1. Introduction

2. Results and Discussion

2.1. Method Development: Lys-C Digestion Method 1

Lys-C digestion Method 1 was developed to be suitable for Ab-1 DS or any protein sample with a concentration ≥50 mg/mL. Our experiment design covered several considerations for the enzymatic digestion of the proteolysis-resistant protein Ab-1. (1) In this method, Ab-1 was denatured in 6 M guanidine HCl (GuHCl), reduced with dithiothreitol (DTT), and carbamidomethylated using iodoacetamide (IAM). (2) We chose DTT over tris(2-carboxyethyl)phosphine (TCEP) as the reducing agent because TCEP can reduce oxidized Met residues [29] on the molecule prior to digestion, resulting in less accurate quantitation of that attribute. (3) The most commonly used alkylation reagents are iodoacetic acid (IAA) and IAM. We used IAM because IAA is typically dissolved in 1 N NaOH, and that would increase the pH of the sample mixture and introduce a negative charge to peptides [30,31]. (4) The reduced and alkylated protein was digested with Lys-C enzyme after a simple dilution step to lower GuHCl to ≤2 M. GuHCl concentrations greater than 2 M affect enzyme activity. At a concentration ≤2 M GuHCl, Lys-C retains 90% of its activity while keeping the protein in a more denatured condition to help digestion of the protease resistant Ab-1, whereas trypsin only retains 50% of its activity and requires a desalting/buffer-exchange step to remove GuHCl prior to protein digestion [32,33]. Therefore, with a dilution step rather than a desalting/buffer-exchange, this Lys-C digestion protocol was simplified without the need for the additional desalting step.

To optimize the Lys-C digestion conditions, we focused on the following parameters: reagent concentration, buffer pH, incubation time and temperature during reduction, alkylation, and digestion. Table 1 provides a detailed list of the conditions tested. It is important that the antibody is sufficiently denatured, unfolded, and reduced prior to digestion, allowing Lys-C to easily access the molecule for a complete digestion. For the protein reduction to break its disulfide linkages and improve its digestion, we optimized the level of the reducing agent DTT to achieve complete reduction without an excess of DTT. We examined the amount of time required to fully reduce the protein, and we evaluated and selected a pH that would ensure complete reduction and minimize artificial deamidation. The optimal condition determined for each parameter is shown by a bold, underlined value in Table 1. The best reduction condition was achieved using 3.5 mM DTT and pH 7.5 at 37 °C for 1 h. Following reduction, alkylation of the sulfhydryl groups on the cysteine residues was required to prevent reformation of the disulfide bonds. IAM was used in 2.5-fold excess of DTT to ensure complete alkylation. However, if too much IAM was used, it would over-alkylate the protein, which would alkylate amino acid residues other than cysteine (e.g., lysine). We tested the alkylation conditions including IAM concentration and incubation time. The optimized condition for alkylation was 8.5 mM IAM for 15 min at 37 °C in the dark. The remaining (little) IAM after reaction with DTT was unstable under light and in the presence of excess water from a three-fold dilution immediately after the alkylation step. Therefore, there was no need to use additional DTT to quench the alkylation step.

Table 1. Method 1 optimization of test conditions.

Table 1. Method 1 optimization of test conditions.

	Parameter	Test Conditiona	Note
Reduction	DTT (mM)	3.5, 5	6 M GuHCl for protein denaturation at 37 °C. Use Met (10–20 mM) as a scavenger throughout the procedure to reduce artificial oxidation
	Buffer pH	7, 7.5, 8
	Incubation time (min)	30, 45, 60, 90
Alkylation	IAM (mM)	6.8, 8.5	Alkylation at 37 °C in the dark
	Incubation time (min)	15, 30
Dilution	Scheme 1. Dilute 3× before digestion and then dilute 3× before LC/MS injection Scheme 2. Dilute 9× before digestion		Dilution to lower GuHCl level (≤2 M) in digestion and more dilution (<1 M) before LC/MS injection
Digestion	Lys-C to protein ratio	1:20, 1:10	Digestion at pH 7 to minimize artificial deamidation Digestion temperature: 37 °C
	Incubation time	30 min, 1 h, 2 h

^a Bold, underlined value indicates the optimal test condition. DTT, dithiothreitol; GuHCl, guanidine HCl; IAM, iodoacetamide; LC/MS, liquid chromatography-mass spectrometry; Met, methionine.

As this protocol eliminated the desalting step, a simple dilution of the sample into the digestion buffer lowered the GuHCl concentration (≤2 M). Two dilution schemes were examined as shown in Table 1. Between them, Dilution Scheme 1 with a 3-fold dilution (before digestion) performed well, while the digestion efficiency was decreased using a 9-fold dilution (Dilution Scheme 2). After the dilution, the Lys-C digestion was carried out at pH 7 to minimize artificial deamidation occurring during protein digestion [34]. Lys-C to protein ratios (enzyme-weight: protein-weight) of 1:20 and 1:10 and digestion times were evaluated. The results revealed that efficient protein digestion was achieved after 2 h of Lys-C digestion at 37 °C. One important aspect when developing such a method was to choose the conditions that will minimize the degree of artificial modifications induced, balancing maximum digestion efficiency with minimum artificial modifications. Besides digestion at neutral pH for minimal artificial deamidation, free Met was added as a scavenger throughout the sample preparation to reduce artificial oxidation [35].

2.2. Method Optimization: Lys-C Digestion Method 2

The Ab-1 DP is formulated in a capsule form, i.e., an enterically coated solid form. After dissolving the capsule in liquid for analysis, the concentration is 3 mg/mL. With protein at such low concentration, any digestion using a dilution scheme is especially challenging; as the protein concentration in the digestion step is very low (0.03 mg/mL) if Method 1 was used. Our main focus was to find lower concentrations of the denaturing agent that were still effective for protein denaturing, so that a smaller dilution factor could be used. Therefore, lower concentrations of GuHCl (2–5 M) were tested, and the minimum concentration used to get complete reduction was 4.7 M. Another method modification was to dissolve DTT in Tris HCl buffer instead of water to avoid further dilution of the sample. Other conditions remained the same as those used in Method 1, including reduction and alkylation reagent concentrations, Lys-C enzyme-to-protein ratio, and incubation times. In parallel, Table 2 lists the reagent and the protein concentrations after each step for both methods. Using Method 2, the final protein concentration after the digestion was 0.2 mg/mL. Note that the Met levels were maintained in the range 10–20 mM to prevent protein oxidation during the sample preparation [35]. As the recommended injection amount for a protein sample is 2.5–5.0 µg for LC/MS analysis, the injection volume was modified accordingly. This Lys-C digestion protocol was suitable for use with Ab-1 DP lots or for samples with low concentrations (≥3 mg/mL).

Table 2. Stepwise procedures and the concentration of the protein sample and reagents.

Table 2. Stepwise procedures and the concentration of the protein sample and reagents.

	Method 1	Reagent	Concentration	Method 2	Reagent	Concentration
Reduction	8 M GuHCl 85µL	GuHCl	6.0 M	8 M GuHCl 67µL	GuHCl	4.7 M
	1 M Tris pH 7.5/0.2 M Met 6 µL	Tris HCl	0.05 M	1 M Tris HCl pH 7.5/0.2 M Met/65 mM DTT 6 µL	Tris HCl	0.05 M
		Met	11 mM		Met	11 mM
	40 mM DTT 10 µL	DTT	3.5 mM		DTT	3.5 mM
	50 mg/mL Ab-1 12 µL	protein	5.1 mg/mL	3 mg/mL Ab-1 40 µL	protein	1.0 mg/mL
	Incubate at 37 °C for 1 h			Incubate at 37 °C for 1 h
Alkylation	200 mM IAM 5 µL	IAM	8.5 mM	200 mM IAM 5 µL	IAM	8.5 mM
	Incubate at 37 °C for 15 min in dark			Incubate at 37 °C for 15 min in dark
Digestion	Take 25 µLof the alkylated sample (125 µg) into a new tube			Take 68 µL of the reduced sample (68 µg) into a new tube
	Reduced sample 25 µL	protein	1.5 mg/mL	Reduced sample 68 µL	protein	0.4 mg/mL
	Add H2O 39 µL(GuHCl conc. <2 M)	GuHCl	1.8 M	Add H2O 74 µL (GuHCl conc. <2M)	GuHCl	2.0 M
	1 M Tris pH 7/0.2 M Met 6 µL	Tris HCl	0.1 M	1 M Tris HCl pH7/0.2 M Met 12 µL	Tris HCl	0.1 M
		Met	18 mM		Met	19 mM
	0.5 mg/mL Lys-C solution 13 µL	Lys-C: protein	1:20 (w:w)	0.5 mg/mL Lys-C solution 7 µL	Lys-C: protein	1:20 (w:w)
	Incubate at 37°C for 2 h			Incubate at 37 °C for 2 h
Dilution & Quench	Dilution (3x)	protein	0.5 mg/mL	Pipette 79 µL digested sample, dilute 3×	protein	0.2 mg/mL
	Add H2O 140 µL (GuHCl conc. <1M)	GuHCl	0.6 M	Add H2O 120 µL(GuHCl conc.<1M)	GuHCl	0.7 M
	Add 20 µL0.2M Met	Met	16 mM	Add20 µL 0.2M Met	Met	18 mM
	Add 10% TFA 5 µL to quench digestion	TFA	0.2%	Add 10%TFA 5 µL to quench digestion	TFA	0.2%

DTT, dithiothreitol; GuHCl, guanidine HCl; IAM, iodoacetamide; Met, methionine; TFA, trifluoroacetic acid.

2.3. Method Performance Evaluation

To evaluate the peptide map method performance, we focused on several aspects, such as protein reduction completion, alkylation efficiency, and digestion completion. Disulfide bonds are present in many proteins including Ab-1. Protein reduction and alkylation conditions play important roles for the complete reduction of disulfides. When optimizing the conditions for protein reduction, the reduction completion of disulfides was assessed and confirmed. Alkylation efficiency was also thoroughly evaluated. Efficiency decreased when alkylation of cysteine was not complete, i.e., <100% cysteine was alkylated. Over-alkylation was measured when the N-terminus or other amino acids besides cysteine (e.g., lysine) were alkylated. Therefore, we adjusted the alkylating agent and incubation time to balance under and over alkylation. The average alkylation efficiency was measured to be >95%. Optimizing reduction and alkylation conditions was also essential for improving protein digestion.

Protein sequence coverage is the key measurement when evaluating the performance of a peptide map method. In this study, the digestion conditions were optimized to achieve maximized protein sequence coverage. For Ab-1, Lys-C, as the enzyme choice, generated 100% sequence coverage as expected. However, the biggest challenge for this molecule in Lys-C digestion was the high level of missed cleavage peptides due to the enzymatic resistance of this molecule. The issue was the mis-cleaved peptide, L1+L2, which ranged from 9% to 15% in different sample preparations by different analysts or on different days. Mis-cleavage at such a high level leads to an inconsistency in method quantitation. Note that other mis-cleaved peptides (L3+L4 and L2+L3) were below 2%, which is less of a concern. Figure 1 shows the MS total ion current (TIC) chromatograms analyzed using the newly developed Lys-C peptide map method (Method 1) and illustrates the protein wild-type peptides L1 to L4 with some mis-cleaved peptide peaks. Note that the peptide mis-cleavages are common for peptide mapping. They are useful in some cases to improve sequence coverage. For example, a short peptide, usually three amino acids and less, is too hydrophilic to be retained in a reversed-phase (RP) high-performance liquid chromatography (HPLC) column. It often is washed out in the void volume with small molecule reagents used in sample preparation. With the peptide mis-cleavage event, the short peptide can be retained in a RP column and observed in LC/MS analysis when it attaches to another peptide. Therefore, the protein sequence coverage is improved. In contrast to mis-cleavages that cause incomplete digestion, over digesting the protein produces unspecific cuts, which is also undesirable. To minimize both mis-cleavages and unspecific cuts, we carefully optimized the digestion conditions and tested with higher amounts of enzyme and longer digestion times. In the end, with the optimized digestion conditions, we were able to bring down the level of mis-cleaved peptide L1+L2 to less than 5% from the 9–15% range.

Figure 1. MS total ion current chromatograms of Ab-1 by Method 1 (L1-L4 =peptides by Lys-C).

Figure 1. MS total ion current chromatograms of Ab-1 by Method 1 (L1-L4 =peptides by Lys-C).

Figure 2 displays typical MS TIC chromatograms of Lys-C digested Ab-1 following the Method 1 protocol. Both methods provided comparable results, providing a choice for analysts to use. Moreover, using these methods, we tested seven other molecules, including mAbs, bispecific antibodies, an antibody drug conjugate, and a Fab antibody fragment. Figure 3 shows the TIC chromatograms of Ab-2 (a mAb) as an example. Therefore, these two newly developed Lys-C map methods can be applied to various antibody formats.

Figure 2. MS total ion current chromatograms of Ab-1: Method 1 (bottom) and Method 2 (top). *Peaks in blank digest. (L1-L4 = peptides by Lys-C).

Figure 2. MS total ion current chromatograms of Ab-1: Method 1 (bottom) and Method 2 (top). *Peaks in blank digest. (L1-L4 = peptides by Lys-C).

Figure 3. MS total ion current chromatograms of Ab-2: Method 1 (bottom) and Method 2 (top).

Figure 3. MS total ion current chromatograms of Ab-2: Method 1 (bottom) and Method 2 (top).

2.4. Method Robustness

The optimized Lys-C digestion methods were evaluated and demonstrated to be robust. To assess the robustness, a half fractional factorial design of experiment (DoE) with five factors and two levels was performed (Table 3). Digest buffer pH, time, temperature, and reduction buffer pH were included because they are known to affect deamidation, pyroglutamate (pE) formation, isomerization, and succinimide formation. Injection volume changes would affect quantitation as sample load changes; therefore, it was also included in the DoE study. A total of 20 runs, including an additional 2 replicates and 2 center points, were completed for each method.

Table 3. The DoE design with factors and levels.

Table 3. The DoE design with factors and levels.

DoE Factor	Condition Specs	DoE Level (low/high)
Digest buffer (pH)a	7.0 ± 0.1	6.9 / 7.1
Digest time (min)a	120 ± 10	110 / 130
Digest temperature (°C)a	37 ± 2	35 / 39
Reduction buffer (pH)a	7.5 ± 0.1	7.4 / 7.6
Injection volume (µL)b	5 for Method 1 17 for Method 2	4 / 6 15 / 19

• ^a This DoE factor can affect deamidation, pE formation, isomerization, and succinimide.
• ^b This DoE factor can affect quantitation as the load increases.

The DoE study demonstrated the robustness of Method 1 and Method 2. Table 4 lists the statistics for the DoE study with relative abundance maximum (Max), minimum (Min), difference (Max-Min), average, standard deviation (SD), and relative SD (%RSD). Based on these results, both methods are robust for Ab-1 PTM quantification, as the RSD was 5% or less.

Table 4. DoE results: the statistics of relative abundance for Method 1 and Method 2.

Table 4. DoE results: the statistics of relative abundance for Method 1 and Method 2.

Statistics	Method 1		Method 2
	Signal Peptide (%)	N-terminal pE (%)	Signal Peptide (%)	N-terminal pE (%)
Max	13.2	7.1	13.9	7.3
Min	11.7	6.3	11.9	6.1
Difference (Max-Min)	1.5	0.8	2.0	1.2
Average	12.5	6.7	12.8	6.7
SD	0.4	0.2	0.5	0.3
% RSD	3.3%	2.8%	4.2%	5.1%

Min, minimum; Max, maximum; pE, pyroglutamate; SD, standard deviation; %RSD, relative SD.

2.5. Suitability for Ab-1 Stability Study

The major PTMs of Ab-1 from the manufacturing process were N-terminal signal peptide and N-terminal pE. In the stability studies, Ab-1 samples were subjected to chemical and physical stresses including oxidation by 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), elevated temperatures, and low pH. AAPH is a free radical generator that produces alkoxyl and alkyl peroxyl radicals. The AAPH stress model is used as an indicator of oxidation susceptibility for antibodies. Such stress often induces protein oxidation of Met or tryptophan residues [8,9]. The stress under elevated temperatures and low pH may produce protein deamidation of asparagine [7,17], or pE formation at N-terminal glutamic acid [19,20], etc. Even if it is not known whether these PTMs would affect biological activity, monitoring PTMs occurs as part of routine quality testing because such quality control testing can ensure the safety and efficacy of the DP. Using the newly developed Lys-C maps, we can identify and quantify these modifications of Ab-1. Figure 4 illustrates the protein wild-type peptides L1-L4 with well-separated PTM-modified peptides.

Figure 4. UV chromatograms of the protein digest of Ab-1 (L1-L4 = peptides by Lys-C; prepared using Method 1). M, methionine; W, tryptophan.

Figure 4. UV chromatograms of the protein digest of Ab-1 (L1-L4 = peptides by Lys-C; prepared using Method 1). M, methionine; W, tryptophan.

The Lys-C digestion method is stability indicating. Unstressed (control) and stressed samples were analyzed in parallel using the new method. The results of force-stressed Ab-1 DS samples using AAPH, high temperatures, and low pH are summarized in Table 5. The Ab-1 samples subjected to thermal stress conditions (storage at 40 °C and 50 °C) demonstrated an increase in N-terminal pE compared to the control sample. Deamidation was not detected at either of the two asparagine residues of Ab-1. It is worth noting, Ab-1 is particularly tolerant to high temperatures, and therefore, significant degradation upon thermal stress is not expected. The Ab-1 samples subjected to 10 mM AAPH demonstrated an increase in oxidation at two tryptophan residues, (W₁, W₂) in peptide L4 compared to the unstressed sample. No significant increase in oxidation was observed at other tryptophan or Met residues. The analysis of stressed samples demonstrated the Lys-C digestion method is stability indicating for attributes at critical sites for Ab-1. Relative levels of induced modifications were calculated as:

$$\%\mathrm{Attribute}=\frac{\sum \mathrm{Peak}\mathrm{Areas}\mathrm{of}\mathrm{modified}\mathrm{peptides}}{\sum \mathrm{Peak}\mathrm{Areas}\mathrm{of}\mathrm{wild}\mathrm{type}\&\mathrm{modified}\mathrm{peptides}}\times 100$$

Table 5. Attribute quantitation of the stressed Ab-1 DS (by Method 1).

Table 5. Attribute quantitation of the stressed Ab-1 DS (by Method 1).

Attribute	Relative Abundance (%)
	t=0	4 weeks at 40 °C	2 weeks at 50 °C	pH Control	pH 3.25	AAPH Control	10 mM AAPH
L1 + Signal peptide	9.6	9.0	9.3	10.3	10.2	10.6	10.0
N-terminal pE	5.7	7.3	9.5	6.4	7.0	7.3	6.7
L1 M oxidation	2.3	2.3	2.2	2.2	2.1	2.2	2.1
L1 W oxidation	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
L2 W oxidation	<0.1	ND	ND	0.1	0.2	0.1	1.1
L3 N1 deamidation	ND	ND	ND	ND	ND	ND	ND
L3 N2 deamidation	ND	ND	ND	ND	ND	ND	ND
L4 W1 oxidation	0.3	0.3	0.4	0.3	0.2	0.3	3.7
L4 W2 oxidation	1.3	1.0	1.3	0.9	0.8	0.9	11.2

AAPH, 2,2’-azobis (2-amidinopropane) dihydrochloride; L1-L4 = peptides by Lys-C; ND, not detected; W, tryptophan.

3. Materials and Methods

4. Conclusion

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