Sequencing a Bispecific Antibody by Controlling Chain Concentration Effects When Using an Immobilized Nonspecific Protease

Abstract

Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor. Low concentration samples resulted in the elimination of large peptide products due to a greater number of enzymatic cleavages. A competitive inhibitor rich in arginine residues reduced the number of enzymatic cleavages to the protein and retained large molecular weight products. The digestion of a bispecific antibody with competitive inhibition of aspergillopepsin I maintained large peptide products better suited for sequence reconstruction, resulting in complete sequence coverage from a single LC-MS/MS analysis.

Graphical Abstract

Characterizing the primary structure of a protein is essential to drug development. For instance, accurate identification of the primary structure of antibodies ensures their safety and effectiveness as therapeutic agents. The number of antibodies approved as biologic therapeutic drugs has increased in recent years.¹ Bispecific antibodies (bsAbs) are a growing class of heavily researched antibody therapeutics.^2–5 While monospecific antibodies are produced naturally in the body and target a single epitope with both variable regions, bsAbs are highly engineered molecules that target a different epitope with each variable region.⁶ A wide variety of bsAb structures have been engineered; some mimic the IgG structure of monospecific antibodies, while others are a single chain containing two variable regions connected by a linker.⁶ Extensive characterization of these highly engineered bsAbs is necessary to ensure their correct production.

Mass spectrometry is the principle method for determining the primary structure of antibodies.⁷,⁸ Intact analysis of antibodies by charge variant methods are used to determine the molecular weight and possible variants or post-translational modifications (PTMs) present on the molecule.^9–12 However, fragmenting large molecules does not generate complete fragment ion coverage to validate the entire sequence.^13–16 Typically, specific protease digestions complement the intact analysis to generate higher sequence coverage and site-localize PTMs. Generally, proteins are digested overnight with trypsin and analyzed by LC-MS/MS. The observed peptides are then stitched back together following a database search to reconstruct the original protein sequence. However, trypsin digests often result in incomplete sequence coverage. The peptides generated by trypsin do not overlap in sequence and are sometimes not observed on a reverse phase column due to their extreme high or low hydrophobicity.¹⁷ Additional specific protease digestions are necessary to unambiguously confirm the protein sequence.¹⁸,¹⁹ Multiple digestions require multiple instrument analyses, increasing the time necessary to obtain complete coverage.

Nonspecific proteases are a novel alternative to specific proteases for antibody analysis.²⁰,²¹ Aspergillopepsin I is a nonspecific aspartic protease that functions in 8 M urea and acidic conditions.²² Our lab demonstrated the implementation of an immobilized aspergillopepsin I enzyme reactor to digest monospecific antibodies and analyze them in a single LC-MS/MS analysis.²³,²⁴ Although the enzyme is nonspecific, the resulting chromatographic profiles and peptide coverage maps are highly reproducible under consistent working conditions (i.e., digestion time, sample concentration, buffer conditions, etc.). Aspergillopepsin I is bound to beads and packed into a column to control the amount of time the protein is in contact with the enzyme. The digestion stops once the sample elutes from the column. Digesting a protein with aspergillopepsin I for hundreds of milliseconds results in full characterization of the protein from a single LC-MS/MS analysis, reducing preparation and analysis time. Multiple overlapping peptides are observed within this single analysis and provide unambiguous sequence coverage of a molecule.²⁴ Varying the flow rate through the column dictates the peptide size distribution; as flow rate increases, the interaction time between enzyme and protein decreases, resulting in fewer cleavages and longer peptides that overlap in sequence.²³

The structures of bispecific antibodies present new challenges for analysis with the enzyme reactor. Here, we analyze an IgG-like bsAb with a common light chain.²⁵ This molecule retains the ~150 kDa structure of a monospecific IgG antibody (i.e., two heavy chains and two light chains). However, each heavy chain has a different variable region and a knob-in-hole mutation.²⁶,²⁷ Therefore, the relative concentration of each heavy chain is half that of the light chains, which are identical to reduce mispairing.²⁸ The differing chain concentrations were found to affect the rate of digestion by the immobilized nonspecific enzyme reactor. This work uses standard proteins to investigate the effect of protein concentration on the rate of digestion using the aspergillopepsin I enzyme reactor. A competitive inhibitor mitigates the concentration dependence and is applied to a bispecific antibody with a common light chain.

EXPERIMENTAL PROCEDURES

Materials.

Equine apomyoglobin from skeletal muscle, lysozyme chloride from chicken egg white, β-lactoglobulin from bovine milk, aspergillopepsin I, N-(2-aminoethyl)-maleimide trifluoroacetate salt (NAEM), tris(2-carboxyethyl)-phosphine hydrochloride (TCEP), and sodium cyanoborohydride were purchased from Sigma (St. Louis, MO). Protamine sulfate salt from salmon was graciously provided by Dr. Juan Ausió but can also be purchased from Sigma (St. Louis, MO). One micrometer aldehyde/sulfate latex beads were purchased from Invitrogen (Carlsbad, CA). The IgG-like bispecific antibody biosimilar of ACE-910 (emicizumab) was purchased from Creative Biolabs (Shirley, NY).²⁹,³⁰ IdeS (FabRICATOR) was purchased from Genovis (Cambridge, MA). All other materials were purchased from Sigma unless otherwise noted.

Enzyme Reactor Assembly.

Conjugation of aspergillopepsin I to 1 μm aldehyde/sulfate solid sphere beads was performed as previously described.²⁴ Briefly, ~10 μg of aldehyde functionalized beads were conjugated with aspergillopepsin I. The Schiff base formed during the reaction was selectively reduced with sodium cyanoborohydride. Free aldehyde groups on the beads were blocked with tris and reduced in the same manner. The beads were washed with water between each step and stored dry at 4 °C. Beads were benchmarked against previous ~1 s digestions of 0.2 μg/μL apomyoglobin, as previously described.²⁴

Sample Preparation.

One nanomolar aliquots of equine apomyoglobin samples were reconstituted to the desired concentration using 8 M urea in 50 mM ammonium acetate buffer at pH 4 (digestion buffer). Stock solutions of chicken lysozyme, bovine β-lactoglobulin, and bovine trypsinogen were prepared at ~5 μg/μL using 0.1% acetic acid in water. Individual protein samples were prepared by evaporating an aliquot to dryness. Samples were reduced by reconstituting in 15 mM TCEP in 8 M urea and 0.5% acetic acid in water and alkylated with 10 mM NAEM in 8 M urea and 0.5 M ammonium acetate in water at pH ~7.²⁴ The samples were then brought to the desired concentration and pH ~4 using digestion buffer.

A stock solution of protamine sulfate from salmon was prepared at ~10 μg/μL in water. When used to treat a sample, the final concentration of protamine was 0.2 μg/μL.

bsAb Preparation.

Approximately 8.5 μg of bispecific antibody were first digested with IdeS to cleave below the hinge region, creating subunits similar in molecular weight.³¹ This is not necessary for the efficacy of the enzyme reactor, but ensured that experimental differences were a function of concentration effects and not weight differences. The results of the digestion were evaporated to dryness. Reduction and alkylation of the bsAb with TCEP and NAEM were carried out as described above (Figure 1). The final volume was diluted to 0.2 μg/μL with digestion buffer. This procedure was repeated for a protamine treated digestion such that the final concentration of both bsAb and protamines were ~0.2 μg/μL.

Figure 1.

Representation of bsAb preparation prior to digestion with the enzyme reactor. The green represents the common light chain. The blue and pink indicate the variable regions of the heavy chain. The black and gray represent the knob-in-hole modification. The white regions are sequence identical between the two heavy chains. The light chains are twice as abundant as the other subunits following this protocol.

Enzyme Reactor Digestion.

A 2 mm packed bed of aspergillopepsin-conjugated beads was prepared in an in-house fritted 360 μm O.D. × 150 μm I.D. fused silica capillary.³² Protein samples at various concentrations were digested for a range of calculated digestion times as described previously (see Supplemental Table S1 for exact measurements).²⁴ Following digestion, samples were diluted with digestion buffer to ~1 pmol/μL if necessary.

Chromatography and Mass Spectrometry.

An Agilent Technologies (Palo Alto, CA) 1100 Series binary HPLC system coupled to an in-house modified Thermo Scientific LTQ-FT Ultra mass spectrometer (San Jose, CA) was used for evaluation of all standard protein digests.³³ The ESI source voltage was biased at 2.5 kV, and the heated capillary temperature was 250 °C. Approximately 400 fmol of digestion products were pressure-loaded onto an analytical column packed with 10 cm of 3 μm diameter, 1000 Å PLRP-s packing material (Polymer Laboratories; Amherst, MA) within a 360 μm O.D. × 75 μm I.D. fused silica capillary.³²,³⁴ Samples were washed with 0.3% formic acid in water (Solvent A) for 1 h for desalting. Peptides were eluted with a gradient of 0–25–55–100% Solvent B in 0–5–35–40 min. (Solvent B = 72% ACN, 18% IPA, 10% water, 0.3% formic acid.) Following a 100 000 resolution MS¹ scan, the top 3 peptides were dissociated by collisionally activated dissociation (CAD) and 45 ms of electron transfer dissociation (ETD). All MS² scans were analyzed in the ion trap.

For bispecific antibody digestions, the HPLC system was coupled to a Thermo Scientific Orbitrap Fusion tribrid mass spectrometer (San Jose, CA). The ESI source voltage was biased at 2.5 kV, and the heated capillary temperature was 275 °C. Approximately 400 nL (~400 fmol) of the digestions were pressure-loaded onto the same PLRP-s column. The column was rinsed for ~45 min with 100% A to remove salts. Peptides were eluted with a gradient of 0–25–50–100% B in 0–5–80–85 min at 50 °C. A tiered data dependent method was employed to detect and fragment the eluting peptides as described previously.²⁴ In short, all ions in the MS¹ scan were sorted into three priorities for subsequent fragmentation. The dissociation method was chosen based on the charge density of the precursors (high-capacity ETD for high charge density species, collisional dissociation for low charge density species).³⁵ Large ions were first priority and were fragmented with ETD (10–24 charges; 500–925 m/z; calibrated ETD reaction time) and higher-energy collisional dissociation (HCD) (8–24 charges; 1100–1500 m/z; 25 ± 3% normalized collision energy (NCE)) for a high resolution (120 K) MS² scan with one additional microscan. Medium ions were second priority and were fragmented by ETD (5–9 charges; 300–925 m/z; calibrated ETD reaction time) and HCD (4–7 charges; no m/z restraints; 25 ± 3% NCE) for a high resolution (60K) MS². Small ions were the lowest priority and were fragmented by ETD (3–4 charges; no m/z restraints; calibrated ETD reaction time) and CAD (2–3 charges; no m/z restraints; 30% NCE) in the ion trap for low resolution MS² scans.

Data Analysis.

For standard protein digestion results, data files were searched using Byonic version 3.3.3 (Protein Metrics; San Carlos, CA) against the known sequence by individual dissociation type following conversion to.MGF files. Manual verification of peptides was performed on a subset of peptides from each analysis. Spectra were averaged using Thermo Qual Browser version 4.0.27.10.

For bispecific antibody digestion results, data files were searched using Byonic within Proteome Discoverer (version 2.2.0.386; San Jose, CA) as described previously.²⁴ Manual verification of peptides was performed to confirm the fragment ions observed. ProSight Lite (version 1.4) was used to produce fragment ion maps after manual verification. Calculation of all peptide abundances was performed using Proteome Discoverer. Peptides identified at 1% FDR in the Byonic searches were grouped using a 10 ppm tolerance, a minimum S/N ratio of 5, and quantified by mass area. Only peptides with a confidence value of High from Proteome Discoverer were used. Pairwise ratios were calculated of the treated over untreated samples with a maximum ratio value of 500.

RESULTS AND DISCUSSION

Enzymatic Cleavages Increase as Concentration Decreases.

Apomyoglobin was used as a standard to explore concentration effects on digestion with the aspergillopepsin I enzyme reactor. Previously, proteins were digested at a concentration of 0.2 μg/μL.²³,²⁴ Here, concentrations of 0.1, 0.05, and 0.02 μg/μL apomyoglobin were digested to evaluate changes in the resulting peptide mixture. At constant digestion time, decreasing the concentration of apomyoglobin led to an increase in smaller peptides at the expense of the large peptides necessary for overlapping sequence coverage (Figure 2). This implies that as the protein analyte concentration decreased, the number of cleavages by aspergillopepsin I increased. In other words, the reactor is diffusion limited at concentrations below 0.1 μg/μL.

Figure 2.

Total ion current chromatograms for the digestion of apomyoglobin at (A) 0.2, (B) 0.1, (C) 0.05, and (D) 0.02 μg/μL. All digestion times are ~1 s. Generally, smaller peptides eluted at ~12–20 min, while larger peptides eluted at ~25–35 min. The stars in (A) and (B) denote undigested apomyoglobin.

Diffusion limited enzyme reactors are dependent on the ability of the substrate to diffuse to the enzyme and for the products to diffuse away from the enzyme.^36–40 Aspergillopepsin I presents an additional layer of complexity. Because aspergillopepsin I is nonspecific, all products of an enzymatic cleavage are also substrates for additional cleavages. This accounts for the increase in abundance of smaller peptides. Immobilized reactors can also be catalytic limited, in which all active sites of the enzyme are occupied by a substrate.⁴⁰ Products have time to diffuse away under these conditions and therefore do not react repeatedly. The reaction is closer to being catalytically limited at concentrations from 0.1–0.2 μg/μL, as evidenced by the preservation of large peptides. These concentration effects were reproduced with other proteins, such as chicken lysozyme, bovine β-lactoglobulin, and bovine trypsinogen (see Supplemental Figures S1–3).

The chromatograms appear to indicate that apomyoglobin is most affected by the change in starting concentration. Many factors likely affect the digestion with the aspergillopepsin I immobilized reactor. The structures of apomyoglobin (Protein Data Bank (PDB) entry: 1YMB⁴¹), chicken lysozyme (PDB:1DPX⁴²), β-lactoglobulin (PDB: 1BEB⁴³), and bovine trypsinogen (PDB: 1TGB⁴⁴) may contribute to how sensitive the proteins are to these concentration effects. Apomyoglobin consists primarily of α-helices with no β-sheets, whereas chicken lysozyme, β-lactoglobulin, and bovine trypsinogen all contain β-sheets to some extent. We hypothesize that α-helices are more effected by the starting concentration than β-sheets. The accessibility of the enzyme to the cleavage site may be impacted by the secondary structure of the protein of interest, causing these structural effects.

Although it may be ideal to use this enzyme reactor with a sample concentration at 0.2 μg/μL, not all biological samples can be so ideally concentrated. Therefore, methods of mitigating these concentration effects were explored to make the enzyme reactor amenable to a variety of sample concentrations. One way to compensate for the concentration effects is to decrease the amount of time the sample is in contact with the reactor, i.e. increase the flow rate. Digestion times of ~0.7 and ~0.4 s for the 0.1 and 0.05 μg/μL samples, respectively, were comparable to that of a ~1 s digest at 0.2 μg/μL (Supplemental Figure S4). A comparable digestion for the 0.02 ug/uL sample was estimated to be ~0.2 s. This short digestion time requires higher flow rates or shorter bed lengths. However, bed lengths shorter than 2 mm would be difficult to reproduce, and the frit would not be able to withstand the higher pressures required for the higher flow rates. Consequently, other methods of compensating for the concentration effects need to be explored. A competitive inhibitor was evaluated for this purpose.

Protamines from Salmon as a Competitive Inhibitor.

Previous experiments showed that aspergillopepsin I favors arginine or lysine in the P1 position.²³,²⁴ Therefore, an ideal inhibitor would be a peptide/protein with many basic residues so the enzyme preferentially cleaves the basic inhibitor instead of the sample protein. Salmine, a mixture of protamines from salmon, was used to test this hypothesis. Protamine is a member of sperm nuclear basic proteins that replace histones toward the end of spermatogenesis.^45–47 These proteins are typically less than 100 amino acids in length. The four major protamines in salmine are 30–32 amino acids in length with ~70% of those residues being arginine (see Supplemental Table S2 for sequences of salmine).⁴⁸ This mixture of protamines was added to variously concentrated apomyoglobin samples such that the final concentration of protamine was 0.2 μg/μL. The chromatograms in Figure 3 show a ~1 s digestion of apomyoglobin samples treated with protamines.

Figure 3.

Total ion current chromatograms for the digestion of protamine treated apomyoglobin at (A) 0.2, (B) 0.1, (C) 0.05, and (D) 0.02 μg/μL. All digestion times are ~1 s. The starting concentration of protamine was 0.2 μg/μL for each sample. The abundance of protamine in each sample differs because the sample was diluted to ~0.017 μg/μL (~1 pmol/μL) apomyoglobin prior to analysis. The differing degrees of dilution resulted in a higher abundance of protamine at lower apomyoglobin concentrations. The stars in (A), (B), and (C) denote undigested apomyoglobin. In all chromatograms, the first peak at ~12 min is attributed to protamine.

A comparison of the chromatograms of apomyoglobin digestions before (Figure 2) and after (Figure 3) protamine treatment shows that the abundance of high molecular weight products increased after treatment with protamine, including the abundance of undigested apomyoglobin. The introduction of protamine to the diffusion limited system (Figure 3C and D) resulted in digestion profiles similar to the profiles generated by more highly concentrated digestions (Figure 2A). Adding protamine to almost catalytically limited digests resulted in a high abundance of undigested apomyoglobin (Figure 3A and B). This implies that protamine is slowing the rate of digestion of apomyoglobin regardless of concentration. This phenomenon was reproduced for 0.05 μg/μL digestions of chicken lysozyme and β-lactoglobulin (Supplemental Figures S5–6). Figure 4 depicts this trend by tracking the abundance of a selection of peptides across the concentrations tested for all proteins. Adding protamine enables the use of the aspergillopepsin I enzyme reactor on samples an order of magnitude lower in concentration than previously possible.^23,24 Ultimately, this allows a greater range of samples with a known concentration to be digested.

Figure 4.

Comparison of selected peptides from ~1 s digestions of (A) apomyoglobin, (B) β-lactoglobulin, and (C) chicken lysozyme across different concentrations as well as treatment with 0.2 μg/μL protamine. As concentration decreases in the absence of protamine, the larger peptides decrease in abundance, while smaller peptides increase in abundance regardless of basicity. For example, peptides G2-L280 and T71-L138 from apomyoglobin are similar in size but have different pI values of 4.98 and 9.26, respectively. Therefore, this trend is independent of the hydrophobicity of the peptides. Treatment with protamine inhibits the production of smaller peptides while preserving larger peptides. However, at 0.02 μg/μL with protamine, the larger peptides were not recovered at the same abundance as that in the 0.2 and 0.05 μg/μL digestions.

Protamine was digested by itself for ~1 s at 0.2 μg/μL to evaluate the digestion (Supplemental Figure S7). Undigested protamine and its digestion products eluted at the start of the gradient. These digestion products were also observed when protamines were used as a competitive inhibitor.

Protamine must be competing with the analyte for access to the active site on aspergillopepsin I. Protamine is capable of diffusing to and from the active site faster than the analyte protein due to its lower molecular weight.^49–51 The high diffusion rate of protamines in combination with the high abundance of preferential cleavage sites allows it to take up active sites and prevent the protein of interest from being continually digested.

Because the peak associated with protamines elutes from the column first, rinsing the column with 4% solvent B eliminated peaks associated with protamines (Supplemental Figure S8). Some peptides from the protein of interest were also lost during this rinse. However, these peptides are typically low in molecular weight and are usually found within larger peptides, which increased in abundance by adding protamines. As shown in Figure 4, treating samples with protamine reduced the rate of digestion for most proteins. However, the rate of reduction varied between proteins. The 0.05 μg/μL bovine trypsinogen digest with protamine resulted in a high abundance of undigested trypsinogen and a low abundance of digestion products, suggesting that protamine prevented trypsinogen digestion. (Supplemental Figure S9). Trypsinogen should not be active because it was reduced, alkylated, and is in a denaturing buffer. This lack of digestion was not reproduced on the array of proteins tested. However, it is possible that other proteins may display a similar loss of digestion due to their interaction with protamine.

Unambiguous Sequence Coverage of a Bispecific Antibody.

Recently, the digestion and unambiguous sequence coverage of adalimumab was obtained using a ~1s digestion at 0.2 μg/μL.²⁴ A tiered decision tree instrument method on the Thermo Orbitrap Fusion was implemented to optimize the fragmentation of eluting peptides. The decision tree dictated if a precursor was fragmented either by ETD or collisional fragmentation, and whether it was analyzed in the Orbitrap or the ion trap.²⁴ Extending this methodology to bsAbs was driven by the increased research on bispecific antibodies in drug development.⁴ Generally, each chain of an IgG-like bsAb is different, making their concentrations half that of a monospecific antibody. Therefore, the digestion of a bsAb should be diffusion limited and result in a greater number of enzymatic cleavages in comparison to a monospecific antibody. The biosimilar of emicizumab was used to demonstrate the diffusion limited digestion of a bsAb.²⁹,³⁰ Emicizumab employs a common light chain with a knob-in-hole mutation in the Fc/2 region. The effect of concentration should be evident between subunits because the concentration of the light chain is twice that of the heavy chains following reduction and alkylation. Figure 5A shows the chromatogram of the bsAb digested at 0.2 μg/μL. The majority of ion current lies within the first 15 min of analysis, which is indicative of a high abundance of small to medium sized peptides (~0.8–5 kDa). However, larger peptides are more desirable for analysis because they ensure more confident connectivity.

Figure 5.

Total ion current chromatograms for the digestion of a bsAb at 0.2 μg/μL (A) without and (B) with protamine. Digest times for both chromatograms were ~1 s.

Protamine was introduced to the bsAb sample following reduction and alkylation such that the concentration of both bsAb and protamine was 0.2 μg/μL. This mixture was digested for ~1 s to give the chromatogram shown in Figure 5B. Qualitatively, the ion current is not as concentrated in the beginning of the gradient (~20–28 min) compared to the untreated digestion. The abundance of larger peptides increased in the treated sample as evidenced by the increase in ion current later in the gradient. All identified peptide abundances were calculated. Figure 6A shows that a majority of peptides (~61%) were observed in both digestions at similar abundances (within a 4-fold change). Minor differences in the experimental setup, such as changes in chromatography, flow rate, and differing ion suppression, could result in slight changes in peptide abundance. Additionally, sample quantity constraints only allowed for one digestion with and without protamine. For these reasons, a 4-fold threshold was used to classify a change in abundance as substantial. Over 200 peptides were observed in the treated digestion that met the threshold. Conversely, only 20 peptides were more abundant in the untreated digestion. While these peptides were still identified in the untreated sample, increasing the abundance of peptides is desirable because it can result in a higher quality fragmentation spectrum. Substantially, 145 unique peptides were observed in the treated digestion and 62 of those peptides were above 5 kDa. Comparatively, only 15 out of the 94 unique peptides in the untreated digestion were above 5 kDa. Figure 6B plots the log of the ratio of abundance between the treated and untreated digestion against the molecular weight. The points between the dashed lines are considered similar because they were within a 4-fold change in abundance. Peptides across the molecular weight range increased in abundance after treatment with protamine and are depicted in Figure 6B as the points outside the dashed lines. Importantly, peptides >5 kDa significantly increased in abundance. Therefore, treating the sample with protamine provided the expected results: larger peptides are retained at higher abundances due to a reduction in the number of enzymatic cleavages. These larger peptides provide less sequence ambiguity than the smaller peptides produced from the untreated sample.

Figure 6.

Comparison of peptides observed in the bsAb digestions. (A) Histogram depicting the number of peptides grouped by difference in abundance. (B) Scatter plot of the log of the ratio of treated to untreated abundances by molecular weight. Points between the gray dashed lines represent peptides that were within 4× variation. The colors of the points correspond to the colors in (A).

Figure 7 shows the peptide map as well as observed fragment ions by ETD (red) and collisional fragmentation (blue; both CAD and HCD): 1096 of 1101 fragment ions were observed, resulting in ~99.5% sequence coverage. Although protamine treatment successfully retained larger peptides, it did not alter the preferential cleavage of particular residues. For example, as shown in Figure 7C and E, there is a missed fragment cleavage at D29-V30. No peptides were observed that contain these residues within a single peptide. This was likely a result of aspergillopepsin I preferentially cleaving here prior to other sites along the molecule, as previously reported.²⁴ These minor preferential cleavages account for two of the five missed cleavages.

Figure 7.

Peptide and composite fragment ion coverage maps of (A) common light chain, (B) Fd′ A, (C) Fc/2 A, (D) Fd′ B, and (E) Fc/2 B. The colors of the peptide lines correspond to the colors represented in Figure 6A. Red fragment ions represent c and z· ions, while blue fragment ions represent b and y ions. The yellow N61 in maps (C) and (E) show the localization of a G0F or G1F glycan tree.

CONCLUSIONS

The analyte concentration substantially changed the molecular weight distribution of peptides produced from a digestion with the aspergillopepsin I enzyme reactor. These concentration effects must be controlled to broaden the utility of the enzyme reactor because biologically relevant samples are variable in their protein distributions. For instance, each chain of an IgG-like bispecific antibody is generally half that of a monospecific antibody. Introducing protamine to the sample mitigated the effect of concentration on the resulting digestion. Protamines were an effective competitive inhibitor to shift the system closer to a catalytically limited state. Protamines improved the characterization of an IgG-like common light chain bispecific antibody. Unambiguous sequence coverage of the antibody was obtained from a ~1 s digestion and a single chromatographic analysis. While this work used primarily ~1 s digestions to demonstrate these concentration effects, controlling the flow rate through the reactor can easily further tune the peptide size distribution. For example, very large proteins (>30 kDa) can be digested with protamine at very short digestion times (~400–600 ms) to provide high abundance peptides containing a significant percentage of the molecule. These large peptides can then be targeted for extensive characterization with new techniques.⁵²,⁵³