Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control

Figures & data

Figure 1. Generic MAM workflow for biotherapeutic modalities that enables targeted attribute quantitation and new peak detection.

A schematic graph illustrating the basic MAM workflow for biotherapeutic modalities using the LC-MS-based peptide mapping method.

Table 1. General product quality attributes for monitoring or control by conventional methods and by the standard MAM.

	Intact Level Conventional Method	Attributes Contributing to the Product Variant	Peptide-Based Standard MAM (Reduced, Trypsin*)
Product Variant	To monitor or control product variant		For monitoring or control of the PQA?
Charge	ICIEF/IEC	Deamidation	Yes
		Oxidation	Yes
		Hydroxylation	Yes
		Fab glycan	Yes
		Lys succinylation	Yes
		Pyroglutamate	Yes
		Glycation	Yes (might underestimate^Citation19)
		AGEs	Yes
		O-glycan	Yes
		C-terminal lysine	Yes
		Isomerization	Yes
		Succinmide	Yes
		VHS extension	Yes
		High Order Structure	No
		LMWF	Potential (depending on the fragment sequence, e.g., fragments that include tryptic cleavage sites may not be captured due to masking by the digested peptides,^Citation11 and may not be as easy to quantitate ladder cleavages such as the common hinge-region fragmentation^Citation20)
Size	R-CE-SDS	NGHC	Yes
	NR-CE-SDS	LMWF	Potential, see above
	SEC	HMWF	No
Identity	Peptide mapping by UV or QDA	Identity	Yes
Oxidation	HPLC	Oxidation	Yes
Glycosylation Related	CE or HILIC-Glycan	Glycosylation related	Yes
Cysteine Related	HPLC, NR-CE-SDS	Cysteine related	No (NR peptide map might be needed)
Sequence Variant	NGS or LC-MS peptide mapping	Sequence variant	Yes
Host Cell Protein	ELISA or LC-MS peptide mapping	Host Cell Protein	Potential (challenging for low-level host cell proteins)
BsAb-Related Impurities, Homodimer, etc.		BsAb- related impurities, homodimer, etc.	No
New Peak		Purity by MAM	Yes (no new peaks)

*Trypsin is the standard enzyme for peptide-based MAM. Lys-C or another enzyme digest might be needed for some modifications and modalities based on the sequence coverage.

Table 2. Considerations of MAM trypsin digestion for minimized artifacts.

Step	Considerations	Purpose
Denaturing	Keep Met at 10 to 20 mM	Minimize oxidation artifact
Reduction	Buffer* at pH 7 to 7.5, 37°C	Minimize deamidation artifact while keeping effective reduction
Alkylation	Buffer at pH 7 to 7.5, 37°C or room temperature	Minimize deamidation artifact while keeping effective alkylation
Buffer Exchange	Keep Met at 10 to 20 mM, pH 7 to 7.5 buffer	Minimize oxidation artifact
Trypsin Digestion	Buffer* at pH 7 to 7.5, 37°C, 1 h to 2 h	Minimize deamidation artifact while keeping effective digestion
Quenching	Acidify with TFA (to ~pH 3) and~20 mM Met	Minimize post-digestion oxidation artifact and ensure auto-sampler stability

*Phosphate or bicarbonate buffer should be avoided since it introduces deamidation artifacts.^30,37.

Table 3. Considerations of SST controls of bracketing reference standards for MAM targeted variant analysis*.

SST Evaluation Criteria	Rationale for Provided Control
1) Mass accuracy for designated peptides	Detector mass accuracy performance across the retention time range of the method and the run sequence.
2) The absolute peak height of designated peak heights is within the given ranges	Assures reference standard sample preparation, chromatographic separation, and mass spectrometry performance across the retention time range of the method and the run sequence.
3) The relative peak height of designated peptides is within the given ranges	Replaces visual inspection^Citation10 for overall pattern consistently (peak intensity relative to the most abundant peak intensity). Covers the remaining peaks not covered by SST evaluation criteria 1) and 2). Ensures overall pattern is consistent with historical pattern.
4) The relative abundance of designated peptides is within the given ranges	Assures the quantitation of attributes. Specific peptides are identified with acceptance criteria that assure method sensitivity, column separation/column age, sample preparation (alkylation and oxidation), and glycan quantitation.

*The acceptance criteria can be established based on the statistical analysis of many data points acquired during method development/qualification/validation.

Table 4. Mean value, standard deviation (SD), and % relative standard deviation (RSD) of % abundance of a subset of representative attributes for mAb4 from the intermediate precision study (n = 24, using three instruments, three column resin lots, with six independent sample preparations by two analysts on two different days).

Attribute	Mean (%)	SD	% RSD
Methionine Oxidation in Fc	2.3	0.2	10.3
N-terminal Pyroglutamate	1.0	0.1	13.5
N-terminal VHS Extension	0.8	0.1	7.2
Tryptophan Oxidation	1.7	0.2	13.7
Aspartic acid Succinimide formation	0.7	0.1	8.9
Internal clip	0.5	0.1	13.0
Lysine Glycation	2.9	0.5	17.7
Asparagine Deamidation	1.1	0.1	11.6
NGHC	2.6	0.3	10.2
M5	1.6	0.2	12.9
G0F	66.7	1.1	1.7

Figure 2. Comparison between acidic variants measured by MAM and IEC in stability and stress samples of mAb4.

A schematic graph illustrating the comparison of MAM and IEC methods in measuring acidic variants in stability and stress samples of mAb4.

Table 5. Current examples and potential MS applications in QC beyond the peptide mapping-based MAM.

Quality Attribute	Method	Reference
scFc Oxidation	Samples were treated with IdeS, EndoS, and dithiothreitol to generate three individual IgG subunits, LC, Fd’ and single chain Fc, RP-MS in commercial QC lab	Sokolowska et al.^Citation2
NGHC LC and HC Identity LC Glycation N-Linked Glycosylation Profile	A rapid intact MS-based MAM, directly out of bioreactors	Lanter et al.^Citation62
CDR Oxidation Sites	Used the IdeS protease-digested and reduced co-formulated mAbs	Xu et al.^Citation63
Conjugated Species of ADC DAR	Subunit and intact MS-based MAM	Martelet et al.^Citation64
Chain Assembly Fragments Aggregates Protein Identification	QC-compliant iMAM, through RP-MS, CEX-MS, and SEC-MS	Carillo et al.^Citation60
BsAb Homodimer	Standard addition-based absolute quantitation, intact MS, potential use in QC	Macchi et al.^Citation61

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