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Cell Line Development Interlot & Biosimilar Comparability Lot Release & Stability Process & Product Impurities Antibody Drug Conjugates

Assay Packages: Process and Product Impurities

Many different impurities are present in or generated during the mAb manufacturing process. We work to make sure your biotherapy’s development program isn’t derailed by process or product contaminates that can arise from raw input materials, occur as residual processing agents, or formed as reaction by-products. We have years of experience with routine to high complexity assays for both R&D and GMP purposes to support data requirements at every phase of the development cycle. With state-of-the-art instrumentation and fast turnaround times, we provide you reliable results to push your project forward.

Ideal during:
Phase I
Phase II
Phase III

Process impurity types that need to be assessed include:

Host-cell contaminants

During mAb manufacturing, a significant proportion of process impurities arise from the host cell used to express the drug. Detection of these species requires highly specific techniques to accurately quantify even low levels of contamination.

Host cell protein (HCP) impurities, present at PPM-levels in biotherapies, are a major immunogenicity risk because they can elicit an unpredictable immune response in patients. Their complex and diverse nature makes them challenging to detect or monitor.

With acceptance criteria for host residual DNA usually set at a very low level (often ≤1.0 pg of DNA per mg of drug substance), effective removal techniques and sensitive methods of detection are critical.

Manufacturing additives

A wide variety of manufacturing additives must be monitored during mAb development and manufacturing;

  • Detergents
  • Protein A
  • Transfection reagents, including polyethylenimine (PEI)
  • Antibiotics
  • Anti-foam agents
  • Growth factors

Microbial battery

Many mAbs are produced in microbial organisms to benefit from rapid growth rates and high yields, making it important to monitor and control the presence of microbial contaminants. A component of the cell wall of Gram-negative bacteria, called endotoxin, produces responses ranging from fever and chills to fatal septic shock, making its removal from your mAb critical and a regulatory requirement.

Used to monitor the presence of potentially harmful microbial contamination, bioburden testing should be employed throughout the entire mAb manufacturing process. Sterility testing is also important to inform the integrity of mAb production.

Assay families that satisfy testing needs include:

  • Process contaminants
    • Host cell protein and DNA
    • Process additives

Product impurity assay families that satisfy testing needs include:

  • Charge distribution

    Occurring as a result of post-translational modifications (PTMs) during the manufacturing process, or due to chemical modifications throughout purification and storage, charge variants can have a considerable impact on the biological activity and pharmacokinetics of a mAb. A regulatory requirement for mAbs, charge variant analysis can be evaluated by techniques including cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF).

  • Size distribution

    While a single mAb product is desired, the initial production material often contains size variants such as aggregates, fragments, and biomolecules exhibiting additional light chains. Since these have the potential to affect immunogenicity and potency, it is important to monitor their presence. Size exclusion chromatography (SEC) is the most commonly used method to evaluate size distribution, while techniques such as dynamic light scattering (DLS) and capillary electrophoresis sodium dodecyl sulfate gel (CE-SDS) are also highly informative.

  • Molecular weight analysis

    Molecular weight is a major indicator of mAb identity, influenced by the nature of the light and heavy chains as well as by PTMs. Typically evaluated by intact mass (IM) analysis, mAb molecular weight can also be investigated by deglycosylating the heavy chains to remove one of the major sources of protein heterogeneity.

  • Sequence mapping

    A widely used sequence mapping technique to determine mAb identity, peptide mapping ranges from the generation of single enzyme peptide maps to more complex analysis such as data-dependent acquisition (DDA) of collisionally activated dissociation (CAD) methods. This information can be enhanced using N- or C-terminal sequencing, or ion mobility spectrometry.

  • Modification analysis

    Understanding and controlling any modifications that occur during the manufacturing process help generate a consistent mAb product. For example, N-glycan mapping can inform mAb stability, bioactivity and immunogenicity, while sialic acid quantification is key to preventing excess sialylation. It is also wise to perform disulfide mapping since manufacturing-induced disulfide reduction is known to result in poor yields during mAb harvest.

  • Physical attributes testing

    Many different physical properties of a mAb require testing during manufacturing. These include pH, concentration, and osmolality, all of which are features essential to the manufacture of a consistent drug product.

Assay families that satisfy testing needs include:

  • Product contaminants
    • Size variants
    • Charge variants
    • Microbiology
      • Viable
      • Non-viable

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