Quantitative Mass Spectrometry

Quantitation of API, impurities, HCPs and more

Quantitative Mass Spectrometry (MS)

The highly complex process of manufacturing biopharmaceuticals requires the use of both biological and chemical systems to successfully manufacture and purify the drug product. This inevitably means the final product will contain more than just the active pharmaceutical ingredient (API). Quantitative mass spectrometry provides a mechanism for assessing the amount of a particular component (e.g. the Active Pharmaceutical Ingredient, an impurity or a contaminant) in a sample.

An assessment of the process related impurities that are present in the drug product is required and both a qualitative and quantitative assessment of the components in the final drug product must be performed. This information is also vital for development of appropriate production and purification strategies.

Regulatory Guidance

According to US and European regulatory guidelines (e.g. the FDA’s Development of therapeutic protein biosimilars: Comparative analytical assessment and other quality-related considerations. Guidance for Industry published May 2019 and the EMA’s Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: quality issues (revision 1) published Dec 2014), the impurity profiles of a product must be investigated. Appropriate control strategies are critical to ensure process performance and product quality. Product and process testing across early manufacturing steps will help to ensure final product quality and consistency.

For this reason a qualitative and quantitative assessment of the components in the final drug product must be performed. This information is also vital for development of appropriate production and purification strategies, with information from quantitative MS based analyses helping shape data-led decisions.

In order to make an assessment of the amount of any particular component present in a sample, quantitative mass spectrometry investigates the degree of response of specific components identified in the sample against the response of standards. This type of analysis can be performed using different types of mass spectrometers and with different analytical approaches, depending on the precise requirement and nature of the component under investigation. Some examples of the application of quantitative mass spectrometry as described below.

Methods and Instrumentation

BioPharmaSpec uses gas chromatography and liquid chromatography approaches for quantitative MS, depending on the nature of the analysis being undertaken. Mass spectrometric instrumentation covers single quadrupole, quadrupole time-of-flight and triple quadrupole machines. Chemical derivatization strategies are used where necessary in impurity or residual analysis and heavy (cold) labelled peptides may be generated for identification and quantitation of specific peptides in Host Cell Protein (HCP) work. Drug conjugated peptides may be prepared from Antibody Drug Conjugates (ADCs).

Applications of Quantitative Mass Spectrometry

Measurement of Active Pharmaceutical Ingredient (API)

Marker peptides from the API can be generated and monitored quantitatively. This can be used to measure drug clearance (i.e. investigate drug half-life) or accumulation of the API in biological materials such as plasma.

Monitoring Impurities and Contaminants

The use of a wide range of chemicals during protein production, selection and subsequent purification, in addition to any chemical modifications that may be made to the naturally produced biologic, such as PEGylation, results in a large number of chemicals coming into contact with a drug product.

All of these extraneous chemicals can end up in small amounts in the final product (despite the significant purification strategies applied during downstream processing) and may be capable of generating a biological response such as an immune reaction or other harmful event in a patient, therefore putting them at risk. The product itself may also be susceptible to modification resulting in a decreased shelf life, increase in aggregation or other adverse effects, leading to a loss of efficacy.

As part of our Process Related Impurities assessments, BioPharmaSpec scientists can develop both qualitative and quantitative analytical strategies for residual chemicals of interest in your sample.

Host Cell Protein (HCP) Assessments

BioPharmaSpec’s approach to the assessment of Host Cell Proteins using mass spectrometry employs a phased strategy with two methods of quantitation.

  1. Relative Quantitation

The early phase quantitative analysis is performed using either Waters or SCIEX quadrupole time-of-flight instrumentation, both of which offer high resolution and mass-accuracy when coupled with direct on-line liquid chromatography.

Although the precision of the quantitation method allows for confident comparison of the relative protein values, the differences in peptide ionization efficiency in these complex sample mixtures will influence the peptide intensities and will vary between proteins. More accurate quantitation of key HCPs of interest can be achieved through the use of absolute quantitation methods.

  1. Absolute Quantitation

Absolute quantitation is performed using carefully selected heavy (cold) labeled peptides on triple quadrupole mass spectrometers (Waters TQS). Triple-quadrupole mass spectrometers are ideal instruments for absolute quantitative analysis because they have a large linear response range and high sensitivity and selectivity.  Multiple Reaction Monitoring transitions (MRM) transitions are assessed using this instrumentation.

Characterization of Antibody Drug Conjugates (ADCs)

BioPharmaSpec is highly experienced in providing Drug Antibody Ratios (DARs) for ADCs in a variety of matrices to support PK studies and in vitro stability studies.

Need a quantitative MS method?

Our scientists can develop quantitative mass spectrometry methods for a wide range of uses, such as impurity analysis and detection of host cell proteins. Contact our experts today to find out more. CONTACT US TODAY