Apart from on-target binding, antibodies also have other parts that need to be engineered before they develop into a drug. This is particularly tricky for the plethora of fusion antibodies that are non-natural. Here I jotted down some notes as I read around this topic.

In vitro assays

What it does: Degradation when stored in formulation buffer; impact potency and increase aggregation rate.
Check: Size in stressed environments e.g. elevated temperature, high and low pH.
Experiments: SDS-PAGE, CE-SDS, SEC-HPLC and LC-MS

SDS-PAGE (Sodium-Dodecyl Sulfate Polyacrylamide Gel Electrophoresis):

  • Samples are denatured with SDS, separated by mass on polyacrylamide gel matrix using an electric field.
  • If degradation is present: “smeared” band.

CE-SDS (Capillary electrophoresis):

  • Sample is mixed with SDS-gel buffer then electrophoresed through an SDS-gel filled capillary (injected into the capillary inlets using high voltage).
  • Quantitative detection near the distal (anodic end) of the capillary using UV absorbance detection

How it does: Post-translation modifications (PTM) of charged residues, e.g. deamidation, isomerisation, succinimide formation, oxidation, sialylation, N-terminal pyroflutamic acid, C-terminal Lys clipping.
What it does: During formulation and manufacturing processes: Asn deamidation, Asp isomerization, Met oxidation, and Lys glycation. Affect antibody’s biological activity and stability, pharmacokinetics (PK) and tissue distribution.
Recommendation: Avoid large charged patches in CDR. Important for purification and formulation platform fit.
Experiments: cIEF.

cIEF (Capillary isoelectric focusing):

  • Determine isoelectric point (pI) governed by the heterogeneity of charged species.
  • More basic pI links to increased tissue uptake and blood clearance.
  • Most therapeutics have pI >8.

What it does: Aggregation is an irreversible process. Determines long term stability (high temperature and low pH stress) during viral inactivation, manufacturing, storage (shelf life), patient administration and immunogenicity.
Experiments: SEC-MALS, DLS.

SEC-MALS (Size exclusion chromatography-Multiangle light scattering):

  • Proteins in solution are separated by molecular size on SEC.
  • UV detector and Refractive index concentration detector, then MALS.
  • Monomer, dimer and trimer peaks: mono-disperse. Larger aggregates are polydisperse.
  • +ve: SEC+MALS enables absolute molar mass measurement of the eluting species independently of retention time; MALS has high sensitivity to detect low-level large aggregates.
  • -ve: limited molecular weight separation range, potential adsorption of aggregates to the resin that lowers detection, dilution and shear force can affect the equilibrium of oligomers in solution.

DLS (Dynamic light scattering):

  • Analyse unseparated sample
  • Give overall size distribution (low intensity/high mass signal for monomer; vice versa for aggregates), sample polydispersity, hydrodynamic radius, average molecular weight.
  • Diffusion interaction parameter (kD) calculated by a linear fit of measured diffusion coefficients vs concentration.
  • +ve: DLS is compatible with most buffers, that makes it a suitable technique for examining buffer and pH effects on aggregation in high throughput.
  • -ve: lack of separation of monomer and oligomer species so poor resolution and only yield qualitative data.

What it does: Denaturation at high temperature (measure melting temperature Tm)

Recommendation: Tm 50 degrees Celsius

Experiments: DSF/DSC, DLS (surrogate)

Differential scanning fluorimetry (or calorimetry; DSF / DSC):

  • Thermal denaturation analysis of the whole IgG, complementary thermal stability binding assay
  • Assess retention of binding after incubating at a range of increasing temperatures, with a fluorescent dye that is highly fluorescent in a hydrophobic environment: As protein gradually unfolds upon increasing temperatures, more hydrophobic pockets become accessible for the dye and the signal increases until the protein is completely denatured. Melting temperature (Tm) = half maximal of fluorescence peak.
  • -ve: Require purified protein, at high concentration (>100mg), low throughput (1-2 hour per sample)
  • Plot: SYPRO Orange Fluorescence vs Temperature

Surrogate: DLS (Dynamic light scattering):

  • Mechanism as above, but against increasing temperature (not concentration)
  • Measure aggregation onset temperature (Tagg)
  • Low Tagg tends to have low thermostability and colloidal stability (i.e. less desirable)

What it does: Antibody aggregate or binds to “off-targets”

Experiments: SIC / CIC, SMAC, AC-SINS, CSI-BLI, ICC

SIC / CIC (Self-interaction chromatography / Cross-interaction chromatography):

  • Measures the retention time of an antibody flowing across a chromotography column conjugated with the same antibody (“self”) or polyclonal human serum antibodies (“cross”).
  • Long retion time means strong self-interaction, hence low solubility
  • -ve: low throughput (100+ antibodies)

SMAC (Standup monolayer adsorption chromatography):

  • SEC column-based
  • Resin: Hydrophobic standup monolayer with terminal hydrophilic group (mimic exterior of protein)

AC-SINS (Affinity-capture self-interaction nanoparticle spectroscopy):

  • Measure protein self-interaction by capturing mAb on the surface of a gold colloid that displays surface resonanceoscillations in frequency with visible light.
  • As the immobilised mAb self-interact, the colloids aggregate, changing the oscillation frequency to absorb at a longer wavelength.
  • Analyse the red-shifting of the maximum of absorption peak compared to buffers and mAb controls. Red-shifting and its strength is indicative of the self-interaction propensity of the tested mAb sample.

CSI-BLI (Clone self-interaction biolayer interferometry):

  • Calibrate with antibodies captured on a biosensor. Use lights to detect self-binding.
  • +ve: higher throughput

Context: Concentration of antibody in solution needs to be high (>100mg/mL; ~200mg/mL if intravitreous injection), but viscosity should be low so that high doses can be delivered through a small volume (1.0-1.5mL) into the subcutaneous space.

Measurement: Osmotic second virial coefficient (B22) and diffusion interaction parameter (kD).

How to measure: Solution viscosity (η) in rheometers, diffusion coefficients (kD) from DLS profiles, retention time in chromatographic column determined via hydrophobic interaction chromatography (HIC), standard monolayer adsorption chromatography (SMAC) or cross-interaction chromatography (CIC).

Impact: Poor solubility can be related to hydrophobicity and aggregation.

Recommendation: For subcutaneous delivery: 50-100mg/ml (for dosing volumes down to 0.5ml).

How to measure:

  • Direct measurement of solubility requires significant amounts of purified antibodies (rare at early candidate screen): centrifugal ultrafiltration, quantify protein concentration by UV spectrometry. However, the shear forces and pressure-induced stress can harm the antibodies.
  • Alternative: static solvent absorption concentrations (selective permeability backed by a pressure resistant absorbent that pulls solvent and salts through the membrane).
  • Non-specific protein-protein interaction assay can give some indication of relative solubility in ranking.
  • Measure: B22 by DLS and PEG (Polyethylene Glycol) / ammonium sulphate-based precipitation assays.

PEG (Polyethylene Glycol):

  • Measure midpoint at which half of the protein population was precipitated and was determined via the derivative of the precipitation curve.

Impact: Effects on physicochemical properties of antibodies: deamidation, methionine oxidation, cysteine pair shuffling, fragmentation, aggregation, altered antigen binding.

Experiments:

  • Serum samples from in vivo experiments. (Or, for early-stage screening, incubating antibodies in human serum.) Analysed for intact antigen binding properties by ELISA/flow cytometry and BVP (baculovirus particle); and for specific amino acid modifications, by MS. Binding PSR (poly-specificity reagent).
  • The serum effects on self-association and aggregation can be measured by AC-SINS.

How it does: Post-translational modification. Arises from switching between expression host cell lines or changes in culture conditions.

What it does: Non-glycosylated antibody (in Fc) shows: reduced Fc gamma receptor binding, lower thermostability, higher proteolytic susceptibility, aggregation, structural changes; undesired.

Impact: In V-region, glycosylation alters antigen binding properties: should avoid.

Experiments: CZE-LIF.

CZE-LIF:

  • Glycans are cleaved from the antibody, labelled with fluorophore, separated by CZE and identified from reference standards or MS.
  • More Tyrosine –> increase affinity without losing specificity
  • More Arginine –> lose specificity
  • More Serine (hydrophilic) –> will not lose specificity
  • Specificity: measure by evaluating their relative propensity to interact with milk proteins

Computational predictions

Based on the premise that: ΔΔG = ΔGMut–ΔGWT .

DeepDDG features:

  • Sequence-based: amino acid types (wild type, mutant, neighbours), PSSM, Fitness score from Multiple Sequence Alignment (MSA), Protein design probability
  • Structure-based: backbone dihedral angles (phi, psi, omega), secondary structures, solvent-accessible surface area, number of hydrogen bonds (backbone-backbone, backbone-side chain, side chain-side chain), distance and orientation between mutated residues and neighboring residues

Fraction of native contacts (Q-value) computed from high temperature (400K) MD simulations correlates with the melting temperature of sdAb.

Aggregation is irreversible but solubility is reversible process, but prediction tools tend to work on both.

To measure rates of aggregation: sequence composition, propensities e.g. hydrophobicity and secondary structure (β-strand tends to aggregate more than α-helix).
Other observations: Amount of positively charged residues on the surfaces (more +ve charge, less soluble), increased ratio of Lys:Arg. 
Tools: SOLart, SAP, DI, CamSol.

ML-based SOLart features:

  • Sequence-based: amino acid compositions, protein length
  • Structure-based: solubility-dependent statistical potentials, secondary structures, solvent-accessible surface area. 

Spatial aggregation propensity (SAP):

  • Quantify exposure of hydrophobic residues derived from a crystal structure/averaged over snapshots from MD simulations.
  • Better colloidal stability if point mutations are introduced to the predicted hydrophobic patches to become more hydrophilic.

Developability Index (DI):

  • SAP & net charge of target proteins.

CamSol:

  • Linear combination of physicochemical properties of amino acids. Score smoothed over a window of 7 residues.

AggScore:

  • Energetic contribution of each residue to hydrophobic and electrostatic surface patches
  • Asn deamidation probability features
  • Sequence-based: pentapeptide deamidation half-life, C-terminal flanking residue
  • Structure-based: Backbone and side chain dihedrals, Asn local secondary structure, Percent solvent accessibility, solvent-accessible surface area, side chain hydrogen bonds, nucleophilic C-N attack distance.

Further readings

  • https://www.drugtargetreview.com/article/32916/developability-assessment-of-therapeutic-antibodies
  • Jain, T. et al. (2017) Biophysical properties of the clinical-stage antibody landscape. Proceedings of the National Academy of Sciences. 114(5), 944-949.
  • Jarasch, A. et al. (2015) Developability assessment during the selection of novel therapeutic antibodies. Journal of Pharmaceutical Sciences. 104(6),1885-1898.