Therapeutic monoclonal antibodies (mAbs) harness the highly evolved specificity of adaptive immunity to fight disease. mAb-based therapeutics have grown exponentially with the advent of mammalian cell culture, process, and formulation technology. At the same time, state-of-the-art and emerging analytical and biophysical methodology provides very detailed process and product information. Although such a battery of methodology and wealth of information is critical to product understanding, the accuracy, precision, robustness, and suitability of such techniques are also of critical importance. Performance specifications have previously been set on a product-specific basis and continued suitability verified with trending and comparability to in-house product-specific reference standards. This mechanism is likely irreplaceable due to the highly individual yet heterogeneous nature of mAb therapeutics. However, a representative and widely available material, coupled with detailed historical data, would greatly supplement characterization efforts throughout the drug product lifecycle. To this end, a first-of-its kind qualitative and quantitative biopharmaceutical reference material to supplement drug substance/product characterization is described. The NISTmAb IgG1κ is intended to provide a well-characterized, longitudinally available test material that is expected to greatly facilitate development of originator and follow-on biologics for the foreseeable future.

The primary structure of a protein, including therapeutic monoclonal antibodies (mAbs), is a critical quality attribute that determines a great deal of its structure, function, and stability. Significant effort is devoted to determining the complete amino acid sequence of recombinant proteins of potential therapeutic benefit. The focus of this chapter is to demonstrate current, state-of-the-art, mass spectrometry-based primary structure confirmation using the recombinant human IgG1ҝ NISTmAb as a representative example. A combination of intact mass analysis, top-down sequencing, IdeS fragment mass analysis, IdeS fragment (or “middle-down”) sequencing, and peptide mapping are discussed with respect to their orthogonality and limitations. A historical review of the methods employed to determine the primary structure of proteins is also included for completeness.

Therapeutic monoclonal antibodies (mAbs) harness the highly evolved specificity of adaptive immunity to fight disease. mAb-based therapeutics have grown exponentially with the advent of mammalian cell culture, process, and formulation technology. At the same time, state-of-the-art and emerging analytical and biophysical methodology provides very detailed process and product information. Although such a battery of methodology and wealth of information is critical to product understanding, the accuracy, precision, robustness, and suitability of such techniques are also of critical importance. Performance specifications have previously been set on a product-specific basis and continued suitability verified with trending and comparability to in-house product-specific reference standards. This mechanism is likely irreplaceable due to the highly individual yet heterogeneous nature of mAb therapeutics. However, a representative and widely available material, coupled with detailed historical data, would greatly supplement characterization efforts throughout the drug product lifecycle. To this end, a first-of-its kind qualitative and quantitative biopharmaceutical reference material to supplement drug substance/product characterization is described. The NISTmAb IgG1κ is intended to provide a well-characterized, longitudinally available test material that is expected to greatly facilitate development of originator and follow-on biologics for the foreseeable future.