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European Biopharmaceutical Review

Compare and Contrast

The EMA guideline covering analytical considerations of biosimilars recommends the application of extensive state-of- the-art characterisation studies, in order to demonstrate with a high level of assurance that the quality of biosimilars is comparable to their reference medicinal product.

The EMA recognises that: “For some analytical techniques, a direct or side-by-side analysis of the biosimilar and reference medicinal product may not be feasible or give limited information – for example, due to the low concentration of active substance and/or the presence of interfering excipients, such as albumin. Thus samples could be prepared from the fi nished product – for instance, extraction, concentration, and/or other suitable techniques. In such cases, the techniques used to prepare the samples should be outlined, and their impact on the samples should be appropriately documented and discussed, for example, comparison of active substances before and after formulation/ deformulation preparation.”

This is the first major hurdle that has to be overcome in terms of providing analytical data to support a claim of biosimilarity. A number of innovator products are formulated at low concentration with relatively high concentrations of protein, amino acid, mono- and di-saccharide and/or surfactant stabilisers. All of these common excipients can – and will – interfere with the analytical data obtained. For example, protein and amino acid excipients impede amino acid composition analysis and, potentially, peptide mapping, whereas saccharide and Tween surfactant-based excipients hamper monosaccharide composition analysis. In addition, a number of surfactant-based excipients can affect mass spectrometric intact molecular weight analyses and peptide mapping. It is therefore necessary to remove these excipients to a level where any interference is minimised.

Purification Methods

A number of techniques are available for purification, including, but not limited to: chromatography, based on affinity, hydrophobicity, size or charge; size/ molecular weight-based filtration; and dialysis. It is important to try a variety of approaches to choose the most appropriate method.

Recovery of product through the process should always be checked. Once a satisfactory regime has been developed, a more thorough assessment of the product prior to and post purification should be performed. These techniques should demonstrate that the active biological ingredient has not changed significantly in a structural/physicochemical sense, and is suitable for side-by-side analysis to measure comparability.

The chosen techniques should examine:
  • Intactness
  • The protein backbone, including analysis of disulphide bridges and post-translational modifi cations (PTMs)
  • Oligosaccharide profile
  • Secondary and tertiary structure
Measuring Molecular Weight

Assessing the intact molecular weight profile of a biopharmaceutical product can be performed relatively quickly and provides significant data to assess any impact on the product during or following purification. On-line liquid chromatography electrospray mass spectrometry (LC/ES-MS) analysis of proteins and glycoproteins using quadrupole-time of flight (Q-TOF) instrumentation has revolutionised the measurement of intact molecular weight. For instance, the type of data that can be obtained from analysis of a monoclonal antibody (mAb), intact and following reduction, can be seen in Figures 1
(see page 18), 2a and 2b.

The major peaks observed are 162 mass units apart, which is consistent with variation in galactosylation of the N-linked oligosaccharides present on the heavy chains of the molecule.

The signal shown in Figure 2a is consistent with that expected for the light chain, and the signals observed in Figure 2b are consistent with the heavy chain – including the expected major N-linked oligosaccharides.

mAb products are often formulated at relatively high concentration with a mixture of amino acid, sugar and surfactant-type excipients. It is therefore usually possible to obtain the above quality of data for samples analysed prior to and following purification. This is not necessarily the case for blood circulating hormones – for example, erythropoietin and follicle stimulating hormone – which are formulated at relatively low μg amounts/mL with significant levels of interfering excipients.

The Protein Backbone

Peptide mapping involves MS analysis of specific protease digests of the biopharmaceutical product, usually utilising on-line reverse-phase high-performance liquid chromatography separation with ultraviolet (UV) and mass spectrometric detection (LC/ES-MS). This provides molecular weight information for the peptides released from a protein or glycoprotein by the protease of choice. The data obtained are able to provide a good assessment of the protein backbone prior to and following purification. The technique allows for the observation of the presence or absence of predicted peptide masses using a template protein – or nucleotide – sequence; therefore allowing a first order confirmation of the expected protein primary structure, together with the detection of PTM events leading to a shift in mass. However, it does not provide confirmation of the amino acid sequence itself, which must be obtained by further experiment, including MS/MS fragmentation analysis.

Where possible, LC profiles with UV and MS detection for material prior to and following purification should be obtained and compared. Surfactant stabilisers – where present – may interfere with mass spectrometric ionisation of the eluting peptides and, in such cases, a comparison of the UV profiles obtained from analysis of product digests should be used for the assessment.

During the comparison, an assessment of free sulfhydryl groups and disulphide bridges should be considered. Analysis of each digest before and after reduction can provide the data necessary for measurement of disulphide bridges and free thiols. The overall MS data should also be used to look for differences in PTMs. Extensive purification could have an impact on the deamidation and oxidation state of the product in particular, and these data should be used, for example, to assess any changes in Asparagine, Glutamine and Methionine residues.

Isoform profiling can be used to provide an overall full molecule assessment of deamidation and other charge-based changes occurring during purification. Imaging capillary isoelectric focusing (cIEF) relies on free solution isoelectric focusing (IEF) in a capillary column to detect focused protein zones using a whole column UV absorption detector. This technology has the comparable resolution of traditional gel IEF, but incorporates the advantages of columnbased separation technology, including quantitation (using UV at 280nm) and automation. The raw data obtained from cIEF profiling of a mAb is displayed in Figure 3.

The data obtained allows for accurate determination of the pI of each isoform, and quantitation of each isoform based on the UV peak area. An increase in acidic isoforms following purification could, for example, suggest that the purification process is inducing deamidation.

Oligosaccharide Profile

Oligosaccharides present on glycoproteins are prone to degradation and losses of sialic acid content (desialylation). Therefore, it is important to demonstrate that no significant changes in oligosaccharide profile have been induced by the purification process. Products such as mAbs have one N-linked glycan consensus sequence (Asparagine-Xxx-serine or Threonine, where Xxx can be any amino acid except Proline) within each heavy chain located in the hinge region. In most cases, the intact molecular weight data and information obtained from analysis of the glycopeptide(s) in mass spectrometric peptide mapping experiments will allow for assessment of the comparability of oligosaccharide profiles prior to and following purification. The glycans must, however, be released and analysed to allow an assessment of the full population, including minor species. Data obtained from analysis of the N-glycans released from a mAb are shown in Figure 4. The major signals observed are consistent with the commonly encountered mAb glycans G0F, G1F and G2F.

The hydrophilic interaction liquid chromatography (HILIC) trace obtained from analysis of the N-glycans released from a mAb product (following 2-AB labelling) can be seen in Figure 5. Both sets of data can be used to provide a relative quantitation of the N-linked oligosaccharides observed and suggest the structures of the N-linked glycans present, therefore allowing for assessment of oligosaccharide population pre- and post-purification.

Secondary and Tertiary Structure


It is important to examine products for secondary and tertiary structure, as a significant change in tertiary structure can suggest degradation of the product during or following the purification process. Circular dichroism (CD) analysis is one technique commonly used, and the data obtained from CD analysis of a mAb is displayed in Figure 6. Furthermore, assessment of the raw data using CDSSTR software allows the relative percentages of various secondary structures to be estimated.

Summary

A range of analytical techniques can be used to prove that the structural integrity of an active pharmaceutical biological ingredient has been retained following any necessary purification. These analyses should assess the intactness, protein backbone, PTMs, oligosaccharide profile and secondary/tertiary structure of the product, pre- and post-purification.

Where possible, samples should be analysed as received. Analysis of purified product should only be performed where significant interference from excipients is observed. Full structural and physico-chemical examination of a number of batches of biosimilar, alongside innovator, will be needed to provide the required assessment of structural comparability.


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Professor Howard Morris is Founder, Chairman and Chief Scientific Officer of BioPharmaSpec. He has over 45 years of research experience in the field of biomolecular mass spectrometry, which has included a number of seminal discoveries at the structure and method development levels, ranging from the first successful mass spectrometric protein experiments, through to the conception of the novel Q-TOF geometry mass spectrometer for ultra-high sensitivity unambiguous de novo sequencing.

Dr Andrew Reason is Founder, Chief Executive Officer and Managing Director of BioPharmaSpec. He has 25 years of experience in analysis of protein and glycoprotein biopharmaceuticals and has been involved in the commercialisation of a number of analytical methods for characterising biopolymers. In addition to his scientific and managerial duties, Andrew has contributed to many industry publications and is a regular presenter at conferences. He is also a Visiting Professor at the University of Warwick.
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Howard Morris
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Andrew Reason
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