Multi-Attribute Method (MAM): An Emerging Analytical Workflow for Biopharmaceutical Characterization, Batch Release and cGMP Purity Testing at the Peptide and Intact Protein Level

Figures & data

Figure 1. An overview of the MAM workflow for analysis of biotherapeutic proteins from process development to product characterization. Sample preparation using enzymatic digestion can be accomplished by manual handling or automated workflows involving robotic platforms with different proteases. The peptide mapping experiment comprises the discovery phase to assess sequence coverage and identify the product quality attributes (PQAs). A list of CQAs is created in a peptide workbook format which is then used for routine GMP-compliant monitoring of the selected targets, detection of new features, and reporting.

Figure 2. Schematic overview of advanced MAM method transfer, from research and development to manufacturing and QC using an eWorkflow procedure that contains all the associated methods and the reporting template required to set up the injection sequence, acquire, process the data, and summarize the results in a report with a visual pass/fail representation. Data lake allows for software integration, instrument management, data storage, seamless data and workflow transfer, accelerated data processing and advanced reporting.

Table 1. Manual sample preparation protocols used for MAM approach.

Sample	Manual Protocol	Authors, Year
IgG1 mAb IgG2 mAb	100 µg Ab denatured and reduced with 10 mM DTT in 7.5 M GdnHCl and Tris at pH 8.3, for 30 min at room temperature. Alkylation with 20 mM IAA for 20 min at room temperature in the dark. Reaction quenched with 11 mM DTT. Buffer exchange into Tris pH 8.0 using a BioSpin-P6 columns. Digestion with trypsin 1:10 ratio at 37 °C for 30 min. Digestion quenched with 10% TFA at a 1:10 ratio.	Rogers et al. 2015
IgG mAbs	Conventional tryptic digestion: 100 µg mAb denatured and reduced with 50 mM Tris at pH 8.0 with 6.0 M GdnHCl and 20 mM DTT at 56 °C for 30 min. Alkylation with 50 mM IAM for 30 min at room temperature in the dark. Reaction quench with 11 mM DTT. Buffer exchange into 50 mM Tris pH 8.0 with 8 M urea using Zeba spin column. 50 mM Tris pH 8.0 was added into the buffer-exchanged sample to dilute the 8 M urea to 2 M. Digestion with trypsin 1:20 ratio at 37 °C for 4 h. Digestion quenched with 10% TFA at a 1:10 ratio.	Wang et al. 2016
	Ultrafast tryptic digestion: 50 µg mAb diluted with 20 µL of the reduction buffer (8 M urea in 20 mM ambic and 10 mM DTT) and denatured and reduced at 70 °C for 3 min in a water bath. Sample diluted 5X using 20 mM ambic. Digestion with trypsin at 1:20 ratio at 37 °C for 5 min. Digestion quenched with 10% TFA at a 1:10 ratio.
IgG1 mAb (CHO)	Sample dilution to 1 mg/mL in 100 mM NaP pH 7.0 buffer. Then 100 µg of sample was alkylated and denatured at 37 °C for 30 min in a 100 mL mixture of 5.5 mM NaP pH 7.0, 0.2 mM NEM, 50 mM NaCl and 3.2 M GdnHCl. Sample was diluted with 100 mM NaP pH 7.0 and 0.1 mM EDTA to reduce guanidine concentration to 1.7 M. Digestion with Lys-C at 1:5 ratio at 37 °C for 4 h.	Xu et al. 2017
IgG1 DP IgG1 biosimilar	Sample dilution (0.5 mg/mL) with 7.0 M GdnHCl, 100 mM Tris pH 8.3. Reduction with 500 mM DTT for 30 min at room temperature. Alkylation with 500 mM IAA for 20 min at room temperature in the dark. Reaction stopped with 50 mM DTT. Buffer exchange to remove excess GdnHCl, DTT and IAA with 50 mM Tris pH 7.9, using Bio-spin 6 columns. Digestion with trypsin at 1:10 ration for 30 min at 37 °C. 10% FA added to quench the digestion.	Jakes et al. 2021
BsAbs	Denaturation in 6 M Gdn, 50 mM Tris, 1 mM EDTA, pH 8.0. Reduction with DTT for 15 min at 37 °C. Alkylation with IAA for 15 min at room temperature in the dark. Reaction quenched with DTT. Buffer exchange into 50 mM Tris, 1 mM CaCl2, pH 7.0 solution using Sephadex G-25 columns. Digestion with trypsin at 37 °C for 2 h.	Evans et al. 2021
IgG1, IgG4, BsAb	Sample reduction (1 mg mAb) with 16 mM DTT for 60 min at 37 °C in 6 M GdnHCl, 360 mM Tris, and 2 mM EDTA at pH 7.0. Alkylation with 39 mM of IAA for 15 min at room temperature in the dark. Buffer exchange into a digestion buffer (50 mM Tris, 2 mM CaCl, pH 7.5) using NAP-5 desalting columns. Digestion with trypsin at 1:20 ratio for 1 h at 37 °C. 80% FA containing 54 mM L-methionine added to quench the digestion.	Hao et al. 2021
IgG1, IgG4, msAbs, fragments	Sample dilution in 6 M Gdn, 250 mM Tris-HCl pH 8.5 solution. Reduction with 1 M DTT at 60 °C for 30 min. Alkylation with IAA for 60 min at room temperature. Sample desalted using BioSpin 30 Tris columns and eluted with 25 mM Tris-HCl 7.1 digestion buffer. Digestion with Trypsin/LysC mixture at 1:25 ratio for 2 h at 37 °C in a water bath. 2% TFA added to quench the digestion (final concentration 0.17%)	Song et al. 2021
Fc-fusion protein	Denaturation in 7.5 M guanidine HCl, 250 mM Tris, 2 mM EDTA, pH 7.5. Reduction with 500 mM DTT for 30 min at room temperature. Alkylation with 500 mM IAA for 20 min at room temperature in the dark. Reaction quenched with 50 mM DTT. Buffer exchange into 100 mM tris, 50 mM methionine, pH 7.5 solution using NAP-5 column. Digestion with trypsin at 1:10 ratio. Digestion quenching by acidification.	Guan et al. 2022
IgG1	1. Sample (15 µg mAb) denaturation in 6 M GdnHcl, 25 mM Tris-HCl buffer pH 7.1 with 20 mM L-methionine. 2. Reduction with 10 mM DTT for 15 min at 56 °C, 400 rpm. 3. Alkylation with 20 mM IAA for 30 min at room temperature in the dark. Reaction quench by adding 10 mM DTT for 10 min in the dark at room temperature. 4. Buffer exchange to 25 mM Tris pH 7.1 using Zeba Spin desalting plates. 5. Digestion with trypsin at 1:50 ratio supplemented with 20 mM L-methionine for 4 h at 37 °C, 400 rpm. 6. 1% TFA (final concentration) to quench the digestion.	Carvalho et al. 2022

GdnHCl: Guanididine hydrochloride; Ambic: ammonium bicarbonate; DTT: dithiothreitol; IAA: iodoacetic acid; IAM: iodoacetamide; NaP: sodium phosphate; FA: formic acid; TFA: trifluoroacetic acid; CaCl: calcium chloride.

Table 2. Automated sample preparation protocols used for MAM approach.

Sample	Automated Protocol	Authots, Year
IgG1, IgG4, msAbs, fragments	Hamilton robotic system 1. Sample denaturation and reduction with 20 mM DTT in 6 M Gdn, 250 mM Tris-HCl, pH 8.5 for 15 min at 50 °C. 2. Alkylation with IAM at room temperature for 30 min. 3. Sample desalting using Zebra desalting plate and elution with 25 mM Tris-HCl pH 7.1 buffer. 4. Digestion with trypsin/LysC at 1: 25 ratio for 2 h at 37 °C. 5. 12.5% FA to quench the reaction (0.4 % final concentration).	Song et al. 2021
IgG1	Hamilton robotic system 1. Sample denaturation and reduction with 10 mM DTT for 30 min at room temperature. 2. Alkylation with 20 mM IAA for 25 min in dark. 3. Buffer exchange of samples into 50 mM Tris pH 7.9 using SizeX100 IMCStips. 4. Digestion with trypsin at a 1:10 ratio for 60 min at 37 °C. 5. 2.5% TFA was added at a 1:10 ratio to quench the digestion.	Ogata et al. 2021
IgG1 IgG2	Hamilton robotic system 1. Sample denaturation with 7.2 M GdnHCl, 90 mM Tris, 0.1 mM EDTA and reduction with 30 mM DTT at pH 7.4, for 30 min at 37 °C. 2. Alkylation with 500 mM IAM at room temperature for 30 min. 3. Desalting using dialysis plate for 2 h. 4. Digestion with trypsin at 37 °C for 4h. 5. 10 % TFA to quench the reaction.	Qian et al. 2021
IgG1 IgG2 IgG4	Hamilton robotic system 1. Sample denaturation with 7 M GdnHCl and reduction with 500 mM DTT for 30 min at 30 °C. 2. Alkylation with 500 mM IAA at 25 °C in the dark. Reaction quenched with 100 mM DTT. 3. Buffer exchange to remove excess GdnHCl, DTT and IAA using SizeX100 IMCStips. 4. Digestion with trypsin at 1:10 ratio for 60 min at 37 °C. 5. 0.2% TFA (final concentration) to quench digestion.	Sitasuwan et al. 2021
IgG1	Kingfisher Duo purification system 1. Protein of interest is diluted 1 mg/mL and placed in deep well 96-well plate. Protein is unfolded using heat denaturation. 2. Reduction with 5 mM TCEP at low pH. 3. Digestion with heat resistant trypsin on a magnetic robotic platform for 30 min at 70 °C using MES buffer at pH 6.5. 4. Samples transferred to samples were transferred to 300-μL vials and 1 μL of 10% TFA was added to quench the digestion (0.1 % final concentration).	Millán-Martín et al. 2021
mAb	ProSIA flow injection system (customized) 1. Reaction chamber 1: denaturation, reduction, and alkylation 2. Reaction chamber 2: digestion with 0.5% trypsin solution at 1:20 ratio for 20 min at 37 °C.	Liu et al. 2022
mAbs	Kingfisher Duo purification system (2-step digestion) 1. Protein of interest is diluted 1 mg/mL and placed in deep well 96-well plate. Protein is unfolded using heat denaturation. 2. Reduction with 5 mM TCEP at low pH 3. 1st trypsin digestion at 75 °C for 15 min. 4. 2nd trypsin digestion at 40 °C for 30 min. 5. 20% TFA and 8 M GuHCl were added to the sample to a final concentration of 1% and 2 M respectively.	Kristensen et al. 2022

GdnHCl or GuHCl: Guanididine hydrochloride; DTT: dithiothreitol; IAA: iodoacetic acid; IAM: iodoacetamide; FA: formic acid; TFA: trifluoroacetic acid; TCEP: Tris(2-carboxyethyl) phosphine hydrochloride.

Figure 3. New Peak detection workflow for Chromeleon CDS and BioPharma Finder software for identification purposes. PR (pattern recognition) Element = 0, indicates only features with monoisotopic mass will be included. PR size > 1, indicates that at least one additional isotope must be detected. Ratio allows for a 1000-fold change (either more or less) in terms of average intensity.

Figure 4. NPD for the detection of HCPs spiked into a commercial chimeric IgG1 drug product (DP): (a) new peaks (red trace) detected in the HCP 1000 ppm spiked sample compared to the commercial DP (blue trace) and (b) new peaks (red trace) detected in the HCP 100 ppm spiked sample compared to the commercial DP (blue trace). Corresponding defined frame detection and filtering parameters are summarized on the right-hand side for each frame plot.

Table 3. Identified HCP peptides as new peaks in commercial DP spiked with 1000 ppm of HCPs by comparing to the reference sample (unspiked DP) using BioPharma Finder.

Frame peak	Mass (m/z)	Rt (min)	Ratio	PR Elem.	PR Size	Charge	Avg. Intens.	Ref. Avg. Intens.	HCP Peptide Identity
1	294.1794	10.01	99999.9	0	1	2	4.6e5	0.0e0	VIAER (rhLPL)
2	231.6406	11.80	99999.9	0	1	2	3.3e5	0.0e0	ALMK (cathepsinL)
3	418.2245	17.78	99999.9	0	1	2	4.9e5	0.0e0	QVVNGYR (cathepsinL)
4	429.2459	20.27	99999.9	0	1	2	7.6e5	0.0e0	LVGQDVAR (rhLPL)
5	331.6665	24.13	99999.9	0	1	2	6.0e5	0.0e0	SVDWR (cathepsinL)
6	253.6579	26.20	99999.9	0	1	2	6.8e5	0.0e0	FALR (rhLPL)
7	389.2474	32.74	99999.9	0	1	2	8.6e5	0.0e0	LVAALYK (rhLPL L)
8	434.1891	33.32	99999.9	0	1	2	4.8e5	0.0e0	GLCLSCR (rhLPL)
9	354.7078	42.62	99999.9	0	1	2	1.0e6	0.0e0	YWLVK (cathepsinL)
10	522.2907	43.01	2011.7	0	2	2	8.7e5	4.3e2	GLGDVDQLVK (rhLPL)
11	717.6838	47.22	99999.9	0	3	3	6.4e5	0.0e0	NLDHGVLLVGYGYEGTDSNK (cathepsinL)
12	822.3614	56.89	99999.9	0	1	2	4.0e5	0.0e0	NSWGSEWGMEGYIK (cathepsinL)

Table 4. NPD kit contents (for more details see reference 49).

Sample	Contents	IgG Conc. (μg/μL)	Cal Mix Conc. (pmol/μL)	Volume
Calibration sample (Cal)	Pierce™ Peptide Retention Time Calibration Mixture	N/A	0.50	20 μL
Blank (Blk)	0.1 % Formic acid	N/A	N/A	100 μL
Reference sample (Ref)	Trypsin digested NISTmAb RM 8671	0.25	N/A	100 μL
Spike sample (Spk)	Trypsin digested RM 8671 spiked with Pierce™ Peptide Retention Time Calibration Mixture	0.25	0.04	100 μL
pH stress sample (pH)	pH stressed and trypsin digested NISTmAb RM 8671	0.25	N/A	100 μL
Unknown sample (Unk)	Trypsin digested RM 8671 with unknown additional treatment	0.25	N/A	100 μL

Table 5. Critical NPD parameters and tolerance windows selected based on InterLab study.

NPD parameter	Tolerance	Comment
Rt window	0.65 min	Based on the community-derived ± 3 sr of the least repeatable peptide (SFANQPLEVVYSK)
Mass accuracy	< ± 5 ppm	An instrument’s mass accuracy average plus three standard deviations is often used to set the mass tolerance window for database searching and identification. Based on the least reproducible peptide (HVLTSIGEK), limit would be 10 |ppm|. All participants employed the use of high mass accuracy instrumentation, and as a community the average ± 1 sx̅ values were < 5 |ppm|
Fold change detection (FCD) threshold	< 5-fold (for unk sample) < 10-fold (for spk and pH sample)	A community FCD threshold may be calculated from the least reproducible (LSSEAPALFQFDLK) peptide using the ± average 3 sR method and here results in a minimum absolute FCD threshold value < 4-fold
NPD threshold	0.1 % (BPC) User-defined	No universal specification for NPD thresholds can be offered as they may differ between instruments, samples, and software platforms; however, general guidelines may apply across the industry as a starting point for further NPD threshold optimization.

sr: repeatability standard deviation; sx̅: Interlaboratory standard deviation; s_R: reproducibility standard deviation.

Table 6. Sources of non-conformity and mitigation strategies for NPD experiments.

Source of non-conformity	Mitigation strategy
Chromatographic artifact	1. Pre-injection column conditioning by running additional blanks and performing multiple injections of a complex protein digest prior to the first used of the column. 2. Rigorous column washing step to prevent carryover between sample injections. 3. Effective autosampler needle wash between samples.
Sample degradation	Adherence to GLP to always keep sample integrity.
Contamination	Ideally, instrumentation used for MAM would at a minimum be dedicated to analysis of tryptic digests and/or utilize only mobile phases used specifically for MAM analysis. If not possible, a rigorous cleaning of the LCMS system between experiments is recommended.
NPD threshold	Setting a high NPD threshold to define true peak signal will reduce false positive results caused by low abundant species but runs the risk of false negative results where true differences between the test and sample and reference go unreported. NPD threshold to be empirically defined as it will depend on instrument sensitivity, software platforms, and data pre-processing methods (background subtraction)
FCD threshold	A single FCD threshold may not be appropriate for all samples and thus may need to be determined in a product-specific manner. In addition, specific process and/or product-related impurities (corresponding to individual peaks) may have higher degrees of clinical relevance and/or risk, and thus may require separate FCD thresholds.
Co-eluting peak/peak picking challenge	Improve chromatographic separation and/or better mass resolution or utilized alternative data processing parameters that allowed for differentiation of peaks with highly similar retention times and m/z values.

Figure 5. Example of iMAM workflow as an in-process test to monitor the behavior of biotherapeutic product quality attributes. Depending on the therapeutic modality of interest, sample preparation may require just dilution, a desalting step or cell removal. Multiple chromatographic separation techniques are available for native intact protein analysis: SEC (size exclusion chromatography), IEC (ion-exchange chromatography), RP (reversed-phase chromatography), ProA (protein A affinity chromatography), HIC (hydrophobic interaction chromatography), HILIC (hydrophilic interaction liquid chromatography) or μCE (microfluidic capillary electrophoresis by ZipChip™). Data processing involves a characterization step to define a list of proteoforms which will be used in the target intact workbook for the monitoring phase. NPD is used for new entities searching. A final report summarizes all monitored attributes and their relative abundances and a pass/fail condition to indicate they meet the established acceptance criteria.

Figure 6. Summary of software and hardware requirements for MAM at the different stages.

Figure 7. V-model diagram for CSV planning and execution.