Analytics and Characterization

SysGene supports analytical testing and characterization of peptides, recombinant proteins and monoclonal antibodies. We tailor each analytical programme to the molecule, its development stage and the quality attributes relevant to its intended application.

Our approach combines chromatographic and electrophoretic analysis with mass spectrometry and peptide mapping to assess identity, purity and molecular integrity. Binding and activity assays provide insight into biological function, connecting the molecule’s structural characteristics with its performance.

Targeted impurity testing supports the assessment of purification effectiveness, including evaluation of host-cell proteins, residual DNA and endotoxin where relevant to the expression system and process.

Analytical findings inform upstream and downstream optimization, support comparisons between batches and help assess the impact of process changes on product quality. Through documented methods, clear reporting and analytical transfer support, we provide an evidence base for continued development, scale-up and technology transfer.

Analyst loading samples onto a chromatography system in a laboratory

Characterization toolkit

Methods by molecule class and structural level

Analytical methods and the parameters they report, across peptides, proteins and monoclonal antibodies.

Fewer than 40 amino acids

Peptides

MW ~0.5–5 kDa

Short linear or simple cyclic chains.
Lack tertiary folding; highly flexible in solution.

Analytical focus: Molecular weight and LC-MS purity.

More than 40 amino acids

Proteins

MW ~10–100+ kDa

Complex folded 3D structures (α-helices, β-sheets).
Susceptible to denaturation and aggregation.

Analytical focus: Secondary and tertiary folding, and activity.

Heterotetramer

Monoclonal antibodies

MW ~150 kDa

Y-shaped four-chain glycoprotein (two heavy, two light).
Complex post-translational modifications (PTMs).

Analytical focus: Glycosylation, charge variants and binding.

The US FDA defines a protein as any alpha amino acid polymer with a specific, defined sequence that is greater than 40 amino acids in size; a polymer of 40 amino acids or fewer is a peptide.

Primary structure and molecular characterization

Molecular mass, sequence, disulfide connectivity, post-translational modifications, identity and purity.

Test	Method	Parameters
Molecular mass	LC-MS / MALDI-TOF MS	Molecular weight, mass accuracy, mass variants
Amino-acid sequence	LC-MS/MS, MS/MS sequencing	Sequence coverage, sequence identity
Sequence confirmation	Peptide mapping–LC-MS/MS	Sequence coverage, peptide identity
Amino-acid composition	Amino Acid Analysis (AAA)	Amino-acid composition, molar ratio
N-terminal sequence	Edman sequencing / MS	N-terminal identity
C-terminal characterization	LC-MS/MS / enzymatic methods	C-terminal identity
Disulfide bond mapping	Reduced/non-reduced LC-MS/MS	Disulfide connectivity, free cysteines
PTM characterization	LC-MS/MS	Oxidation, deamidation, glycation, clipping
Identity	Intact mass + peptide mapping	Expected vs observed mass, sequence match
Purity / related substances	RP-HPLC / UPLC	% Purity, impurities, degradation products

Secondary structure characterization

Secondary-structure composition, structural fingerprint, thermal stability and stress-induced change.

Test	Method	Parameters
Secondary structure	Circular Dichroism (CD)	α-helix, β-sheet, random coil
Structural fingerprint	FTIR spectroscopy	Amide I/II bands, secondary-structure profile
Protein conformation	Raman spectroscopy	Structural/conformational fingerprint
Thermal stability	DSC	Tm
Structural change	CD / FTIR	Change in secondary structure
Refolding / unfolding	CD / DSC	Reversibility, unfolding profile
Stress-induced changes	CD / FTIR / DSC	Structural alteration after stress

Tertiary and higher-order structure characterization

Tertiary conformation, folding, aggregation, fragmentation, oligomeric state and particle size.

Test	Method	Parameters
Tertiary structure	Fluorescence spectroscopy	Intrinsic fluorescence, conformational changes
Higher-order structure	CD / DSC	Structural conformation
Protein folding	DSC / spectroscopic methods	Tm
Aggregation	SEC-HPLC / SEC-MALS	% Monomer, % HMW, % LMW
Fragmentation	SEC	% Fragments, molecular-size distribution
Oligomeric state	SEC-MALS	Molecular mass, oligomeric state
Particle size	DLS	Hydrodynamic diameter

Biological characterization: cell-based assays and SPR

Binding affinity and kinetics, potency, receptor activity and Fc-mediated function.

Test	Method	Parameters
Binding affinity	Surface Plasmon Resonance (SPR)	K_D
Binding kinetics	SPR	k_a, k_d
Binding specificity	SPR	Specific vs non-specific binding
Biological potency	Cell-based bioassay	EC₅₀ / IC₅₀, relative potency
Functional activity	Cell-based assay	Dose-response, activity
Receptor activation	Cell-based assay	EC₅₀, E_max, relative activity
Receptor inhibition	Cell-based assay	IC₅₀, inhibition %, potency
Cell proliferation / viability	Cell-based assay	% Viability, proliferation rate
Cytotoxic activity	Cell-based assay	EC₅₀ / IC₅₀, % cytotoxicity
Mechanism of action	Cell-based functional assay	Target-specific response
Fc-mediated function	Cell-based assay	ADCC / CDC activity, relative potency

Strategy

Characterization strategy: peptides vs proteins vs mAbs

How the analytical approach changes with the molecule class.

Characterization	Peptide	Protein	mAb
Primary structure	LC-MS/MS	Peptide mapping	Peptide mapping + LC-MS
Mass	LC-MS	LC-MS	Intact/subunit LC-MS
Secondary structure	CD / FTIR	CD / FTIR / DSC	CD / DSC
Tertiary structure	Limited	Fluorescence spectroscopy	Fluorescence spectroscopy
Purity	RP-HPLC	RP-HPLC / SEC	SEC
Size variants	HPLC / MS	SEC	SEC
Charge variants	IEX	IEX	cIEF
Glycosylation	If applicable	If applicable	HILIC / RP-HPLC / LC-MS
Aggregation	SEC	SEC / DLS	SEC-MALS
Binding	If applicable	SPR	SPR
Biological activity	Functional assay	Cell-based assay	Cell-based potency
Key parameters	Mass, purity, sequence	Structure, purity, stability	CQAs, PTMs, potency, binding

Discuss analytical support

Tell us what needs to be confirmed, and at what stage.

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