Biological medicines now account for the majority of newly registered medicines worldwide. They are also among the most profitable. When their patent expires, pharmaceutical companies have the opportunity to develop equivalents, i.e. biosimilar medicines. In order for these to be registered, they must pass so-called biosimilarity studies.
Biosimilars vs. generics
Biosimilar drugs are many times more complex than small-molecule drugs. Their manufacturing process typically uses genetic engineering techniques and cell lines. By mimicking the action of natural molecules, these drugs have biological properties that can significantly affect patient safety. Not surprisingly, registration of biosimilar medicines is much more complicated than registration of generic medicines. In order to register a biosimilar drug, it must be proven that it does not differ significantly in terms of efficacy and safety from the original (also called reference medicinal product). To do this, a series of tests called biosimilarity studies are performed.
What is biosimilarity?
Manufacturing drugs using micro-organisms (e.g. E. coli) or cell lines (e.g. CHO) makes the manufacturing process extremely complex. Moreover, it is subject to a certain degree of variability related to the specificities of the in vitro culture and purification process. Proteins are large molecules and come in many variants. An active substance can therefore have many variants that still retain their therapeutic capacity. These are usually due to various post-translational modifications that occur in the cells used for production, or arise at the purification or storage stage. The most common include glycosylation, deamidation, oxidation, loss of the C-terminal lysine or N-terminal modifications of the protein chain.
This has a huge impact on production, as it means that tranches in a single plant can vary significantly from one to another. The degree of variability depends, among other things, on whether the drug is produced on one production line or on many, as well as whether production takes place at the same or different sites. The final properties can also be influenced by the raw materials used, the age of the cells as well as the cycle of use of the chromatography resin.
What is biosimilarity? How to prove that a medicine is biosimilar? To demonstrate biosimilarity, the drug must be compared with a sufficient number of batches of the originator and it must be shown that they do not differ significantly in critical quality attributes (CQA’s).
What are critical quality attributes?
Any physicochemical, structural or biological characteristic that can influence the efficacy, immunogenicity, safety of a drug is a critical quality attribute. Criticality is assessed few times at different stages of product development based on the knowledge we have about the product and the process. How critical it is depends on the damage it can cause and also on how good it can be controlled in manufacturing process.
As a result, some CQA’s need to be tested during product release, others during manufacturing. There are also some that need to be compared to the reference drug for the registration of a biosimilar drug. There is then no need to test them routinely. [JA1]
How are biosimilarity studies carried out?
The reference product monitoring
The development of a biosimilar medicinal product start with the acquisition and characterization of the reference drug from the market. It is good practice to obtain several to a dozen batches of the product. The acquired drug is analyzed for all identified critical quality attributes. Based on the results, a so-called quality target product profile (QTPP) is created. In brief, this is the range of values for critical quality attributes within which a biosimilar should fit. The QTPP is an indicator for scientists developing the manufacturing process. Their job is to guide the process so that the resulting product is comparable. The number of batches of the reference product is not insignificant. Recommendations from regulatory agencies are min. 7-10 batches or 3 for less critical attributes. However, the more batches tested, the more markets considered, the higher quality of a QTPP.
- Binding to receptor/antigen
- Mechanism of binding
- In vitro activity (on a suitable cell model)
- Confirmation of amino acid sequence
- Level of post-translational modifications
- Glycosylation profile
- Charge variants
- Secondary and tertiary structure
- Level and size of aggregates
- Higher-order structures
Biosimilar drug testing
Testing of a biosimilar drug begins as soon as the process is scaled up to industrial or semi-industrial scale. Engineering, confirmation and clinical batches are tested. In this way, the initial biosimilarity studies necessary to apply for clinical trials are obtained. In subsequent stages, the batches used to characterize the process and validate it are also accounted. The complete set of results includes full physicochemical characterization, structure elucidation, binding and functional assays and comparative clinical trial results.
What analyses are typically performed in biosimilarity studies?
Amino acid sequence confirmation.
Sequence coverage is one of the key tests to confirm the identity of a biosimilar drug. It is performed by peptide mapping with LC-MS/MS detection. The protein is cut into peptides by enzymes (usually trypsin, chymotrypsin, Lys-C). The peptides are separated chromatographically. In the mass spectrometer, the molecular weight of the peptide is determined and a fragmentation spectrum is acquired. The individual peptides are then assigned to the spectrum using appropriate software.
An alternative method used to confirm the amino acid sequence is Edman degradation sequencing. It has some advantages over peptide mapping with LC-MS/MS detection. It relies on the fact that it is more suitable for sequence analysis without prior knowledge of the sequence and can also distinguish leucine from isoleucine, which most mass spectrometers cannot handle. The disadvantage of Edman sequencing is low throughput and duration of the test.
The isoelectric point is the pH at which a protein is not charged to any extent. If there are structural differences on the molecule, which e.g. block amino acid functional groups, a change in the isoelectric point occurs. The test is performed using capillary electrophoresis, so-called cIEF.
Post-translational modifications can have different implications for pharmacodynamics and pharmacokinetics. If they occur at sites responsible for binding to an antigen or receptor, they can directly decrease the activity of the drug. In addition, some may increase immunogenicity. Which modifications and where they occur in a protein depends on its amino acid sequence, secondary and tertiary structure, cell line. Among the most commonly monitored are deamidation, oxidation and dioxidation, N-terminal amino acid cyclization, isomerization of aspartic acid, loss of C-terminal lysine or methylation.
The post-translational modification study is based on peptide mapping with LC-MS/MS detection (similar to sequence coverage). Modified peptides differ from native ones in retention time, mass-to-charge ratio and fragment spectrum. With the appropriate software, it is possible to determine the relative level of modification.
Molecular weight and subunit mass
Molecular weight and subunit mass confirmation is one of the most important tests to confirm the identity of the molecule, the profile of the most important glycans and modifications and also the structure of the molecule.
The analysis is performed using high-resolution mass spectrometers.
Charge variant monitoring allows a rapid comparative assessment of proteins in the reference and biosimilar product. The resultant protein charge is influenced by many factors: amino acid composition, post-translational modifications, glycan structure, secondary and tertiary structure and signal peptide presence.
The test involves chromatographic separation of the protein on a column containing an ion exchanger (ion exchange chromatography). A chromatographic profile is thus obtained, in which peaks can be grouped according to their charge.
Proteins undergo N- and O-glycosylation. The glycan profile is one of the most critical attributes. Some glycans may be immunogenic, causing adverse effects on the patient. Others may affect the activity and ability to bind to a receptor or antigen.
Glycan profile should be looked at as early as possible in the development of a biobased drug. Specific enzymes are required for the formation of some glycans and not all cell lines possess them. Knowing the glycan profile of the reference drug therefore allows the host to be selected and the process to be guided accordingly.
The glycosylation profile is determined by various techniques. Routinely, these substances are released enzymatically, derivatized and analyzed chromatographically using a fluorometric detector. Often, a mass spectrometer is used to identify the detected glycans. For high-throughput screening MALDI-TOF can be applied.
Proteins undergo spontaneous folding after translation. A secondary structure is formed as a result. This can be investigated using spectroscopic techniques: circular dichroism in the near ultraviolet and also Fourier transform infrared spectroscopy (FTIR). Both methods are used as orthogonal due to the fact that they differ in their sensitivity to other secondary structures.
The tertiary structure is an extremely important element. Stabilized by disulphide bridges, the correct conformation determines the appropriate biological properties of the molecule. The tertiary structure is analyzed by spectroscopic techniques: circular dichroism in the far UV, intrinsic fluorescence and NMR.
If a molecule has a quaternary structure, it must then be determined whether it is compatible with the reference drug. External fluorescence analysis, FRET, HDX, NMR, DSC, X-ray crystallography and even electron microscopy (e.g. for Virus Like Particles) are used for testing. The choice depends on the molecule size and the properties of the expected quaternary structure.
Aggregate level and size
Determining the level of aggregates is extremely important. Firstly, aggregated molecules are inactive. Secondly, they can cause immunogenicity and tissue degeneration. Depending on the size of the particles, different measurement techniques are used. For small aggregates size exclusion chromatography (SEC) is the best choice. Larger particles are analyzed using techniques based on light scattering measurements, e.g. DLS, MALS or even analytical ultracentrifugation (AUC). Combining several techniques together, e.g. SEC-MALS, is a popular practice.
A popular analytical method in which the aggregation of molecules and its nature can also be compared is Differential Scanning Calorimetry. With this method, it is possible to determine, for example, whether aggregation is reversible or not.
Comparing the activity of two products is one of the most important tasks of bioavailability studies. The method must be tailored to the mechanism of action of the molecule and be investigated at both the molecular and functional level.
Testing for binding to a receptor or antigen is performed by various analytical methods. ELISA and SPR (surface plasmon resonance) are among the most commonly used. Depending on the molecule, other techniques such as FRET, radioligand binding assay and thermophoresis can also be used.
We can divide functional tests into two types: biochemical and cellular assays. From a regulatory point of view, cellular tests are more valuable than biochemical ones.
As with binding assays, functional tests should be tailored to the mechanism of action. In some cases this will be activation of the molecule (e.g. phosphorylation), signal transduction or interaction with the receptor. In the case of cell-based assays, this include signaling pathway activation, antibody-dependent cell cytotoxicity (ADCC) or activation of specific biochemical pathways.
For biosimilar drugs, in vitro assays are paramount over animal studies. For this reason, the choice of appropriate molecular, biochemical and cellular assays is crucial. In most cases, it is not necessary to perform additional animal studies prior to the clinical phase.
How to present results in biosimilarity studies?
There is no single answer how the results should be compared. Here again, an analysis of the criticality of the quality attributes, performed several times, is helpful. If the criticality is low, one may be tempted to use broader acceptance criteria or even no acceptance criteria at all. For the most critical attributes the similarity should be strict.
The so-called interval method is most commonly used. The monitoring of the reference drug results in a range of variability, which should coincide with the range of variability of the biosimilar. A range based on 3 standard deviations is usually adopted. To prove biosimilarity, 95% of the batches of the biosimilar drug should be within the interval created by originator monitoring.
How many batches should be tested in biosimilarity studies?
The answer to this question depends on the criticality of the quality attributes and their variability. For structure elucidation, it is sufficient to compare three batches. For attributes that correlate closely with efficacy or safety, regulatory agencies recommend min. 7-10 series. If the attribute is highly variable or age dependent, the higher number of batches should be considered.
At this stage, it is worth mentioning the impact of analytical methods on the statistical power of the comparison. The methods used should be designed to detect potential differences between two products. Great importance should be attached to the precision and accuracy of the method. All methods used should be qualified and their precision and accuracy estimated.
Biosimilarity studies are a long-term process and start early in product development by monitoring the reference drug product. The ideal situation is to come to market with a biosimilar drug on the day the original drug’s patent expires. This is quite a challenge for companies. It could be said that they are in a real race. However, the stakes are high and definitely worth the effort. It is no secret that biologic medicines are very expensive and this is what makes a whole lot of patients all over the world waiting for replacements.
It may be that the amount of analysis that needs to be done to demonstrate biosimilarity exceeds a company’s resources. However, this does not derail their chance in the market. Nowadays, many companies outsource some or even all of their analyses to contract organizations. Some of the most commonly outsourced include protein structure analysis, functional and cellular assays or even sequencing. This solution allows the company to start without preparing its own spaces in advance. This is a way of saving time and resources, making the biosimilarity studies easier.