Quality Control (QC) is a process to ensure that a produced product or performed service adheres to a defined set of quality criteria or meets customer needs. Quality control of biological and biosimilar drugs includes various tests, some of which are summarized below:

Routine Quality Control Tests

Identification test (IEF, IEX-HPLC, ELISA, peptide mapping, glycosylation profile ciIEF)

Appearance (color, clarity)

Assay (ELISA, SEC-HPLC, RP-HPLC) and dosage uniformity

Purity (CE-SDS – reduced and non-reduced, SEC-HPLC, RP-HPLC)

Protein Content (UV, 280nm, Bradford, BCA, Lowry)

Dosage of excipients (HPLC)

Physical determinations (pH, osmolality)

Moisture (Karl Fisher)

Residual solvents (volatile organic compounds, organic volatile impurities)

Impurities identification (HPLC/UHPLC, LC-MS, GC-MS)

Process-related Impurities determination (ELISA, RT-qPCR)

Elemental impurities (AAS, ICP/MS)

Nitrosamine impurities (LC-MS-MS, HS-GC-MS)

Particulate matter (visible and subvisible particles)

Extractables & Leachables

Enzymatic and chromogenic tests (heparins and others)

Determination of enzyme activity

Biological potency assays (CBA, ELISA, flow cytometry)

Extractable volume

Physical properties of injection devices (glide and brake force for syringes)

Other CMC tests with specific departments

Characterization of New Biological Products

The characteristics of a biotechnological or biological product, based on ICH Q6B guidelines, include determining its physicochemical properties, biological activity, immunochemical properties, purity, and impurities using appropriate techniques to ensure safety, quality, and efficacy.

Comparability Studies

3-1-General Analytical Techniques for Biosimilars

These tests are used to assess the quality attributes of the biosimilar molecule in the early development stages, such as:

Electrophoresis (PAGE, SDS-PAGE, Bioanalyzer, IEF)

Western Blot

Extinction Coefficient, Bradford, Lowry, BCA (total protein quantification)

HPLC AccQ-Tag® (Waters) and OPA® (Agilent) for amino-acid analysis

Sulfide quantitation by Ellman method

Antibody isotyping

3-2-Structural Analysis of the Biosimilar

These tests are used to fully identify the biosimilar molecule for strain selection, bioprocess, and final product attributes:

Intact protein mass (Electrospray MS, MALDI-TOF)

Peptide mapping, amino acid sequencing (LC-UV-MS/MS, QTof, QTRAP)

N and C terminal sequencing of intact protein (MALDI-TOF and Edman degradation)

Glycosylation and phosphorylation sites (LC-UV-MS)

Glycosylation Profiles (GC, LC, MS/MS, capillary electrophoresis)

Monosaccharide profile (LC and GC)

3-3- Conformational Analysis of the Biosimilar

Protein folding and its 3D structure determine the biological activity and immunogenicity of the biosimilar. Related tests include:

Circular Dichroism (CD)

Ultraviolet spectroscopy (UV)

Fluorescence spectroscopy (FL)

Infrared spectroscopy (FTIR)

Identity, Content, Protein Impurities

Chromatographic patterns are essentially quality indicators of the purification process:

Liquid chromatographic patterns (RP, SEC, Ion Exchange, and Affinity-HPLC/UPLC plus LC-UV-MS)

Capillary electrophoresis (reduced and non-reduced)

Imaged capillary isoelectric focusing (icIEF)

ELISA, ECLA, RIA

Process-Related Impurities

These tests are quality indicators related to the purification process and are associated with safety concerns of the product:

Host Cell Proteins (specific ELISA)

Chemical contaminants (HPLC, GC)

Elemental impurities (AAS, ICP-MS)

Nitrosamine impurities (LC-MS/MS, HS-GC-MS)

DNA (qPCR and colorimetric commercial kits)

Mycoplasma (PCR commercial kits)

Endotoxins (colorimetric or gel-clot test)

Bioburden

Biological Activity of the Biosimilar

These tests confirm the biological activity of biosimilars:

Binding studies (ELISA, ECLA, RIA, SPR Biacore®)

Potency assays (Cell-based assays)

Competitive inhibition ELISA assays for vaccines