Proteins & Peptides Process-related Impurity Analysis

The biomanufacturing process of recombinant proteins, monoclonal antibodies, therapeutic peptides, etc. involves the use of various chemicals that may become unwanted process-related impurities in the final product. Analytical guidelines such as ICH Q6B require that these components be evaluated and monitored to ensure that they are removed or reduced to an acceptably low level through effective purification processes. CD Formulation's process residue analysis team follows the ICH Q6B guidelines and applies a series of highly specific and sensitive techniques to identify and quantify these process-related impurities to ensure that the product meets the specification limits of these process residues, supporting process validation and GMP batch release.

What is Biopharmaceutical Process-related Impurity?

Process-related impurities refer to harmful chemicals related to the production process that may exist in the biopharmaceutical process. According to the ICH Q6B guideline, process-related impurities may come from raw materials, production equipment, production environment, and the production process itself, including but not limited to microorganisms, cell substrates (such as host cell proteins), cell culture medium components, metabolites, impurity proteins, residual reagents, chemical reagents used in purification, water-soluble impurities, etc. Even after purification, these foreign chemicals will still remain in the final drug in small amounts, affecting the stability, purity, activity, and safety of the product, and even causing adverse biological reactions. Therefore, these related impurities need to be strictly monitored and controlled during biopharmaceutical process development and production.

Explore Our Proteins & Peptides Process-related Impurity Analysis Services

Effective removal of process-related impurities or process residues is critical to protein/peptide biopharmaceutical development. At CD Formulation, our process residue analysis team designs and develops a wide range of analytical methods to identify and quantify these residues, allowing you to demonstrate that your downstream processes are effectively reducing or removing these chemicals.

With our process-related impurity analysis services, you can fully understand the process residues that may be generated early in development and establish acceptance criteria for ongoing testing requirements, including process validation and batch release testing.

Our process-related impurity analysis services include but are not limited to the identification of the following possible impurities.

Fig. 1 Proteins/peptides process-related impurity analysis. (CD Formulation)

Impurities From Cell Substrates

These impurities are derived from proteins/nucleic acids from the host organism, such as host cell proteins and DNA (HCP/DNA).

• Host Cell DNA Quantification Assays

We utilize real-time qPCR or digital droplet PCR (ddPCR) to quantify the levels of any residual host cell DNA in a drug substance or product. Our services can be applied to viral clearance studies to demonstrate DNA removal.

• Host Cell Protein (HCP) Quantification and Identification Assays

We utilize enzyme-linked immunosorbent assays (ELISA) and high-performance liquid chromatography-mass spectrometry (LC-MS) to identify and quantify HCPs.

1) ELISA: The industry standard for quantitative HCP analysis in biopharmaceutical products and process development, providing total HCP measurements (ng/mg) in a simple and reliable assay format with broad HCP coverage. Our HCP-ELISA supports batch release testing of bulk drug substances, as well as step-by-step confirmation of downstream process performance.

2) LC-MS: An orthogonal approach to support and complement ligand binding assays (LBAs). LC-MS/MS allows for the identification and quantification of individual HCPs and helps ensure that no HCPs escape HCP-ELISA detection above reportable limits (e.g., 10 ng/mg). If one or more residual HCPs are present in the final drug substance at levels above 10 ng/mg, the individual HCP information from LC-MS/MS can be used to facilitate multi-sectoral risk assessment.

Cell Culture Impurities

These impurities include a variety of chemicals such as inducers, antibiotics, serum, and process enhancers such as glutathione, and dithiothreitol (DTT).

• For trace antibiotics such as kanamycin, tobramycin, gentamicin, amoxicillin, and chloramphenicol, our process residue analysis team uses highly selective methods such as LC-MS/MS and multiple reaction monitoring (MRM) to identify and quantify these possible impurities.
• For solubilizers and stabilizers (surfactants and antifoaming agents） such as Triton-X, Tween 20, Tween 80, Pluronics, and polyethylene glycol P2000, our process residue analysis team uses MS, as well as HPLC-evaporative light scattering techniques to identify these possible impurities.
• For process inducers and enhancers such as redox agents (glutathione), solubilizers,  residual solvents (phenol, isopropanol), and chelating agents (EDTA), our process residue analysis team uses LC-MS, gas chromatography-mass spectrometry, HPLC-evaporative light scattering, and other techniques.

Downstream Impurities

These impurities include enzymes, chemical and biochemical purification, and processing reagents (such as cyanogen bromide, oxidizing and reducing agents, inorganic salts, solvents, ligands, etc.).

We use a range of highly sensitive techniques, such as liquid chromatography tandem mass spectrometry, and gas chromatography-mass spectrometry, to identify and quantify downstream derived impurities.

Impurities Specific to A Particular Production Process

Methotrexate, a cytotoxic compound, is commonly used as a cell culture additive to effectively suppress the cell's immune response or as a selection marker for clone screening to help screen out high-yield recombinant protein production cells. Due to its toxicity, regulatory agencies require that the removal rate must be ensured to reach an extremely low allowable limit of 10 ppb.

For methotrexate, we can develop highly sensitive HPLC-MS analytical methods to overcome these complex matrix challenges to meet regulatory requirements.

Other Process Related Impurities

To address any challenges you may face during the protein/peptide biopharmaceutical process, our residue analysis team also offers the following residue-level analysis services.

• Residual Solvents: Including residual solvents (such as DMF, DMSO) and other organic volatile impurities (OVI), we use pharmacopoeial methods such as USP <467>, but we can also develop custom methods to suit your specific samples.
• Elemental Related Impurities: We use inductively coupled plasma techniques such as ICP-OES and ICP-MS to determine elemental impurities such as metal ions, inorganic salts, and arsenic.
• Process Equipment Leachables and Extractables (E&L) Assessment: We provide powerful E&L solutions, ranging from method development and validation, USP <661>, USP <661.1> <661.2>, <665>, regulatory support, material characterization, and risk-based approaches to extractables and leachables testing.

Why Choose Us for Proteins & Peptides Process-related Impurity Analysis？

• Our team of analysts has years of experience in the analysis of proteins and peptide process-related impurities. They have a deep understanding of the complexities involved in these analyses and can provide accurate and reliable results.
• We utilize the latest technology and instrumentation to ensure high-quality and precise analysis of proteins and peptides process-related impurities.
• We adhere to strict regulations and guidelines to ensure that our analysis meets industry standards and provides reliable data for regulatory submissions.
• We understand that each project is unique, and we work closely with our clients to develop customized solutions to meet their specific needs and requirements.
• We offer flexible testing options to meet the needs of your different projects.

Publication

CD Formulation has extensive experience in resolving process-related impurity challenges and can develop pre-analytical purification methods to reduce interferences as needed, as well as develop custom solutions for unusual process additives. Please feel free to contact us if you are interested in our services or have further questions. We look forward to working with you to promote development and innovation in the biopharmaceutical field.

How It Works

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STEP 2

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STEP 3

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