March 15, 2016 (Vol. 36, No. 6)

Greg Kilby Ph.D. COO Protea Biosciences

New Workflows Have Been Developed to Examine the Heterogeneity of mAb Products

Recombinant monoclonal antibodies (mAbs) have been among the most successful pharmaceutical products. Global sales for mAbs reached nearly $75 billion in 2013, representing approximately one-half of sales for all biopharmaceuticals. mAbs comprise about 40% of development-stage biosimilars, according to McKinsey & Company.

Although human IgGs share more than 95% sequence homology, each mAb drug product displays variable heterogeneity due to differences in manufacturing processes, genetic variability, and storage conditions. Among post-translational modifications (PTMs) giving rise to heterogeneity are deamidation, isomerization, oxidation, glycosylation, and terminal cyclization. Heterogeneity of mAbs produced from mammalian cells therefore gives rise to multiple isoforms within a single batch.

Because mAb heterogeneity has biological consequences, analyzing these molecules for quality, consistency, and biological activity demands a level of characterization unknown to the world of small-molecule drugs. The advent of biosimilars underscores the significance of full characterization for biosimilar mAbs, both for batch-to-batch quality concerns and to demonstrate similarity with the innovator drug.

A further consideration regarding heterogeneity arises during process development, when developers must demonstrate their process is well controlled and reproducible. Given the Phase I-through-approval transition probability of 30%, demonstrating batch-to-batch consistency becomes a top priority.

Protea Biosciences has developed rapid, robust workflows to examine the heterogeneity of mAb products at the intact-molecule level, the subunit domain level, and the peptide level, including PTMs. The analysis, based on capillary liquid chromatography and high-resolution/accurate-mass (HR/AM) mass spectrometry, provides high-level characterization to satisfy scientific, therapeutic, and regulatory goals.

Study Rationale

Traditional mAb characterization employs optical detection such as UV or fluorescence that, while sensitive and quantitative, do not provide sequence or structural information essential for designing better products.

Analyzing proteins for intact molecular weight provides sequence confirmation, while glycoform analysis provides clues to biological activity and immunogenicity. PTM analysis also provides insight into the loss of biological activity and/or stability over long-term storage. Toward these ends, mass spectrometry provides useful information in the following areas:

  1. Intact mAb analysis: delivering primary sequence confirmation and relative ratios of expressed glycoforms conferring biological activity or immunogenicity
  2. Subunit domain analysis: In combination with various chemical and enzymatic treatments, intact native mAb’s may be “broken down” into the biologically relevant domains, such as the Fab (antigen binding), light chain, heavy chain, and Fc (immune response) subunits.
  3. Peptide maps using multi-enzyme approaches to ensure 100% amino acid sequence coverage, including sites and prevalence of glycosylation, oxidation, and deamidation

Protea Biosciences developed these workflows to validate a HPLC mass-spectrometry-based approach to characterize protein therapeutics. We tested our methodology first on an intact research-grade immunoglobulin (IgG) to assess the potential for mass spectrometry to elucidate the primary glycoforms. We then performed a series of experiments on the partially digested (IdeS, SpeB) and reduced protein to examine biologically relevant portions of the IgG and to characterize the hinge region of the molecule.

We followed these experiments by using HPLC tandem mass spectrometry approaches on multiple enzyme digestions of the IgG (trypsin, lysC, pepsin, chymotrypsin, gluC, elastase, proteinase K) to produce comprehensive peptide maps. We also characterized sites of PTMs resulting from manufacturing, i.e., the expression system, sample handling, and sample storage.

This approach is particularly useful in forced degradation studies where the mAb is stressed to simulate long-term storage conditions. It is then assessed for either oxidation events, which are implicated in physiochemical stability of the molecule or deamidation events, which are implicated in significant reductions of biological activity or efficacy.

Sample Preparation

For intact protein analysis, human antibody IgG1 kappa (Sigma Aldrich®) was diluted with 0.1% formic acid in water and analyzed by LC/MS. We investigated several approaches for peptide mapping. Initially, the protein was re-suspended in a digestion buffer containing Progenta™ Anionic Acid Labile Surfactant (AALS, Protea Biosciences), tri(2-carboxyethyl) phosphine hydrochloride, chloroacetamide, and ammonium bicarbonate in water. The sample was then processed for 15 minutes in a laboratory microwave digester controlled for temperature, digestion time, and pH.

This approach, while very rapid and producing low levels of sample-handling artifacts, also resulted in some key areas of sequence not being elucidated. An alternative approach was developed using very mild reduction, alkylation, and rapid digestion conditions that also produced very low baseline levels of sample-handling artifacts and provided consistent, reproducible high levels of sequence coverage. When multiplexed with several different proteolytic enzymes, this approach consistently resulted in full 100% sequence coverage.

Conditions of sample preparation were maintained carefully to avoid the introduction of artifactual PTMs resulting from sample-preparation treatment and handling. This provided confidence that changes above a low-percentage baseline were genuine and unrelated to sample prep (Table).

Intact mAb Mass Measurement

Intact human IgG1 was analyzed by reverse-phase LC/MS to evaluate molecular integrity and sample composition. The raw data for the protein peak is shown in Figure 1A. Multiple glycosylation isoforms (glycoforms) were found as shown in the deconvoluted result from Figure 1C. Glycoforms were assigned based on characteristic mass distances of 162 Da, indicating a rising number of galactose sugar units.

Figure 1A is the reverse-phase chromatograph and mass detection for the test protein. Figure 1B, the mass spectrum showing the multiply charged envelope for this protein, in the main peak shown in Figure 1A. The figure comprises a series of multiply charged ions reflective of the intact mass of the molecule where the m/z values represent the molecular weight of the intact IgG, 140 kDa, divided by the associated charge state. Peaks to the right of the graph signify molecules carrying fewer charges than those on the left-hand side. Application of a maximum entropy algorithm to that data to generate Figure 1C, the deconvoluted or neutral (zero charge) mass spectrum.

Positive electrospray ionization (ESI) induces charges on basic amino acid residues. As a rule of thumb, there is approximately one such residue every for every 1,000 Da. Therefore a 140 kDa protein can hold between 50 and 150 charges. The “rule” breaks down for larger, conformational constrained, or partially denatured proteins where basic residues may be charge-inaccessible due to the molecule’s shape.

Figure 1C shows the glycoforms present in the test protein. The first peak in the figure, G0F/G0F, represents the intact molecular weight for the first glycoform, 147,482 Da. The remaining peaks, which differ in molecular weight by 162 Da, represent the remaining four glycoforms, each containing an additional 162-Da galactose sugar residue.

The three sections of Figure 1 illustrate several important points. The test protein is predominantly glycosylated in its native state. Knowing this, and the types of sugar structures normally found on an IgG1, it is possible to obtain a read on the correct intact molecular weight, which in turn confirms the expected amino acid sequence. Note that the glycoform ratios provide a semi-quantitative measure of their abundance within the sample, which for biosimilars is critical for both characterization and comparison to originator proteins. Additionally, glycoform ratio serves as a critical quality attribute within and among several manufacturing batches of the same protein.

Figure 1. Evaluation of molecular integrity and sample composition of intact human IgG1. (A) Reverse-phase chromatograph and mass detection for the test protein. (B) Mass spectrum showing the multiply charged envelope for the test protein. (C) Deconvoluted or neutral (zero charge) mass spectrum.

Characterization of the N-Linked Glycosylation Pattern

Human IgG1 was digested with trypsin for PTMs characterization. The peptide mixture was then separated by reverse-phase chromatography and analyzed by mass spectrometry. Figure 2 shows the LC-MS/MS chromatogram obtained from trypsin-digested human IgG1. The raw data was interrogated using Byonic™ software (Protein Metrics) to identify peptides and PTMs. The result shows glycopeptides were found on the Fc region of heavy chain, which is consistent with previous observations.

The highlighted insert shows the sequence obtained for the chain C subunit sequence of the test IgG. Two short sub-sequences were not covered by our data: the sequence ASPK at the C-terminus (upper left in the graphic), and K (lysine) in the upper right. However, the intact mass determination discussed previously, and the existing sequence information, is sufficient to conclude that the protein is fully characterized.

Figure 2 indicates that the PTMs we were looking for are present as well. Three common PTMs, glycosylation, oxidation, and deamidation, are highlighted. These are labeled in Figure 2 below the sequences on which they were found. Deglycosylation and glycosylation both occurred on the same sequence, TKPREEQYNSTYR. It is apparent from the relative peak dimensions that the native form of this sequence protein is glycosylated, with a very small fraction remaining un-glycosylated.

Also present are peptides that experienced oxidation on the methionine sulfur, and deamidation on the sequence PENNY, a common motif in IgGs that is particularly susceptible to loss of an amide function. The take-home lesson is these PTMs are quantifiable using the HR/AM approach. We found, for example, that oxidation occurred approximately 60% of the time in this motif.

Figure 2. LC-MS/MS chromatograph obtained from trypsin-digested human IgG1. Inset: Sequence obtained for the chain C subunit of the test IgG.

Discussion and Conclusion

These results demonstrate the suitability of HR/AM mass spectrometry for the comprehensive characterization of a human IgG antibody. The combination of intact mass determination and immunoglobulin-degrading enzyme of Streptococcus (IdeS) proteolysis allows routine quantification of glycosylation patterns that potentially affect the drug’s circulating half-life, immunogenicity, and overall clinical efficacy.

Optimized sample preparation followed by HR/AM mass spectrometry represents a novel approach toward solving the complex heterogeneity of mAbs. Specifically, these methods confirmed the correct amino acid sequence and the identity, location, and relative abundance of common PTMs.  

Greg Kilby, Ph.D. (, is vp and COO for Protea

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