Biosimilars can exhibit tremendous heterogeneity in terms of structural and post-translational modifications. Therefore, comprehensive characterisation of biosimilars is necessary to ensure safety and efficacy. The analytical tests to demonstrate similarity of a biosimilar product to a reference drug with respect to protein content, activity, physiochemical integrity, stability, impurities are discussed.
Biopharmaceuticals such as monoclonal antibodies and recombinant proteins have emerged as important life-threatening therapeutics for the treatment of diseases including cancer. Recently 250 products are approved for various human diseases in the United States of America and the European Union. Moreover, 2,000+ biopharmaceuticals are currently in various stages of clinical research and development.
Generally, protein therapeutics are complex and large molecules, heterogeneous and subject to a variety of chemical and enzymatic modifications during recombinant expression, purification and even in long-term storage. This makes a vast analytical challenge than commonly used small drugs. The biopharmaceutical product manufacturer requires comprehensive physicochemical structural characterisation of the therapeutic protein. Especially for biosimilars, this task is extravagant and forms the basis for further comparability. As per regulatory guidelines, characterisation should be performed at different stages of product development. Initially, batches of the target originator molecule should be systematically characterised to determine the exact sequence of the target protein, the post-translational modifications and the other variability of quality attributes in different batches over time. Then, once the biosimilar product is manufactured, systematic characterisation needs to be performed to confirm the structure. Finally, manufacturers must provide data for comparison of biosimilar with the originator molecule. Analyses must conclude the proteins have the same bio-physical/chemical and physiological attributes. Analytical strategies should include a series of physicochemical analytical methods/techniques for primary and higher order structure (ICH Q6B) to detect product related impurities and variants. The present article, describes common analytical strategy for characterisation of biopharmaceuticals.
• 2005, EMEA issued guidelines on ‘Biosimilars’
• 2009, WHO ‘Guideline on Evaluation of Similar Biotherapeutic Products’
• 2010, Biologics Price Competition and Innovation Act (BPCIA) signed
• 2012, FDA issued draft guidance documents to accompany legal acts
• 2013, FDA fourth guidance issued.
Structural characterisation and confirmation:
a) Amino acid sequence: N/C-terminal sequence: Amino acid sequence of protein is compared with theoretical sequence/nature and homogeneity of termini is identified to find truncated forms.
Methods: Mass Spectrometry and Edman degradation.
b) Amino acid composition: Amino acid composition of the protein is determined.
c) Terminal amino acid sequence
d) Peptide map: Primary structure / Identity of protein is confirmed after digestion.
Methods: LC and/or mass spectrometry.
e) Sulfhydryl group(s) and disulfide bridges: Higher order of structure/location and/or connectivity of S-groups is confirmed.
Methods: HPLC and/or mass spectrometry.
f) Carbohydrate structure
a) Molecular weight or size: MW of protein is confirmed.
1D SDS PAGE/CE or mass spectrometry, AUC.
b) Isoform pattern
c) Extinction coefficient (or molar absorptivity): EC of known substance is confirmed. Methods: UV absorption or calculation after determination of amino acid composition
d) Electrophoretic patterns: Identity, homogeneity, purity, aggregates and truncates of substance is established
Methods: 1D SDS PAGE, CGE, IEF or 2D PAGE or CE.
e) Liquid chromatographic patterns: Identity, homogeneity and purity of substance is established.
Methods: HPLC / RPHPLC ,IEX, SEC, AC, HILIC, HIC.
f) Spectroscopic profiles: Higher-order structure is determined.
Methods: IR, Fluorescence, CD and NMR.
Amino acid analysis is extensively used to accurately quantify a protein. In a first step, amino acids are liberated through 6N HCl acid hydrolysis (at 110°C, 24 hr). Liberated amino acids are subsequently subjected to automated pre-column derivatisation. Derivatised amino acids are separated by HPLC and detected by fluorescence (FID) or ELSD detector. In case of FID, commonly the derivatisation is carried out by using o-phtaldialdehyde for primary and 9-fluorenylmethyl chloroformate for secondary amino acids analysis. Finally amino acid composition will be determined.
Peptide mapping is the most widely used identification test for therapeutic proteins. It involves four different important steps:
1. Isolation and purification of the protein
2. Selective cleavage of the peptide bonds
3. Chromatographic separation of the peptides and
4. Analysis and identification of the peptides.
This process involves generally trypsin enzymatic digestion of a protein to produce peptide fragments, separation and identification of peptide fragments.
This helps the monitoring and detection of
• Single amino acid changes
• Oxidation
• Deamidation
• Degradation products.
It also enables the direct detection of common monoclonal antibody variants like
• N-terminal cyclisation
• C-terminal lysine processing and
• N-glycosylation, as well as unexpected variations such as a translated intron.
Generally peptide separations are performed on HPLC-QTOF-MS instrument due to the convenience of HPLC coupling and more structural information, even for larger peptides, due to its mass accuracy and high resolution.
The analysis of charge isoforms in therapeutic protein preparations is very important during the manufacturing processes. The production and purification procedure makes the proteins (mAbs) to exhibit changes in charge heterogeneity. These changes may not only impact stability but also finally on activity and they even cause adverse reactions. Therefore cation exchange chromatography is regularly used to determine the acidic (left of the main peak) and basic charge isoforms (right of the main peak). These isoforms are generally deamidation and glycosylation products. The isoform characterisation involves calculation of the percent area of acidic and the basic forms. Each peak will be analysed on a mass spectrometry for confirmation after desalting and volatile buffer exchange.
Although protein biologics are relatively stable molecules, a number of chemical modifications and degradation reactions can occur during manufacturing, storage and in formulation. Electrophoresis, chromatography and mass spectrometry techniques are most often used to determine the molecular weight of intact proteins and their degradation products. SDS electrophoresis: Ability to visually identify proteins, aggregates and impurities in the migrating bands. Samples can be collected for further analysis by cutting out gel bands. Size-Exclusion Chromatography: advantages: High-resolution separations of aggregates and impurities based on their size. Short analysis time compared to SDS-PAGE. Simple method commonly used for in process monitoring to monitor aggregate purification and removal of impurities. RP-HPLC/QTOF-MS: Provides mass information for intact protein, variants, impurities and non-target proteins derived impurities. Fig. describes formation of Impurities in Synthetic Peptides.
Biosimilars are now recognised throughout the world as safe and effective medicines. Key factors in the determination of Biosimilars product composition and degree of similarity to reference products involves a vast array of analytical methods/techniques. Nearly all characteristics of a biosimilars and its corresponding innovator product has to be as similar as possible. Recent developments in analytical techniques allow a more detailed characterisation of both the biosimilar and the innovator’s product.
Liquid chromatography is already well established for intact protein analysis e.g. size-exclusion, ion-exchange chromatography. Gel electrophoretic approaches remain the gold standard for obvious molecular weight, size heterogeneity, purity, consistency manufacturing process determinations. Capillary gel electrophoresis and capillary isoelectric focusing methods permit the combination of the high resolution of gel techniques and the advantages of the microfluidic format of capillaries. Capillary zone electrophoresis appears to be a good candidate, since its easy coupling with time of-flight mass spectrometry could provide important information with simple and efficient analytical method. The top–down approach of Mass spectrometry and spectroscopy are also widely used to collect complementary structural information regarding 2D and 3D protein conformation. However, several analytical approaches are always needed to cover all other properties. Recent instrument technological progress will contribute to a better knowledge of these parameters and help to understand the impact of changes in manufacturing process on the quality and consistency of biopharmaceuticals.
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