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Proteomics Whitepaper

Chemical Tests You Must Conduct When Developing Novel Biologics

Biologics, especially protein therapeutics, are an intriguing and fast-growing frontier for biopharmaceutical companies of all sizes. The activity of proteins can be modified to have a certain targeted effect in the human body, such as inhibiting an enzyme or binding to a receptor, leading to novel approaches in disease diagnosis and treatment. Because biologic drugs are so precise, they can be used to treat specific conditions without causing as many side effects as traditional small molecule drugs and, although the number of FDA-approved protein therapeutics is relatively small, a number of these biologics are on the market effectively treating diseases ranging from cancer to autoimmune disorders.

Biopharmaceutical manufacturers who have identified candidates for novel protein therapeutics must be prepared to conduct a battery of chemical and biological tests to file a successful Biologics License Application (BLA). Biologics have complex structures and are more susceptible to variation during manufacturing than chemically-synthesized drugs, and even a slight process change can dramatically impact the safety, quality, and efficacy of the final product. Failing to accurately characterize a biologic, identify impurities, and study degradation pathways can lead to costly setbacks in the drug development process.

If your pharmaceutical company is planning to make the leap into biologics, you should familiarize yourself with the analytical approaches and chemical tests you will need to conduct to ensure your products will be safe and effective in a clinical setting.

Analytical Approaches in Biologics

Because protein therapeutics come from living cells, their matrices are complex. Scientists must “clean up” these matrices to analyze protein candidates that biopharmaceutical companies plan to use in novel therapies. Below is a brief overview of a few approaches scientists commonly use when analyzing biologics. It is important to note that these methods are not typically used singly; researchers use orthogonal approaches to gather as much data as possible.


HPLC is a workhorse tool throughout the biopharmaceutical industry. For analysis of proteins, scientists employ variants of the most common HPLC approach called reverse-phase HPLC. These include:

  • Size-exclusion chromatography – Molecules are separated and characterized by mass distribution.
  • Ion-exchange – Molecules are separated based on charge
  • Hydrophobic interaction chromatography – Proteins are separated in a reverse salt gradient to afford separation based upon the substance unique pattern of hydrophobic regions.

Newer UPLC (Ultra Performance Liquid Chromatography) can be used in place of HPLC in many cases. It is faster and is now replacing traditional HPLC in many applications.


Electrophoresis is a technique used to separate macromolecules based on their size and electrical charge. Polyacrylamide gel electrophoresis (PAGE) is often used for the quantitative analysis of protein and nucleic acids. A newer approach, capillary electrophoresis, is often combined with tandem mass spectrometry to characterize a protein’s primary structure and amino acid sequence.


Liquid-chromatography mass-spectrometry (LC-MS) is a technique used to separate and identify compounds from a mixture. Once compounds are separated in the liquid chromatography phase, they are ionized and measured based on the masses of their ions.


Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) was developed to allow for MS analysis of large biomolecules. First, a sample is ionized, and its molecules become charged. The sample is then pulled into the mass analyzer section of the instrument, and the molecules are separated based on the time it takes them to pass through a time-of-flight tube. This can give researchers a peptide map, which they can use to identify target proteins uniquely in the sample.


Enzyme-linked immunosorbent assays (ELISAs) are used to detect highly specific fingerprint regions of individual molecules allowing unique and sensitive quantitation of specific substances in a complex mixture. These assays use polystyrene plates to which antibodies and proteins bind. Non-bound materials can then be washed away, allowing for the highly specific measurement of the specific analytes.

Enzyme Assays

Since many biologics catalyze specific reactions to achieve their therapeutic or diagnostic effect, in vitro enzyme assays are employed to measure specific activity of the therapeutic or diagnostic protein species. Paired with techniques listed above, enzyme assays allow for determination of both a diagnostic or therapeutic product’s identity and purity as well as its potency.

Raw Material Testing

Chemical analyses for biologics should start with the raw materials used in the manufacturing process. There is a risk that raw materials could become contaminated with substances from the bacteria, yeast, viruses, and other agents used in their production. All raw materials should be tested for identity, purity, and stability. Manufacturers must develop and validate GMP-compliant methods for quality control.

Biologics Characterization

Characterization testing for biologics is an important step in developing protein therapeutics. This testing is also challenging because of the diverse materials used in bioprocesses and the complex structures of proteins. Scientists typically conduct a wide range of characterization assays, including tests for:

  • Purity – Purity is defined by the FDA as “relative freedom from extraneous matter in the finished product, whether or not harmful to the recipient or deleterious to the product.” Factors that can affect a product’s purity include moisture, heat, external contaminants, and the manufacturing process itself. Pharmaceutical manufacturers must be able to demonstrate a therapeutic biologic product’s continued purity to get approval from the FDA.
  • Potency – Potency testing is another requirement for regulatory submissions. For biologic products, testing for potency means confirming that the biological activity of the therapeutic is producing a defined result at a defined dosage. Researchers must typically develop new potency assays for novel biotherapeutics.
  • Peptide Mapping – Peptide mapping is a technique used to characterize the amino acid structure of proteins. It is also used as a quality control measure for lot-to-lot testing.
  • Host Cell Protein Detection – Host cell proteins are contaminants that can be co-purified with a therapeutic product during the product process, potentially altering the activity and safety of the therapeutic. Tests to detect and quantify host cell proteins are an important part of quality assurance during the development phase.
  • Glycosylation – Covalently linking a carbohydrate to a target protein or lipid is the process of glycosylation. Some proteins will not undergo protein folding unless they are glycosylated. It can also increase the stability of proteins through the linkage of carbohydrates to certain residues of the protein. Glycosylation creates a variety of modified proteins with tailored and desired functions.

The most extensive characterization testing usually takes place during the development phase of the therapeutic, but additional testing may be required if the manufacturing process changes.

In most cases, researchers will need to develop and validate new methods to characterize novel biologics. The International Council for Harmonization (ICH) has developed a set of guidelines (Q6B) that can be used as a starting point when designing a characterization method panel.

Impurities Characterization

Impurities testing is typically considered part of the characterization process, but it is broken out in its own section here to emphasize its importance in therapeutic development. It is essential for researchers to identify impurities because if they go undetected, they can interact with the therapeutic protein and have a negative effect on the stability, safety, and efficacy of the product.

Manufacturers must perform tests to determine if there is even a small percentage of the wrong protein in the therapeutic they are developing. This typically requires several orthogonal methods. For example, ELISA is commonly used for host cell protein detection but does not provide detailed information on the level of each individual host cell protein, so mass spectrometry may be used to fill in the gaps.

In addition to identifying impurities, manufacturers must consider those impurities’ sources. Impurities typically come from one of two general sources:

Product Modifications

Impurities could come, for example, from unintentional changes to the protein amino-acid sequence that alter the way the protein acts. Impurities can also arise from sub-forms of a therapeutic protein that do not have the same potency as the biologic product.

Materials Used in Production Processes

These may include surfactants, excipients, or additives used to promote growth in cell cultures. Researchers must develop methods to quantitate all additives, excipients, and surfactants so that they can show regulatory agencies what is in the biologic.

It is difficult to completely remove all impurities from a biologic, but manufacturers are responsible for ensuring that the level of impurities in the final product remains safe and consistent. If manufacturers detect unsafe levels of impurities, they must implement purification steps to their development process to reduce those impurities to an acceptable threshold.

Process Impurities

Chemical analyses for biologics should start with the raw materials used in the manufacturing process. There is a risk that raw materials could become contaminated with substances from the bacteria, yeast, viruses, or other agents using in the upstream manufacturing process.  Surfactants and detergents, coupling agents, antifoam agents, and other process impurities must be identified and quantified.

Stability Testing

Stability testing involves evaluating a biologic over an extended period to ensure its quality, safety, and efficacy remain consistent across its life cycle and under different conditions. It is a regulatory requirement that helps determine shelf lives and storage conditions for the final biologic product.

It typically takes several years to carry out stability tests, so manufacturers usually start planning for this testing downstream in the biologic development process. Due to the long-term storage requirements, manufacturers often outsource their stability testing to independent chemical analysis labs that can keep the biologic samples in storage chambers and analyze them throughout their life cycle.

In addition to real-time stability testing, some labs will also conduct accelerated stability testing, which exposes samples to higher temperatures and humidity to speed up their degradation and determine their point of failure. Accelerated stability testing can help manufacturers predict shelf life and identify potential problems early in the development process.

E&L Studies

Extractables and leachables (E&L) testing is used to determine whether any organic or inorganic compounds could or will leach into a drug product from an outside source, such as the product packaging or manufacturing equipment. Because leachants can change the way a drug product works, E&L studies are required for any high-risk medications. Since biologics are usually injected, they are typically considered high-risk and subject to E&L testing requirements.

The FDA and Product Quality Research Institute (PQRI) have developed guidelines for a proactive, risk-based approach to extractables and leachables testing. Pharmaceutical manufacturers need to conduct E&L studies on any packaging or manufacturing equipment that comes into contact with the biologic. This will help ensure that no compounds leach into the biologic product and alter the way it works.

Extraction Studies

Extraction testing is the first part of E&L studies and is used to illustrate a “worst-case scenario.” The goal is to identify all possible leachants that could contaminate the biologic under any condition. Researchers can use the method they develop for extraction studies to help them develop analytical methods for leachable studies.

Leachable Studies

Leachable testing generally takes place during stability testing, since the goal is to identify leachants that could contaminate a biologic under conditions of normal use. Researchers will compare chromatograms from leachables studies to those from extractables studies. The chromatograms should show the same peaks, but the peaks should be smaller for the leachables studies due to the realistic storage conditions. Researchers should not see any new peaks in a leachables chromatogram that were not in a corresponding extractables chromatogram.

While there is no way to eliminate the risk of E&L contamination entirely, taking a proactive approach to E&L studies early in the biologic development process will help manufacturers avoid product contamination and safety issues.

Working with an Independent Analytical Lab

Biologics take years to develop and approve for use in clinical settings. If your pharmaceutical company decides to expand into biologics development, you cannot afford any unnecessary delays from impurities in your final product or methods that cannot be easily scaled.

One way to ensure your biologics development stays on track is to work with a trusted independent lab that has the equipment and skilled chemists necessary to carry out your biologics analyses. Contact Avomeen to learn how our scientists can develop customized testing solutions for your biologics challenges. As a full-service, GMP-compliant testing laboratory, we can develop and validate new testing methods for your novel biologics and conduct all assays required by the FDA and other regulatory agencies.

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