Formulation development of antibody-drug conjugates.

Formulation development of an ADC resembles that of a conventional antibody, but the conjugated form introduces new molecular attributes such as drug-to-antibody ratio and stability of the drug itself that need to be considered. An extended set of analytical tools, coupled with understanding of how ADCs and conventional antibodies differ in terms of their stability, guides formulation selection.

Physicochemical stability of the antibody-drug conjugate Trastuzumab-DM1: changes due to modification and conjugation processes.

In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1.

On developing a process for conducting extractable-leachable assessment of components used for storage of biopharmaceuticals.

Extractables and leachables are product-related impurities that result from product contact with components such as gaskets, stoppers, storage bags, cartridges, and prefilled syringes that are used for processing, storage, and/or delivery of biopharmaceuticals. These impurities are a concern for patients due to potential effects on product quality and safety. It is possible that such an impurity could directly impact the patient or indirectly impact the patient by interacting with the protein therapeutics and forming protein adducts.

Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies.

The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE.

The effect of cosolutes on the isomerization of aspartic acid residues and conformational stability in a monoclonal antibody.

The aspartate residue (Asp 32) located in the complementarity-determining region (CDR) of a recombinant humanized monoclonal antibody (MAb I) is highly susceptible to the isomerization reaction. The modification of Asp 32 residue due to the isomerization reaction results in a significant reduction in the binding affinity of MAb I to IgE. The binding of a MAb I therapeutic to IgE is important for its desired pharmacological effect.

Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization.

The asparagine (Asn) deamidation and aspartate (Asp) isomerization reactions are nonenzymatic intra-molecular reactions occurring in peptides and proteins that are a source of major stability concern in the formulation of these biomolecules. The mechanisms for the deamidation and isomerization reactions are similar since they both proceed through an intra-molecular cyclic imide (Asu) intermediate. The formation of the Asu intermediate, which involves the attack by nitrogen of the peptide backbone on the carbonyl carbon of the Asn or the Asp side chain, is the rate-limiting step in both the deamidation and the isomerization reactions at physiological pH.