Citation: Moore S, Harper K, Marshall S, “Key Considerations for Non-Clinical Safety Assessment of Inhaled Biopharmaceuticals”. ONdrugDelivery Magazine, Issue 80 (Nov 2017), pp 46-49.

Simon Moore, Kirsty Harper and Sylwia Marshall highlight some of the key considerations for the development of inhaled biopharmaceuticals, including non-clinical safety assessment.

Inhalation is the route of choice for the delivery of numerous small molecule drugs, especially for the treatment of respiratory diseases, as it has a number of advantages over parenteral routes. These include faster onset of action due to the large surface area (80–120 m2) and good vascularisation of the lung, improved therapeutic index due to targeted delivery requiring lower doses and improved patient compliance.

Historically, the development of biopharmaceuticals for inhalation has been hindered by challenges such as high drug requirement, manufacturing costs and stability issues. However, technological advances addressing these concerns are now facilitating the development of such modalities. One of the earliest marketed inhaled biopharmaceuticals was Pulmozyme®, which was approved in 1993 for cystic fibrosis. The field has continued to grow since then, and there are an extensive number of inhaled biopharmaceuticals in early development.

The percentage of biopharmaceuticals in the global pipeline has grown from 30% in 2010 to 42% in 2017, and total revenues from their sales increased from 17% of all prescription drugs to 26% over the same period, with the figures expected to reach 30% by 2022.1 It is highly likely that inhaled biologics will contribute to this projected growth, having already been evaluated for numerous indications (Figure 1).


There are of course numerous differences between biologics and new chemical entities (NCEs) and these heavily influence the development strategy, including non-clinical safety assessment.

“Shear forces exerted during the process of aerosol generation can impact the structure and therefore alter bioactivity, with the worst-case scenario being loss of potency. For feasibility studies, one should consider the inclusion of not only a binding assay, but also a cell based potency assay, where the pharmacological activity of the test material may be evaluated and any change in potency following aerolisation noted…”

Biologics are a heterogeneous group of medicinal products that are generated or derived from biological sources and include biopharmaceuticals (proteins including monoclonal antibodies, peptides and oligonucleotides), vaccines and advanced therapies (gene/cell therapies). Each of these product types has specific features as well as specific biology that must be considered when designing non-clinical safety assessment programmes. Biopharmaceuticals are generally much larger than NCEs with many having complex structures, including secondary and tertiary structures, which are intrinsically linked with their function. Therefore, the physicochemical properties of these products must be taken into consideration when designing delivery systems.

The general approach to safety assessment of biopharmaceuticals is described in the ICH S6 (R1) guideline, “Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals”, where the basic principles of safety assessment in pharmacologically relevant species, and inclusion of appropriate pharmacodynamic (PD) endpoints wherever possible, are specified. This approach translates for assessment of biopharmaceuticals delivered by all routes of administration, inhalation included, and will likely determine the required programme of work for an inhaled biopharmaceutical.

Biopharmaceuticals exert their activities through specific interaction with their targets in the recipient patient, and it is therefore essential that all safety assessment studies replicate the clinical situation as far as possible with regard to target expression, binding and subsequent downstream biology. A comprehensive understanding of the pharmacology of the biomolecule in both humans and the candidate preclinical safety species is therefore required, and studies should only be performed in appropriate species. This may mean that a single species approach is sufficient and there are examples of biopharmaceuticals that received subsequent clinical approval following evaluation in a single species.

Due to the strong emphasis on pharmacology, non-clinical safety programmes are product specific and, unless the biopharmaceutical has a chemical modification, may omit some studies that are routinely found in NCE preclinical safety work packages, such as genetic toxicology studies.

Further, for most biopharmaceuticals, safety pharmacology endpoints are undertaken on a risk-based approach and are often incorporated into the design of pivotal repeat dose toxicity studies, with investigations in a single species commonly being acceptable. Depending on the mechanism of action of the biopharmaceutical, respiratory safety pharmacology may need to be supplemented with investigations of other systems which may be targeted, such as the central nervous system. The feasibility of such investigations needs to be carefully considered, especially with reference to the selected pharmacologically relevant species.


Inhaled drugs tend to be either liquid formulations administered via a nebuliser in a hospital environment or with the assistance of an experienced carer, or are self-administered as either aerosols or dry powders via handheld inhalers, which are generally acknowledged to be more efficient, stable and convenient for patients.

In general, most biopharmaceuticals show good aqueous solubility, and in the case of repurposing of existing products, a solution formulation is likely to already exist. For powders, however, more novel manufacturing techniques (lyophilisation, spray drying or vacuum foam drying) are more likely to be used than traditional manufacturing techniques, such as micronisation, as they tend to provide greater stability and ensure structural integrity of the biopharmaceutical. In addition, powders can accommodate the inclusion of various excipients.

There are a number of device types, each associated with their own advantages and disadvantages, that may be used to deliver biopharmaceuticals. Nebulisers can operate with many liquid formulations and are capable of delivering large quantities of drug, which may be needed to ensure sufficient clinical overages for toxicity assessment in the non-clinical setting. Nonetheless, liquid formulations can have limits with viscosity, ionic strength and surface tension which will impact output and drug concentration.

Pressurised metered dose inhalers (pMDIs) are not easily compatible with biopharmaceutical drugs due to the inherent temperature, pressure and excipient aspects, although in some cases there may be viable approaches to stabilise the drug product. An alternative approach to nebulisers and pMDIs are soft-mist devices which provide a pMDI-like dosing experience with an aqueous solution product. However, drawbacks include the requirement for high concentrations and the forces involved in delivering the formulation, which may prove incompatible for drug products where large doses are needed.

In contrast to pMDIs, which require the patient to co-ordinate breathing in with actuating the device by hand, dry powder inhalers (DPIs) generally require little to no hand-breath co-ordination, and they can deliver quite high payloads with a quicker dosing time than nebulisers. However, additional pre-formulation, formulation and device screening is necessary for DPI-based products, to address some of the dry powder formulation and stability characteristics.


Confirmation of the amount of the dosed test material is not only good scientific practice but also a regulatory requirement. To verify the concentration of the delivered dose, samples are collected directly from the exposure system from locations that are representative of the breathing zone for the animals (generally a facemask or restraint tube attachment position) using methodology that provides optimal trapping of the drug and permits chemical analysis of the active component. For most liquid formulations, this comprises a glass sintered sampling trap using an appropriate trapping solvent. For powder or suspension formulations, a quartz fibre filter is used rather than the standard glass fibre filters for NCEs. This is used in conjunction with silanizing analytical glassware prior to use.

For aerosol concentration and particle size assessment, standard Ultra Performance Liquid Chromatography (UPLC) analysis is normally employed. However, alternative methodology may have to be used depending on the biopharmaceutical. As mentioned earlier, biopharmaceuticals have complex structures and in many cases their activity depends on correct folding and subsequent tertiary structure. The shear forces exerted during the process of aerosol generation can impact the structure and therefore alter the bioactivity of the drug substance, with the worst-case scenario being loss of potency. For feasibility studies, one should consider the inclusion of not only a binding assay, but also a cell-based potency assay, where the pharmacological activity of the test material may be evaluated and any change in potency following aerolisation noted. Since such assays tend to be product-specific, early dialogue with the selected non-clinical CRO partner is encouraged to ensure smooth transition from exploratory studies to regulatory GLP safety assessment.

Figure 1: Various indications for which biotherapeutics have been evaluated using the inhalation route.


Non-clinical safety studies with biopharmaceuticals intended for inhaled delivery have a number of additional considerations that are unique to this method of administration. Although confirmation of drug exposure by comprehensive pharmacokinetic/ toxicokinetic (PK/TK) evaluation is expected in all biopharmaceutical safety assessment packages, it is important to consider that for inhaled products systemic exposure may not always be achievable or indeed desired. For instance, there may be limited transport of the delivered biopharmaceutical due to its size (molecules larger than 50 kDa display reduced bioavailability2,3) or targeted delivery, and binding to a receptor in the lung or a specific cell population may lead to retention of the drug in the lung.

Therefore, sampling of the local environment by bronchoalveolar lavage (BAL) to confirm that the intended delivery has been achieved as well as establishing systemic exposure should be considered.

The feasibility of obtaining BAL measurements requires careful consideration as, although possible, in-life sampling carries an inherent risk to the animal. For this reason strict sampling limits are imposed and it is highly likely that a full lung TK profile will not be possible in non-rodent species, with rodent studies requiring additional animals for such assessments.

The analytical approaches required for TK assessment of biopharmaceuticals may differ to those more commonly employed for NCEs, with immunoassays based on ligand binding assessment often required, although liquid chromatography-mass spectrometry (LC-MS) or MS based assays can still be utilised if a signature peptide has been identified, or for smaller products such as oligonucleotides.

As mentioned earlier, the safety profile of a biopharmaceutical can only be adequately assessed in a pharmacologically relevant species, ideally where the intended clinical biology can be replicated. As a result, markers to confirm PD activity should be included in safety assessment studies wherever possible. Appropriate markers should be identified based on the expected pharmacological effect and assessment performed at timepoints relevant to its induction.

A detailed understanding of the intended biology is therefore required and this should include any downstream effects in addition to the direct effect of the drug interacting with its target. The relationship of this biology in the non-clinical species to the clinical situation should also be thoroughly investigated so that any differences in the level or distribution of the target expression can be understood and interpreted.

In addition to PD endpoints, safety biomarkers can also be incorporated into the non-clinical safety studies. These can include markers of immune activation (CRP, cytokines, immune cell activation and/or mobilisation), immunogenicity assessment (discussed later), as well as assessment of “off-target” pathways that have been identified for certain classes of drugs. For example, prolonged coagulation and complement activation have long been associated with oligonucleotides, especially those with a phosphorothioate backbone or products with lipid based formulations.4,5 The exact parameters required for analysis are selected based on the biology and risk specific to the individual product, and if this risk is unknown or theoretical it can be assessed in preliminary studies to determine whether further follow up in pivotal studies is required.


One of the considerations specific to biopharmaceuticals is the development of immunogenicity. Administration of a human protein to an animal species can induce an immune response specific to the drug following delivery by any of the main routes of administration. The lung is predisposed to remove foreign material, and populations of the immune system, such as macrophages, specifically support this, so the potential for immunogenicity responses should be explored.

Although it is accepted that immunogenicity in an animal model is not predictive of immunogenicity in the clinical setting, the recognised consequences warrant at least the collection of samples. Blood samples should be collected prior to treatment and following completion of dose administration to assess the presence of systemic anti- drug antibodies (ADA), should there be any change to the PK/PD relationship during the study. This may be followed-up by more detailed investigations such as assessment of the functionality of the ADAs in neutralising antibody assay and/or immunohistochemistry staining for the presence of immune complexes. Such in depth characterisation is not often needed at the preclinical stage but it should be considered for inclusion in clinical studies.


Drug delivery via inhalation is an exciting and growing field of drug development. Despite the additional considerations associated with the inhalation route in the context of biopharmaceuticals, there is considerable research activity in this field. A detailed understanding of the pharmacology and biology of the biopharmaceutical product and careful execution of appropriately designed non-clinical safety studies, combined with selection of the most appropriate delivery method, can ensure a successful transition from non-clinical to clinical assessment.


  1. “World Preview 2017: Outlook to 2022”. Research Report, EvaluatePharma, 2017.
  2. Patton J, Byron P, “Inhaling medicines: delivering drugs to the body through the lungs”. Nat Rev Drug Discov, 2007, Vol 6(1), pp 67-74.
  3. McElroy M, Kirton C, Gliddon D, Wolff R, “Inhaled biopharmaceutical drug development: nonclinical considerations and case studies”. Inhal Toxicol, 2013, Vol 25(4), pp 219-232.
  4. Shaw D, Rustagi P, Kandimalla E, Manning A, Jiang Z, Agrawal S, “Effects of synthetic oligonucleotides on human complement and coagulation”. Biochem Pharmacol, 1997, Vol 53(8), pp 1123-1132.
  5. Henry S, Jagels M, Hugli T, Manalili S, Geary R, Giclas P, Levin A, “Mechanism of alternative complement pathway dysregulation by a phosphorothioate oligonucleotide in monkey and human serum”. Nucleic Acid Ther, 2014, Vol 24(5), pp 326-335.