How to Choose the Right Analytical Methods

In this article, we will focus on biologics and analytical methods development. Analytical method development is an important component of biologics development and the control of quality attributes that affect product performance and stability is critical to success. Testing performed to release the bulk drug substance or drug product for clinical use should include the following five quality attributes:

• Identity
• Purity
• Potency
• Strength
• Safety

For monoclonal antibodies (mAbs), a set of platform methods are often used—physicochemical methods that are applied to recombinant glycoproteins generally and to mAbs as a class. The platform methods are provided by the CDMO. Additionally, methods measuring biological activity are product specific and must be provided by the sponsor. Taken together, the platform physicochemical and specific biologic activity methods, along with a set of USP/Ph. Eur. general and safety tests, make up the analysis that is reported in the certificate of analysis (CofA).

Biologics Release Testing Measures

Release testing will always include tests measuring one of the five attributes:

Identity

• pI and charge heterogeneity
  Capillary isoelectric focusing (cIEF) or cation exchange (CEX)-HPLC is often used as a release test to confirm identity and assess charge heterogeneity. Because the net charge of a mAb derives from its amino acid sequence and post-translational modifications, these methods can be used to characterize the pattern and cIEF can confirm pIs of charged isoforms that are unique to each mAb product.
• Peptide mapping
  In addition to confirming pI, identity can be established by peptide fingerprinting. In this technique, the IgG molecule is reduced and alkylated and an enzyme cleaves the amino acid chains into short peptides, which are separated using HPLC. The pattern forms a unique “peptide map” for each IgG molecule.

Purity

• Monomer size and aggregation
  These attributes are characterized for release testing and analysis of stability. Size analysis tests include sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis-SDS (CE-SDS) and size exclusion chormoatography (SEC). SDS-PAGE and CE-SDS are denaturing techniques, and the latter is more common because it is quantitative. SEC is a native technique and the principal method for soluble aggregate quantitation. The presence and increase of product aggregates during storage is a main purity concern for the FDA.
• Fragments/IgG4 half-molecules
  Fragments of any subclass of IgG can result from proteolysis or chemical degradation. Due to structural differences in the hinge region, human IgG4 tends to form half molecules both in vitro and in vivo. This can be problematic for drug product stability, so an engineered version (S228P) of the IgG4 heavy chain is typically used for IgG4 therapeutics. To monitor the formation of fragments of any mAb subclass or half-molecules of IgG4, you can use CE-SDS, non-reducing SDS-PAGE and SEC. We recommend including CE-SDS analysis of the reduced mAb (IgG1–2) as a release test to assess formation of product fragments as a purity method.

Potency

• For mAbs, potency can be measured in two fundamental ways. The first is binding to antigen in a plate-based method such as enzyme-linked immunosorbent assay (ELISA). This type of assay demonstrates that the mAb is biologically active insofar as its ability to bind to its target antigen is intact, but it provides no information about whether effector function of the mAb is active. For this, a plate-based method for Fc receptor binding can be used.
  • Alternatively, a cell-based method may be needed—one that couples binding of antigen with effector function to cause, for example, a target cell to be killed, completing the mechanism of action (MoA). Cell-based potency methods can be done in vivo (in an animal) or in vitro (using cells in culture). The purpose of the test is to correlate the chosen bioassay with the clinical response.
  • As noted, the choice of a potency method depends on understanding the product’s MoA. If the MoA cannot be determined completely [e.g., antibody dependent cell-mediated cytotoxicity (ADCC) vs. antibody dependent cell-mediated phagocytosis (ADCP)], additional potency assays should be used.

Strength

• Protein quantitation
  Protein concentration is characterized for release testing by measuring light absorption. How strongly a protein absorbs light at a particular wavelength is an intrinsic property of the protein based on its chemical composition.
  Proteins that contain the aromatic amino acids tyrosine and tryptophan absorb ultraviolet light at 280 nm. Based on its unique absorbance at 280 nm, the proteins are assigned a molar extinction coefficient which determines the concentration of drug in solution following measurement of absorbance at 280 nm. More recently, variable pathlength slope spectroscopy has been used to determine concentration without sample predilution—particularly useful for high concentration protein formulations.

Safety

• These are compendial methods that ensure the product is free of bacterial endotoxin, bioburden and visible or sub-visible particulates.
  • In designing assay methods for release of pharmaceutical products, the assays must pass several tests, including linearity, sensitivity, accuracy, precision, selectivity and robustness. This confirmation process is referred to as “qualification,” and must be done before methods are used to support cGMP production.
  • In addition to tests used for release and stability, other methods are used to characterize the reference standard of the mAb product:
• Molecular weight (MW)
  Analysis of molecular weight is performed for characterization. Confirmation of the predicted molecular weight of the expressed glycoprotein product ensures fidelity of the recombinant protein expression system to produce the intended molecule. Given its excellence in determining mass accurately, ESI-MS is the method of choice here. For glycoproteins and mAbs, glycoform variation may result in multiple species with different MWs, so the expected MW of the non-glycosylated variant can be predicted based on its amino acid sequence and confirmed by reduction and deglycosylation of the glycoprotein.
• Free sulfhydryl groups
  Analysis of free sulfhydryl groups is performed for characterization to ensure correct folding of the protein therapeutic. The 3D structure of glycoproteins is often stabilized by disulfide bonds. The location of sulfhydryls can be predicted by combining amino acid sequence with domain folding patterns and is an indication of proper folding. Ellman’s Reagent—5,5 ́dithiobis (2-nitrobenzoic acid) is recommended.
• Heavy chain glycosylation
  Glycosylation is typically determined for characterization, but it is also monitored for release when it affects the potency of the product. For a mAb, glycosylation may be critical if the effector function [ADCC, ADCP, complement-dependent cytotoxicity (CDC)] has a role in its mechanism of action. There is no preferred test method for this attribute— when qualitative and quantitative lot-to-lot testing of carbohydrates is needed, monosaccharide compositional analysis is advised.
• C- and N-terminal heavy chain heterogeneity
  This attribute is measured for characterization. Invariably, human heavy chains contain a lysine residue at the C-terminus. The residue is often clipped off during biosynthesis, creating the potential for charge heterogeneity of the assembled oligomer. The charge heterogeneity caused by C-terminal Lys and other potential charge variations (deamidation, oxidation) is monitored using CEX-HPLC.
• Disulfide structure
  Disulfide structure is analyzed to characterize the protein and is not required for release. This deeper analysis creates a linkage map of cysteines in the protein sequence. Structure should be determined during process development.

As part of biologics development, analytical methods are used in process monitoring, characterization and release & stability of bulk drug substance and drug product. Careful selection and planning of these methods is key to the overall product development cycle.