Unwanted particles can spell disaster for biologicals. Tara Sanderson, PhD, Formulation Services Manager at SGS M-Scan, explains why and how to formulate to avoid them
The manufacture of biologics is a growing area, as companies are increasingly using the technology to develop better-targeted, more effective and safer drugs. However, biologics are not always as stable as small molecule drugs, and the manufacturing and storage process can introduce particulates. These can affect the shelf life of the drug, but more importantly, can expose already vulnerable patients to potentially life-threatening immunogenic responses. To remain competitive in the field, and to ensure the safety of patients, biologics manufacturers need to remain aware of ways to analyse and control particulates in their products.
Particles in biologics can be intrinsic (introduced as part of the manufacturing process or as aggregated particles of drug during storage) or extrinsic (arising from the drug container or device). By understanding what causes the development of particles and knowing how to control them, manufacturers of biological drug products can increase their chances of getting a product through regulatory approval, reduce the need for potential process changes or reformulation, and improve cost of goods and profit margins.
The detection of particles during product development or storage can cause delays at the approval stage
Some patients have immune systems that will recognise the particles in biologic therapeutics as foreign, and trigger an immune response. This immunogenic reaction can range from simply annoying, such as a slight rash or mild flu-like symptoms, through to serious systemic reactions, and can be a particular issue in patients who are already debilitated.
The detection of particles during product development or storage can cause delays at the approval stage, as the regulators will require extra characterisation steps and evidence of clearance, and may even require changes to the manufacturing and formulation processes. These changes will mean the need for comparability and stability studies and potentially new method validations and reference standards. All of these issues can add cost and increase time to market.
To deal with the problem of particulates in biologics, manufacturers need to understand what kinds of particles are involved and their possible source. These particulates fall into two subcategories – non-proteinaceous particles and proteinaceous aggregates. Non-proteinaceous particles are process-related and can be introduced during production at a number of stages, and include fibres from filters, particles of plastic and rubber from packaging and minute droplets of the silicone oil used to lubricate syringes.
Proteinaceous aggregates, which may or may not be visible to the naked eye, can aggregate spontaneously if high concentrations of proteins are present in the solution, or at air/liquid and surface/liquid interfaces. Aggregates may occur due to unfolding of the protein as a result of degradation or may initiate around non-proteinaceous contaminants that create a catalyst seed for aggregation. Storing biologics in non-ideal conditions where they could be exposed to changes of temperature can trigger aggregation, as can (often unavoidable) vibration and agitation during transport.
Changes in the production process can influence the likelihood of particulate formation, and so manufacturers need to ensure types of particles and levels are monitored at every step, including formulation and filling steps, and use with delivery devices. The techniques for analysing particles vary according to particle size (see Figure 1), which can range from visible, at over 100µm, through to oligomers and down to fragments at less than 1nm.
Figure 1: Analysis of Particles
There are a number of steps throughout the biologics manufacturing process where manufacturers can intervene to reduce particulate levels. These interventions should happen as early as possible in the development process, as changing processes and formulations later on can incur greater costs, and may even affect a drug’s marketing approval.
Sequencing: Software can predict where in the protein sequence aggregation is likely to happen – for example, where free or internal thiols will bind covalently with other protein molecules. This provides an opportunity for protein redesign at an early stage of development.
Expression and purification: Processing steps, such as lowering the pH to inactivate viruses and using ultrafiltration and diafiltration to purify the expressed protein, can introduce contaminants, from fibres shed by filters to denatured and inactive proteins. In-process aggregate analysis will warn of potential problems.
Formulation: Good formulation design can control the development of particles, by stabilising the conformation of the protein API, and is the main approach used by manufacturers to create a product that remains stable during shipment and storage. Any changes that are made to the biologic after initial formulation will mean that it needs to be checked again for particulates, and may need to be reformulated.
Characterisation: Manufacturers need to carry out sub-visible particle testing as part of the characterisation and comparability studies, to ensure that any changes, including scale-up, have not introduced any additional propensity for particle development.
The manufacturer will need to test the formulated protein under simulated conditions that represent as many potential shipping situations as possible
Shipments: However careful carriers are, products will always be exposed to temperature and pressure changes and agitation during shipping. The manufacturer will need to test the formulated protein under simulated conditions that represent as many potential shipping situations as possible.
Drug product fill: Some biologics are lyophilised or frozen for storage, and the reconstitution or thawing process can trigger aggregation. Consequently, the biologic will need to be tested for particulate risk after drying and reconstitution, or after one or more freeze/thaw cycles, and the results compared with a control sample.
Release: Because they can be broken down in the digestive system, most biologics are administered systemically, generally by injection. Filling a delivery device and then administering the drug can introduce particles that trigger aggregation, such as fragments of rubber or plastic from the delivery device itself, or tiny droplets of silicone oil used to lubricate the barrel of a syringe.
Stability studies: By their nature, biologics are not very stable molecules, and often have to be stored under specific conditions to prevent denaturing. Making changes to the sequence and formulation can improve stability, but producers must monitor and characterise any particles that form.
Creating a stable and reproducible formulation process is important to the consistent manufacturing of biologics for therapeutic use and patient safety. The following case study looks at an IgG1 monoclonal antibody that, though it was safe and effective in development, was found to have a propensity to aggregate when it went through freeze/thaw cycles and on shipment, reducing its potential for day-to-day use in patients and affecting its chance of approval for marketing.
The challenge: In this example, the challenge for SGS was to create a more stable formulation that could be transported without aggregation, and that could cope with repeated freeze/thaw cycles. Additionally, this had to be completed within the originator’s limited timeline and budget, and using only the limited sample volumes available.
The solution: The team at SGS created a strategy to design the optimum formulation, beginning by mimicking the worst possible shipment conditions and the most extreme temperatures. This included agitating the sample for 24 hours at room temperature and then cycling the temperature from –20°C to +40°C. The SGS team then compared this degraded material with control samples to find the degradation pathway. While the charge profile and the secondary structure remained unchanged, there was some scrambling of SS bridges and minimal changes in the overall tertiary structure, along with significant changes in the number of subvisible particles, mostly over 2µm.
Creating a stable and reproducible formulation process is important to the consistent manufacturing of biologics
In the next step, the SGS team carried out a pH and excipient screen that included agitation and freeze/thaw cycles. By including known degradation conditions for the product, the outcomes informed the creation of a shortlist of more stable formulations, which were then further assessed using a Design of Experiment (DoE) approach. The team then selected four lead candidates, based on further screening protocols. These candidates were further assessed for thermal stability at temperatures between 20°C and 100°C, as well as being checked for conformational stability and particle counts.
The outcome: These conformational analyses and particle counts led to the selection of a lead candidate, which should be more stable to temperature changes and agitation. The use of highly sensitive, high-throughput analysis techniques allowed the SGS team to stay within the timeline and budget, while still including a range of different imaging and analysis tools, and traditional methods such as SEC and DSC, so that all potential sizes and types of particles could be included in the process.
Because of the issues of patient safety, the impact on shelf life and production, as well as any subsequent increases in development costs, it is essential that manufacturers tackle the issue of controlling particles in biologics manufacturing upfront, and build this into the development process and timeline. By beginning the process as early as possible, they can allow time for thorough evaluation, and reduce the risk of any costly delays to launch.