Novel biotherapeutics can cause infusion-related reactions in patients, which may only become apparent in first-in-man trials. The FDA recommends in its definitive Guidance for Industry for Immunogenicity Assessment for Therapeutic Protein Products: “in vitro assessments of the capacity of such therapeutic protein products to mediate cellular activation, including proliferation and cytokine release in human whole blood or peripheral blood mononuclear cells are recommended” prior to first-in-man use.
The FDA also states in its recent guidance for Nonclinical Safety Evaluation of the Immunotoxic Potential of Drugs and Biologics that “an assessment of the potential for cytokine release syndrome caused by therapeutic proteins using unstimulated human cells in both plate-bound (or other assays that can assess the contribution of crosslinking of receptors) and soluble formats with appropriate positive and negative controls. These assays are considered critical for hazard identification.”
To provide more information to drug developers before drugs reach clinical trials, ProImmune has developed ProStorm®, an in vitro cytokine release assay which can help to predict the likelihood of first infusion reactions. Over several years ProStorm® cytokine release assay has been deployed on a very broad range of biotherapeutic entities, from peptides to proteins, mAbs, ADCs, bi-specifics, vaccines and more complex novel biological entities with data from the assay being submitted routinely to regulatory agencies, such as the FDA.
For generic peptide products following the FDA ANDA (Abbreviated New Drug Application) regulatory pathway, the sponsor must demonstrate that the active ingredient in a generic synthetic peptide is the same as the active ingredient in a previously approved peptide. One key element to this analysis is to demonstrate that any impurities present in the drug product does not induce unwanted innate (or adaptive) immunogenicity. As a sensitive whole blood assay, ProStorm® is ideally suited to address these critical and urgent questions in your product’s development.
With a typical core turnaround time of only 4 weeks ProStorm® is a rapid service helping you make timely decisions in your development program.
For best results we typically source fresh blood samples specifically for your study from a donor cohort chosen to match the needs of your project. Most often, we will use cohorts of at least 20 healthy volunteers, but we are able to source donors with known conditions if this is required.
The assay can be set up with fresh whole blood and/or isolated PBMC, with drug that is either in solution or immobilized, depending on the customer requirements. We routinely perform assays in all of these formats.
We use your drug preparation at a range of concentrations designed to mimic the peak exposure during a first infusion.
We add your drug preparation to the blood or PBMC samples, and after incubation, harvest the serum to measure cytokine levels. Typically, we would measure TNFα, IFNɣ, IL-2, IL-4, IL-6, IL-8 and IL-10, but in consultation with you we will select the best panel. Cytokine levels are measured using our optimized immunoassay technology for accurate quantification. Our team analyses your data to give you results that can be used in informing the next stages of your research.
We present two types of analysis as standard – plots showing the response from your donor cohort by cytokine for each drug/concentration, and a threshold (cut-point) analysis to indicate potential risk.
Figure 1: – Prostorm® results. Cytokine Response Data for IL-6. Responses from a cohort of 29 donors, stimulated with (left to right) Remicade®, aggregated Remicade®, Erbitux®, Campath®, Herceptin® and Vectibix®, at a range of concentrations. Medians are indicated by red lines. Particularly notable are the high responses from Campath®, and from aggregated Remicade®.
In addition to plots showing the cytokine responses of your whole donor cohort to each drug and dose combination tested, we also present a threshold (cut-point) analysis. In this analysis the test article therapeutic is typically compared to Erbitux®(Cetuximab), as this is known to elicit a low incidence of first infusion-related reactions.
Figure 2: example threshold analysis
This analysis approach is particularly powerful when used across a range of drug concentrations and when used with a large donor cohort. The results shown are from 29 healthy donors, and show the IL-6 response to 100µg/ml drug.
Figure 3: Erbitux® responses and 95th centile thresholds applied across increasing drug concentrations, IL-6 responses from a 29-donor cohort.
We will also typically include Campath® (Alemtuzamab) as administration of this mAb is associated with a high incidence of first-infusion reactions, and can serve as a useful high response comparator.
Figure 4: Campath® responses with Erbitux® 95th centile thresholds applied across increasing drug concentrations, IL-6 responses from a 29-donor cohort.
A particularly attractive feature of the assay is its suitability for assessing fully formulated therapeutics. This allows the effects of changes in formulation to be easily assessed. In one of our exploratory studies at ProImmune, we investigated the cytokine responses to Infliximab (Remicade®) and aggregated Remicade®. The results (figure 5) clearly show the increased reactivity of fresh blood to the aggregated formulation.
Figure 5: Remicade® and Aggregated Remicade® responses with Erbitux® 95th centile thresholds applied across increasing drug concentrations, IL-6 responses from a 29-donor cohort. Positive responders are indicated by filled data points.
Figure 6: For a range of well known therapeutic drugs in the market place, where the clinically observed incidence of cytokine release reactions is known a good correlation is seen between the ProStorm® assay results and clinical incidence of cytokine release.
Q: Does unexpected high cytokine release in a ProStorm® project cause problems with the regulatory submission?
A: Understanding the hazard of potential cytokine release is important for any prospective clinical study. If a hazard is identified from the assay, usually numerous options are available for mitigating the hazard going into the clinic. These often include: adjusting the dose range, extending drug release time, changing mode of administration, planning for treatment of clinical effects of cytokine release, another chance to look at manufacturing / fill finish related issues (where relevant).
Q: Which cytokine release assay works best: whole blood or PBMC, soluble or plate bound?
A: The most appropriate assay format will depend on the test articles under investigation. In some situations more than one testing format is advisable and we can implement the assay formats most suitable for your compounds. Some early publications suggested that blood based assays did not produce the sensitivity of PBMC assays. Generally we find that this is not the case and our optimized whole blood assay shows very good sensitivity with commercially available drugs known to cause cytokine release. The lack of manipulation of whole fresh blood as a test system also makes this version an attractive assay variant.
Complex peptide drugs can be produced through recombinant or synthetic manufacturing processes. When developing either novel or generic peptides, the developer must demonstrate that any impurities present in the drug product (these can be host-cell derived, direct peptide-related impurities (i.e. truncated or inserted amino acids), or other non-peptide-related impurities) are not posing risks of immune activation that can lead to immunogenicity of the drug substance.
Where generic peptides are concerned (such as glucagon, semaglutide, liraglutide, teriparatide, teduglutide, nesiritide etc.), the responsibility is on the developer to demonstrate that:
1. for each peptide-related impurity found in both the Reference Listed Drug (RLD) and the generic peptide, the level of impurity in the synthetic peptide is not higher than in the RLD;
2. the generic peptide does not contain any new impurities more than 0.5% of drug substance;
3. for such impurities present, a justification can be provided as to why the that impurity would not be expected to affect the safety of the generic peptide compared to the RLD including with respect to immunogenicity.
This can be a daunting prospect for developers of such generic peptides as the technical capabilities required to provide this evidence are not typically available in-house. With the regulatory requirements constantly evolving as our knowledge deepens, you need to work with a trusted partner. ProImmune has more than 10 years of experience in this specific area and we will deliver high-quality reliable data for you in a rapid timeframe and in a format that is routinely reviewed by global regulatory authorities.
Learn how working with ProImmune will accelerate your regulatory approval for generic peptide products:
There are two key components: assessment of both Innate and Adaptive immunogenicity risk.
Assessment of Innate immunogenicity risk
The innate immune response is the body’s first-line, and usually rapid, defense against pathogens. In the context of peptide-related or process-related impurities, the risk is that these impurities can potentially trigger pattern recognition receptors (PRR) on antigen presenting cells that will activate the innate response pathway leading to cellular activation, production of cytokines and initiation of the adaptive immune response. This is a signature of product immunogenicity.
At ProImmune we deploy our ProStorm® Cytokine Release Assay to assess this risk. This is a fresh, whole blood assay where blood is drawn from a panel of healthy volunteers specifically for your study by a trained phlebotomist and then incubated with the test material within just 3 hours of blood draw. Following a 24-hour incubation period, a set of 7 cytokines associated with innate immune activation are quantified in the plasma. It is important to demonstrate appropriate sensitivity of the assay in the presence of your peptide drug. To this end, we spike in to a subset of the samples a range of Innate Immune Response Modulating Impurities (IIRMIs) at an array of concentrations. These IIRMIs are agonists of different PRRs typically including TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, Dectin-1 and NOD2. It is also vital to confirm the viability of the cells (such as Dendritic cells) that are responsible for responding to these impurities and therefore we can also include a flow cytometric verification of this in the study.
Typically, a number of batches (minimum of 3) of both generic and RLD peptide are compared in this assay. It is also important to consider factors such as different API lots, batch expiry date as well as geographical lot release.
Assessment of Adaptive Immunogenicity Risk
Peptide-related impurities could consist of amino acid sequences that inadvertently have an increased affinity to MHC molecules (class I or class II). If a peptide impurity presented by the MHC is then recognized by a cognate T cell receptor in the context of appropriate co-stimulation, then the T cell adaptive immune response will have been activated. This then can lead to significant immunogenicity.
ProImmune has two assay formats to assess these different elements of risk. The first is the ProImmune REVEAL® MHC-peptide Competition Binding Assay which can determine the affinity of binding of generic peptide versus RLD to a panel of MHC class II alleles.
The second is the functional ProMap® T cell Proliferation Assay which measures total T cell proliferation using a flow cytometric readout over the course of 7 days in a panel of different healthy donor PBMCs following addition of the peptides.
Both assays can be used to look at the impacts of specific impurities, or in comparing batches of generic peptide to RLD.
If you would like copies of sample reports for any of the assays described or would like to discuss the technical details of your own project requirements in a confidential manner and receive a costed proposal, please don’t hesitate to contact our team of immunology specialists who will be delighted to assist you.
The full guidance for the FDA “ANDAs for Certain Highly Purified Synthetic Peptide Drug Products that refer to Listed Drugs of rDNA Origin” can be found here: https://www.fda.gov/media/107622/download