How to document the removal of process-related impurities

There is a terrific method for residual protein impurity detection, with detection limits down to low ppm levels. It can track and quantify the residual proteins through all purification steps and eventually the final biopharmaceutical product.

Before going into details with the method, let’s first look at the residual impurities often seen in process development – and why they increasingly concern the FDA and EMA.

The different types of residual impurities found in biologics

During the development and production of biologics such as cell & gene therapy products (CGTs), process-related impurities follow along with the product.

Typically, we divide impurities into three subtypes depending on their origin:

• Cell substrate-derived / product-related (e.g. host cell proteins, host cell DNA)
• Cell culture-derived (e.g. antibiotics, IPTG, DTT, growth factors)
• Downstream-derived / process-related impurities (e.g. enzymes, buffer components)

The last subtype consists of agents used to express and purify biological protein products. Standard process-related residuals include the Benzonase nuclease, enzymes for site-specific PEGylation, aminopeptidase, and Protein A. Other typical residuals are Tris, carriers, ligands, Tween/Polysorbate, DCA, TCEP, heavy metals, solvents, Triton-X, antifoaming agents, PEI, TFA/Acetate, Imidazole, etc.

Why are residual impurities of concern?

Investigating the levels of residuals is crucial, especially in the final product sample. Because the residuals can influence the stability and efficacy of the active ingredient, and may even pose a risk to the patient’s health [1-3].

With the development in sensitive analytical methods for residuals analysis, the FDA and EMA now seem more and more interested in data that map out the efficiency of the purification process for CGTs and vaccines [1-3].

How to fully document process-related impurity clearance

The FDA and EMA are asking an increasing number of CGT developers to document residual protein clearance before their biologic may into late-stage clinical trials [1-3].

The problem, however, is that traditional methods like ELISA and HPLC are restricted by low antibody-specificity and an insufficient limit of quantification, respectively. Since the residuals in samples are present at low-ppm levels, you need a highly sensitive and reproducible approach to get a detailed overview of the residual protein clearance [4, 5].

Fortunately, SWATH mass spectrometry (LC-MS/MS) offers precisely that. It fragments all peptides using Data Independent Acquisition (DIA). The method divides the defined mass range into small mass windows for MS/MS fragmentation of the peptides. Afterward, comparing the acquired MS data to an ion library of known peptides will identify the proteins [6].