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The author describes techniques that can be used to provide the analytical data required by ICH Q6B for characterization of monoclonal antibodies.
Five of the top seven and six of the top twenty biological products in 2010 (in terms of revenue) were monoclonal antibodies (mAbs) (1). Several new mAb products and mAb biosimilars are in development, and all require extensive characterization to obtain the necessary approvals for clinical trials and to eventually be released onto the market.
The most recent regulatory document covering characterization of mAbs was published by the European Medicines Agency (EMA) in July 2009 (2). This EMA guideline entitled Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related Products states that the mA should be characterized thoroughly (2). This characterization should include the determination of physicochemical and structural properties, purity, impurities and quantity of the mAb, in line with International Conference on Harmoniaation (ICH) guideline Q6B (3).
ICH Q6B provides a uniform set of internationally accepted principles for characterization of biotechnological products to support new marketing applications. The document suggests that analyses be performed to provide the following information for biological or biopharmaceutical products:
The techniques used to provide the analytical data required by these guidelines are discussed below.
ICH Q6B states that "The overall amino acid composition is determined using various hydrolytic and analytical procedures, and compared with the amino acid composition deduced from the gene sequence for the desired product, or the natural counterpart, if considered necessary. In many cases amino acid composition analysis provides some structural information for peptides and small proteins, but such data are generally less definitive for large proteins. Quantitative amino acid analysis data can also be used to determine protein content in many cases."
Amino acid composition analysis using reverse-phase high-performance liquid chromatography (RP–HPLC) with pre-column derivatization or ion exchange chromatography with post-column derivatization are now routinely used to determine the amino acid composition of biopharmaceutical products. Provided that the molar ratios of amino acids per mole of protein and the overall molecular weight of the protein are known, the protein content can be calculated from the amount of each robust amino acid detected in a hydrolysate of the product. Also, if the optical density (at 280 nm) for the solution from which the aliquot(s) for amino acid analysis were taken is known, the Beer–Lambert law (absorbance [OD] = ε.c.l; where ε is the extinction coefficient, c is the concentration in M/L, and l is the path length in cm) allows calculation of the extinction coefficient for the product, meaning that spectrophotometry at 280 nm can be used to quantify the product in solution.
For a mAb, the EMA guideline and ICH Q6B request a deduction of the amino acid sequence from the DNA sequence and confirmation of the DNA-derived sequence experimentally by appropriate methods (e.g., peptide mapping, amino-acid sequencing, mass spectrometry analysis). In addition, the variability of the N-terminal amino acid sequences (whether a free amino acid or a pyroglutamic acid is present) and the C-terminal amino-acid sequences (e.g. the presence or absence of C-terminal lysine(s) on the heavy chain) should be analyzed.
Peptide mapping (i.e., analysis of specific protease digests of the mAb followed by analysis of the products using on-line RP-HPLC with ultraviolet [UV] and electrospray mass spectrometric detection [LC/ES–MS]) provides molecular weight information for the peptides released from the mAb by the protease of choice. The data obtained are able to provide mapping confirmation (or otherwise) of the DNA-derived sequence but does not provide confirmation of the sequence of the light and heavy chains of the mAb. To provide confirmation at the primary amino acid sequence level, a number of protease digests combined with on-line RP–HPLC with tandem MS/MS (LC/ES–MS/MS) analysis are required. ICH Q6B does not actually request sequencing at the primary amino acid level. There is a move, however, initiated by some of the regulatory bodies, to provide confirmation of the primary protein sequence for new mAb products and also provide data showing comparability of the the primary amino acid sequence of mAb biosimilar products to a reference product.
As mass spectrometric-based sequencing is unable (in most cases) to differentiate between isoleucine and leucine (these amino acids have the same molecular weight), automated N-terminal sequencing of purified peptides is required for unambiguous assignment of these two amino acids, particularly within the variable regions of the light and heavy chains.
Terminal amino acid analysis is performed to identify the amino- and carboxy-terminal amino acid sequence(s) of the light and heavy chains of a mAb. If the product exhibits more than one terminal amino acid sequence, the relative amounts of the termini should be determined.
Automated N-terminal sequencing following separation of the light and heavy chains using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotting onto polyvinylidene fluoride (PVDF) membrane is used routinely for analysis of the N-termini of mAb light and heavy chains. Automated N-terminal sequencing uses Edman chemistry which requires a free amino functionality at the N-terminus of a protein for labeling prior to cleavage of the N-terminal and subsequent amino acids. This means that for a number of mAbs (which have a pyroglutamic acid at the N-terminus and therefore no free amino group), N-terminal sequencing using this method will not provide sequence information. In this case pyroglutaminase can be used to enzymatically remove the N-terminal pyroglutamic acid residue before N-terminal sequencing.
There is no fully reliable method analogous to Edman chemistry for determining the C-terminal amino acid sequence for a biological product. Information relating to the C-terminal sequence of a peptide or protein can be obtained using carboxypeptidase digestion and/or mass-spectrometric mapping strategies. In the latter approach, intactness of a protein C-terminus or the presence of ragged ends can be assessed using data obtained from a peptide map and intact molecular weight analysis of the product.
During a peptide mapping/sequencing analysis, free sulfhydryl groups and disulfide bridges should also be considered. Peptide mapping post-digestion with a specific protease, followed by analysis using on-line LC/ES–MS or LC/ES–MS/MS prior to/and following reduction, provides the data necessary for a full assessment of disulfide bridges and free thiols within the mAb. Care must be taken when digesting proteins containing free thiols at basic pH as the free thiol can initiate scrambling within the digest, and the result obtained may therefore not be consistent with the true disulfide bridge/free thiol pattern within the product.
As mAbs are glycoproteins, it is also necessary to characterize the glycan portion of each product. Typically, 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 constant fragment (Fc) region. The light chain component of a mAb is not normally glycosylated. Since additional N-linked glycan consensus sequences may be present within the heavy chains, the presence or absence of glycosylation should be confirmed. ICH Q6B requests that "For glycoproteins, the carbohydrate content (neutral sugars, amino sugars and sialic acids) is determined. In addition, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile) and the glycosylation site(s) of the polypeptide chain is analyzed, to the extent possible."
As shown in Figure 1, monosaccharide composition analysis is usually carried out to determine the carbohydrate content of a mAb. Liquid chromatography and gas chromatrography (often with mass spectrometric detection) are commonly used to define the carbohydrate content of a mAb. The method(s) used should allow identification and quantitation of the levels of the neutral sugars (fucose, mannose and galactose), amino sugars (N-acetylglucosamine, N-acetylgalactosamine) and sialic acids (N-acetylneuraminic acid and N-glycolylneuraminic acid) within the product.
Figure 1: Schematic of the normal approaches employed to analyze the glycosylation of a monoclonal antibody (mAb). LC is liquid chromatography, MS is mass spectroscopy.
Following monosaccharide composition analysis, the next stage of analysis moves into characterization of the oligosaccharides normally present on each heavy chain. An example of the methodology used for releasing, purifying and analyzing the N-linked glycans from a mAb is shown in Figure 1.
Reduction/alkylation and the specific protease digestion are intended to denature the mAb so that the relatively large enzyme PNGase F can do an efficient job at removing the N-linked glycans. Once the N-glycans are released, a simple reverse phase cartridge can be used to separate the hydrophilic glycans from the more hydrophobic peptides. The N-linked glycan fraction can then be analyzed using mass spectrometry and/or liquid chromatography-based techniques.
The matrix-assisted laser desorption ionisation time-of-flight (MALDI–TOF) mass spectrum obtained from analysis of the N-glycans released from a mAb (using the methodology shown in Figure 1) is shown in Figure 2. The major signals observed are consistent with the commonly encountered mAb glycans G0F, G1F and G2F (see Figure 2).
Figure 2: Raw data obtained from matrix-assisted laser desorption ionisation time-of-flight (Maldi-TOF) mass spectrometric analysis of the N-glycans released from a mAb. The N-glycans were permethylated prior to analysis.
The high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) trace obtained from analysis of the N-glycans released from the same mAb product is shown in Figure 3.
Figure 3: Raw data obtained from high-pH anion-exchange chromatography with pulsed amperometric detection analysis (HPAECâPAD) of the N-glycans released from a mAb.
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. Glycosylation is the most common post-translational modification encountered by the regulatory authorities. The regulators are aware that glycosylation can be significantly affected by even simple changes in the manufacturing processes, such as a pH change or a change of growth media. The monosaccharide composition analysis data (in terms of levels of mannosylation, galactosylation and sialic acid) and N-linked oligosaccharide profiling data obtained from the analysis of various batches of a mAb product are therefore used by the regulator as part of the assessment of whether a manufacturer has control of the manufacturing process of a mAb. Comparability of N-glycan profiles from batch to batch suggests that the manufacturing process is under control.
The EMA and ICH guidelines also recommend that the higher-order structure of the mAb should be characterized by appropriate physicochemical methodologies. ICH Q6B recommends the use of "circular dichroism, nuclear magnetic resonance (NMR), or other suitable techniques, as appropriate." Today, circular dichroism (CD) analysis is commonly used to assess the secondary structure of mAbs. The data obtained from CD analysis of a mAb is shown in Figure 4. Assessment of the raw data using CDsstr software allows the relative percentages of the various secondary structures to be estimated as shown in Table I.
Figure 4: Raw data obtained from near ultraviolet (UV) analysis of a mAb.
In addition to the structural characterization requirements outlined above, a number of additional physicochemical properties need to be assessed.
Table I: The result of CDSSTR algorithm assessment of the near UV CD (circular dichroism) data shown in Figure 4.
On-line LC/ES–MS analysis of intact and reduced mAbs using quadrupole time-of-flight (Q–TOF) instrumentation has revolutionized the measurement of the intact molecular weight of mAbs and the released light and heavy chains. The data obtained from intact molecular weight analysis of a mAb using this technique is shown in Figure 5.
Figure 5: Deconvoluted mass spectrum obtained from online electrospray mass spectrometric detection (quantitative time-of-flight) LC/ESâMS (Q-TOF) analysis of a mAb.
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 mass spectrometric data obtained from analysis of the mAb following reduction are shown in Figure 6.
Figure 6A: Deconvoluted mass spectrum obtained from online LC/ES-MS (QâTOF) analysis of a mAb.
The signal shown in Figure 6A is consistent with that expected for the light chain and the signals observed in Figure 6B are consistent with those expected for the heavy chain with the expected N-linked oligosaccharides. Aside from confirming the molecular weight of a mAb and the component light and heavy chains, these data also confirm the intactness of the protein backbones.
Figure 6B: Deconvoluted mass spectrum obtained from online LC/ESâMS (QâTOF) analysis of a mAb following reduction.
In a similar way, imaging capillary isoelectric focusing (cIEF) has revolutionized analysis of the isoforms of mAbs. cIEF is free solution isoelectric focusing in a capillary column. Dedicated systems such as the iCE280 analyser (Convergent Biosciences) detect focused protein zones using a whole column UV absorption detector that avoids disturbing these focused zones. This technology is unique in that it has the comparable resolution of traditional gel IEF, but incorporates the advantages of a column-based separation technology, including quantitation (using UV at 280nm) and automation.
The raw data obtained from cIEF profiling of a mAb is shown in Figure 7. The data obtained allows an accurate determination of the pI of each isoform and quantitation of each isoform based on the UV peak area.
Figure 7: UV (280 nm) response across the capillary obtained following capillary isoelectric focusing of a mAb.
Analysis of mAbs using isoelectric focusing (as discussed above), SDS–PAGE (prior to and following reduction), and capillary electrophoresis are routinely used to provide product distributions based on size and charge.
Chromatographic analysis using size-exclusion chromatography (SEC), RP–HPLC and ion-exchange liquid chromatography provide product distributions based on size, hydrophilicity/hydrophobicity and charge distribution.
Both electrophoresis and chromatography provide information relating to identity, homogeneity, and purity of the product and are often used to purify impurities for identification and structural characterization using the analytical techniques outlined above.
For mAbs in particular, it is essential to assess each product for the presence of multimers and aggregates using a combination of methods. Currently, it is usual for the regulatory authorities to request SEC with multi-angle laser-light scattering (SEC–MALS) and a column free technique such as analytical centrifugation (AUC) to cross check the data obtained from SEC analysis.
Because aggregates can potentially be lost by non-specific binding to the SEC column or, indeed, dispersed by the dilution of the product on the column and the shearing forces during the chromatography, confirmation of SEC results using a column-free technique is required.
Figure 8: Raw data obtained from size-exclusion chromatography with multi-angle laser-light scattering (SECâMALS) analysis of a mAb.
The raw data obtained from SEC–MALS and AUC analysis of a mAb are shown in Figures 8 and 9 respectively. The data obtained from SEC–MALS analysis of the mAb suggests, in this case, the presence of low levels of aggregates (approximately 2.6%). The data obtained from AUC analysis suggests the presence of approximately 5.0% aggregates. It is usual to observe higher levels of aggregation using AUC over SEC–MALS, probably due to the factors potentially reducing the levels of aggregate in the column-based analysis data outlined above.
Figure 9: Raw data obtained from analytical centrifugation (SV-AUC) analysis of a mAb.
The techniques outlined herein are used throughout the product lifeline, in preclinical phases to gain an understanding of the product, to demonstrate that changes to the manufacturing processes, for example scaling up of the process, do not alter the structure and physicochemical properties of the product and to provide data to demonstrate consistency of manufacture of the product. Also, increasingly, characterization-based techniques, such as peptide mapping and oligosaccharide profiling, are being used as part of the release testing regime to release products into clinics for trials and eventually onto the market. In the development of any mAb product at least three and usually several batches will be characterized to the full extent outlined above.
In summary, a battery of analytical techniques are required to fully characterize mAb products. In addition, for submissions to the regulatory authorities, analysis to GLP and cGMP are required. Full characterization of a mAb according to the EMA and ICH guidelines can now be carried out in a matter of weeks, and continuing improvements in instrumentation are constantly allowing analytical laboratories to obtain data with more sensitivity, accuracy, and resolution. The challenge is to remain at the forefront of these technologies and to incorporate the increasing list of other appropriate techniques such as fourier transfrom–infrared (FT–IR) analysis to complement secondary structure analysis using CD and field flow fractionation to provide an additional assessment of aggregation where appropriate, in the characterization of mAbs and other biopharmaceuticals.
ANDREW J. REASON, PHD, IS managing director of life science services at SGS M-Scan, Berkshire, United Kingdom. Andrew.Reason@sgs.com.
1. IMS Midas and Knowledgelink; Pipelinereview.com, Top 30 biologics 2010, special edition, Jan. 2011, www.pipelinereview.com/free-downloads/TOP_30_Biologics_2010_RDPN_Special_Mar_2011.pdf, accessed April 2011.
2. EMA, Guideline on Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related Products (London, Dec. 2008).
3. ICH, Q6B Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (1999).