alternative to synthetic polymers such as poly(ethylene glycol) (PEG) due to its lower toxicity and natural metabolism. Antibodies and their fragments are a good example of the types of proteins which benefit from pharmacokinetic engineering. Here, we chemically attached differing amounts and differing lengths of short (11 kDa) and longer (22 kDa) chain colominic acid molecules to the antitumor monoclonal antibody H17E2 Fab fragment. Different coupling ratios and lengths were seen to alter the electrophoretic mobility of the Fab fragment but have a minor effect on the antibody immunoreactivity toward the placental alkaline phosphatase (PLAP) antigen. Polysialylation generally increased Fab fragment blood half-life resulting in higher tumor uptake in a KB human tumor xenograft mouse model. One H17E2 Fab-PSA conjugate had over a 5-fold increase in blood exposure and over a 3-fold higher tumor uptake with only a marginal decrease in tumor/blood selectivity ratio compared to the unconjugated Fab. This conjugate also had a blood bioavailability approaching that of a whole immunoglobulin.

The emergence of many small, antibody fragment-like mimics will drive the need for such technologies, and PEGylation is still the choice polymer due to its established use and track record. However, there will be a place for many alternative technologies if they can match the pharmacokinetics of PEG-conjugates and bring addition beneficial features such as easier production.