Process-Related Residual & Product-Related Impurity Testing of Biologics

In the course of product manufacturing, a number of chemicals are typically added upstream as part of the bioprocess to improve on product expression and recovery. These chemicals are almost entirely removed during the downstream purification process; however, residual levels of these agents may be left within the bulk drug substance and drug product. To ensure that the level of these agents is within safe amounts, highly sensitive analytical methods need to be used for detection and quantification of such residuals.

Along with process residuals, there is also the possibility for product-related impurities to be carried through the manufacturing and purification process. These impurities, including host cell proteins, host cell DNA, N-terminal truncations and other potential modifications, may cause adverse reactions in animals and humans, and therefore, assays must be employed to ensure that any impurities present are below pre-determined acceptable levels.

  • PROCESS-RELATED RESIDUALS
    Residual Method Description LOQ (ppm)1 LOD (ppm)1
    IPTG HPLC with ELSD/CAD 6 < 1
    PEI HPLC with ELSD/Fluorescence 10/< 2 1-2/< 0.5
    Kanamycin HPLC with UV/Vis 0.03 < 0.01
    Anti-foam HPLC with ELSD/CAD 1 0.1
    TFA/Acetate HPLC with UV/Vis 1 0.1
    Cyanate HPLC with Conductivity/ECD 0.6 0.1
    Imidazole HPLC with Fluorescence 0.13 < 0.1
    Triton X-100 HPLC with ELSD/CAD 5 1
    Polysorbate (tween) 20/80 HPLC with ELSD/CAD 5 1
    Zwittergent HPLC with ELSD/CAD 1-5 < 1
    Pluronic HPLC with ELSD/CAD 1-5 < 1
    Protein A ELISA 0.001-0.10 < 0.001
    PEG HPLC with ELSD/CAD 1-10 < 0.5
    TRIS HPLC 1-10 < 0.5
    Growth Factors (e.g., insulin, interferon) ELISA 0.001-0.10 < 0.001
  • PRODUCT-RELATED IMPURITIES
    Impurity Method Description LOQ (ppm) LOD (ppm)
    Host cell proteins (HCP) Commercial kit or customizedassay 0.001-0.010 < 0.001
    N-terminal truncations N-terminal sequencing > 10%  
    Host cell DNA PCR Method- and product-dependent
    Oxidation/deamidation (PTMs) Peptide mapping LC-MS or LC-MS/MS, IsoQuant HPLC Method- and product-dependent
    Isoform analysis Ion exchange HPLC, cIEF,sialic acid analysis Method- and product-dependent
    Disulfide bridge scrambling(mis-folding); conformational impurities Peptide mapping LC-MS or LC-MS/MS, Ellman’s free sulfhydryl, intrinsic tryptophanfluorescence, ANS dye binding,NMR Method- and product-dependent
    C-terminal integrity Peptide mapping LC-MS or LC-MS/MS Method- and product-dependent
    Aggregation Analytical ultracentrifugation(AUC), SEC/SEC-MALLS,dynamic light scattering (DLS) Method- and product-dependent