Getting Ahead of Industry Expectations for Biological Therapeutics Testing

Emerging trends in biologics testing may come from increasing Agency expectations, advancement in analytical technologies, or the clients' changing business needs in the current economic landscape. One of our strengths at Element is our agility and ability to pivot and develop new tactics to meet industry needs. Throughout this article, you'll learn some insight into three different pressures creating changes in industry standards that ultimately impact safety testing in biological therapeutics. 

Agency expectations: Proteomics for host cell protein analysis and immunogenicity prediction

ELISA (enzyme-linked immunosorbent assay) is the traditional methodology for determining the amount of residual host cell proteins (HCPs) in the bulk drug substance or drug product. In recent years, hyper awareness and vigilance about the safety of gene and cell therapies have led to questions about whether or not coverage of all available host cell proteins by the antibodies is sufficient. One major limitation of any immunoassay is the differences in the affinities of antibodies to their target proteins. If the affinity is low, then the target protein must be present in abundance in order for detection to occur. If affinity is high, then only a small amount of the target protein is needed to give a positive result. An ELISA is essentially the analysis of 10s of thousands of proteins, all with their own independent affinity to different antibodies, yet the method only gives a single averaged signal intensity read out in a 96 dash well plate. If there isn't enough protein present, or if the affinity is too low, the protein could go undetected, even if it is harmful to the safety of the patient.

In contrast, high resolution, high sensitivity bottom up mass spectrometry by proteomics has gained momentum as the more appropriate way to detect host cell proteins in the drug substance or drug product, because LC-MS/MS does not have the same affinity biases¹ as does ELISA. Recently, the FDA accepted an IND submission that only presented proteomics data to show HCPs and no ELISA methodology. The idea of using proteomics is not necessarily to show absolute clearance of HCPs - because it would be very difficult to show true absence of HCPs given the sensitivity of LC-MS/MS, but to use the data to perform a risk-based assessment of the safety of the drug. Specifically, proteomics could be used to determine if putative immunogenic proteins are present. We are still a long way from being able to use this technology for routine QC lot release testing, but a powerful tool used to complement the ELISAs during process development and process performance validations. 

¹NOTE: Bottom-up LC-MS/MS also has its own limitations, but generally could be better controlled in the laboratory setting than antibody-target protein abundance and affinities. 

Advancement in technologies: PCR for mycoplasma and residual host cell DNA testing

Mycoplasma is a large group of bacteria, consisting of over 120 species, and is very difficult to detect because they cannot be gram stained and are super tiny. Mycoplasma could spread and expose patients to a multitude of serious health conditions if present in a biological drug substance or product. USP <63>, EP Chapter 2.6.7 and 21 CFR 610.30 have been the standards for testing mycoplasma, but all three require up to 28 days of cell culturing, require large batch sizes and volume, and would only be suitable to test for a maximum of 6 species. Since the advancement of PCR, up to 90 species of Mycoplasma in a sample could be detected quickly using RT-PCR², or up to 113 species absolutely quantified by a droplet digital PCR (ddPCR) within hours of sample receipt³. This quick turnaround is much more suitable for biological drugs, such as cell therapies or personalized medicines, that might have a short shelf life, and need to be transplanted in a patient within days, or even hours, of being manufactured. 

The advancement of ddPCR is also changing how the industry is analyzing residual host cell DNA, again, a safety component of a biological therapeutic. Quantitative PCR (qPCR) is the traditional methodology for quantifying residual host cell DNA. However, qPCR is a relatively quantitative method that relies on synthesized oligonucleotide standards to be used as standard curves from which DNA from the samples would be quantified against. qPCR is also high variability and often required up to 7-10 replicates per run. Quantitation by ddPCR, on the other hand, is an absolute quantitative method based on the droplet generating technology, and takes away the variability of the test. It does not require standard curves as the quantitation is absolute quantitation of fluorescently labeled probe-positive droplets. The advancement of ddPCR has significantly reduced supply requirements, human hands-on time, and the time that it takes to get a result to demonstrate whether the drug is safe for patients.  

² MycoSEQTM Mycoplasma Real-Time PCR Detection Kit from Thermo Fisher

³ Vericheck ddPCR Mycoplasma Detection Kit from BioRad

Business needs and trends: upstream process raw materials routine testing

Raw materials testing in the biologic space is not trivial. There are no standard USP monographs for most common raw materials used in upstream processes such as cell culture media, media supplements, and more ancillary reagents. If a company wants to begin verifying batches of raw materials with a CDMO or testing lab to prepare for BLA submission, they are often faced with the high up-front cost and long lead time of custom method development for said raw material, followed by method validation, before they are able to analyze the 1-2 samples per year. As we see an increase in small, innovative, advanced therapeutic and/or personalized medicine companies begin to gain momentum, obtain results and the financial support to fast-track to the BLA, we also see the sticker-shock on their faces as they share with us how much it costs - and how long it takes - just to analyze their one batch of raw material.

This major bottle neck in getting safe, efficacious drugs to patients is one that needs to be dealt with immediately. One of my favorite parts of my job is talking to our clients about their patients, the mode of action of their therapeutic, and their current business demands. As a service provider, we take it upon ourselves as our client's partner to alleviate their burden so that they can focus on getting safe, efficacious drugs to patients who need them. Element has taken on the challenge of making non-standard raw materials testing more accessible to more companies working towards making their innovative therapeutic available to patients. Connect with us to learn more about this.

Pressure from the regulatory agencies and the increase in approvals of advanced biologics therapeutics has created an environment that encourages and fosters progress and improvement of advanced analytical technologies. Furthermore, the economic environment of 2024 has added pressure upon companies to get drugs to the bedside table quickly, fiscally efficiently, all while ensuring safety and efficacy. From a testing lab point of view, such pressure provides justification to explore and implement innovative solutions to meet the needs of the clients and the industry.