Qualitative and quantitative analyses of process-related impurities are critical to manufacturing a safe, high-quality drug product. During his 17 June 2020 “Ask the Expert†webcast, Steven Broome (BioPharmaSpec) described using mass spectrometry (MS) for such analyses. Focusing on isopropyl-β-D-thiogalactopyranoside (IPTG), kanamycin, and host-cell proteins (HCPs), Broome overviewed best practices for using MS to detect and quantitate low levels of impurities.
Broome’s Presentation
Chemicals used in biotherapeutic production can be present as impurities in the resulting drug product. These impurities can include inducers, antibiotics, chemical-reaction products, HCPs, and host-cell DNA. The presence of process-related impurities can raise immunogenicity concerns and diminish product potency, efficacy, and longevity. Thus, sponsors should identify impurities and estimate their concentrations early in drug development. Known impurities can be monitored during processing to demonstrate clearance and optimize purification processes.
The high sensitivity and selectivity of MS instruments enable confident identification of process-related impurities. MS relies on detection of molecular ions or specific fragment ions at expected chromatographic retention times. The technique can call out previously unidentified substances in a drug product, and signal-intensity measurements can estimate concentrations of identified impurities.
Case Studies: BioPharmaSpec uses gas chromatography (GC)-MS to detect the inducer IPTG. During GC-MS, sample components are eluted from a column into a mass spectrometer, which fragments those components and measures the resulting fragment ions. IPTG produces a clear fragment ion at m/z: 204.
IPTG is identified in a sample if the known fragment ion signal is detected at the expected retention time, which is determined by first running an IPTG standard. Once identified, a sample’s absolute concentration of IPTG is ascertained by comparing the IPTG peak area of the sample with and without a known concentration of IPTG spiked in. BioPharmaSpec spikes all samples with the same amount of an internal standard (e.g., arabitol) to ensure consistency across assays and account for run-to-run variability.
Kanamycin assays follow a similar process: Analysts spike an aliquot with a known quantity of the antibiotic and then compare its signal response with one gathered from an unspiked sample. Tobramycin internal standards are spiked in all samples to account for run-to-run variability. Analysts use multiple-reaction monitoring (MRM) techniques to improve sensitivity and selectively detect the molecular ions of interest and their key fragment ions.
MRM involves selection of a signal of interest (e.g., m/z: 653.3 for kanamycin) followed by fragmentation of that ion and selective detection of the key fragment ion produced at
m/z: 247.1. High sensitivity allows for detection of low levels of impurities.
HCPs raise unique analytical challenges. A wide range of HCPs can be present in a drug product after purification. It is important to know what the HCPs are both qualitatively and quantitatively because they could have an adverse effect on a patient or product. BioPharmaSpec uses a phased approach that comprises HCP discovery, identification, relative quantitation, and targeted quantitation. This final step requires synthesized, heavy-labeled peptide analogs as standards and MRM to determine an accurate absolute concentration for a few selected HCPs.
MS should play a major role in assessment of process-related impurities. Its versatility, sensitivity, and selectivity enable detection of a wide range of impurities in many sample types. Knowing the types and amounts of impurities in a sample leads to improved manufacturing processes and an improved product.
Questions and Answers
What kinds of product defects derive from process-related impurities? Consider HCPs with protease functions. Such impurities can truncate a protein and diminish its efficacy. Other common problems include deamidation, oxidation, aggregation, and disulfide-bridge scrambling. Product characterization testing (e.g., MS assays for peptide mapping and intact-mass analysis) can determine whether process-related impurities have modified a drug product.
Can residual-analysis methods be used for batch-release testing? This strategy is possible but requires significant method development. BioPharmaSpec can help drug sponsors devise appropriate methods and then perform technology transfer to laboratories with good manufacturing or laboratory practice (GMP, GLP) certification.
More Online
The full presentation of this webcast can be found on the BioProcess International website at the link below.