Background: Antibody drugs have large molecular weight, complex structure and post-translational modification (PTM). Antibody drugs come from living cells and contain a variety of impurities related to products and processes. Different methods are used to analyze the samples from multiple angles to control the structure, purity, activity and impurities of the samples due to the complexity of protein drugs.
Methods: The antibodies were analyzed with dimensions of concentration, content, purity analysis, post-translational modification of primary structure and advanced structure, residue detection, activity detection and so on.
Advantages: Comprehensive and diverse characterization analysis, comprehensive quality management system, international advanced instruments and equipments, and rich team experience to ensure the accuracy, stability, compliance and traceability of analysis results.
Cases: Sanyou Bio has established a multi-dimensional characterization and analysis platform for monoclonal antibodies, multi antibodies and fusion proteins after years of technical precipitation and upgrading. Hundreds of service projects have been accumulated, involving all stages of drug development from early drug development to small pilot process development. A sound standard system has been established according to the pharmacopoeia to provide support and guidance for drug production through accurate and repeatable characterization methods.
Service Overviews
Background: Antibody drugs have large molecular weight, complex structure and post-translational modification (PTM). Antibody drugs come from living cells and contain a variety of impurities related to products and processes. Different methods are used to analyze the samples from multiple angles to control the structure, purity, activity and impurities of the samples due to the complexity of protein drugs.
Methods: The antibodies were analyzed with dimensions of concentration, content, purity analysis, post-translational modification of primary structure and advanced structure, residue detection, activity detection and so on.
Advantages: Comprehensive and diverse characterization analysis, comprehensive quality management system, international advanced instruments and equipments, and rich team experience to ensure the accuracy, stability, compliance and traceability of analysis results.
Cases: Sanyou Bio has established a multi-dimensional characterization and analysis platform for monoclonal antibodies, multi antibodies and fusion proteins after years of technical precipitation and upgrading. Hundreds of service projects have been accumulated, involving all stages of drug development from early drug development to small pilot process development. A sound standard system has been established according to the pharmacopoeia to provide support and guidance for drug production through accurate and repeatable characterization methods.
Service Contents
Service
Service Details
Test Method
Client Provides
Deliverables and Standards
Time
Identification of concentration,
content and purity
Protein concentration
ProA-HPLC、UV280
sample
laboratory report
1-2 days
Protein purity
SDS-PAGE、SEC、CE-SDS
sample
laboratory report
1-2 days
Protein charge isomer
CEX、iCIEF
sample
laboratory report
3-7 days
Isoelectric point of protein
iCIEF
sample
laboratory report
1-2 days
Primary structure analysis
Protein molecular weight
LC-MS
sample
laboratory report
2-4 weeks
Peptide spectrum analysis
LC-MS、MS
sample
laboratory report
2-4 weeks
Post translational modification
N-sugar spectrum analysis
LC-MS
sample
laboratory report
2-4 weeks
Glycosylation site analysis
LC-MS、MS
sample
laboratory report
2-4 weeks
Oligosaccharide chain analysis
UPLC-FLD、MS
sample
laboratory report
2-4 weeks
Sialic acid content
UPLC-FLD
sample
laboratory report
2-4 weeks
Deamidation, oxidation, N-terminal cyclization, etc
LC-MS、MS
sample
laboratory report
2-4 weeks
Advanced structural analysis
Disulfide bond analysis
LC-MS、MS
sample
laboratory report
2-4 weeks
thermal stability
DSF
sample
laboratory report
2-4 weeks
Biological activity detection
Affinity kinetic analysis
BLI、SPR
sample
laboratory report
1-2 weeks
Antibody binding assay
ELISA
sample
laboratory report
1-2 weeks
Antibody blocking assay
ELISA
sample
laboratory report
1-2 weeks
Antibody binding epitope
analysis
BLI、ELISA
sample
laboratory report
1-2 weeks
Biological impurity detection
Protein A residue detection
ELISA
sample
laboratory report
1-2 days
Host protein residue detection
ELISA
sample
laboratory report
1-2 days
Host DNA residue detection
ELISA
sample
laboratory report
1-2 days
Service Highlights
1. Comprehensive and diverse characterization analysis
Analytical Characterization covers concentration, content, purity analysis, post-translational modification of primary and advanced structure, residue detection, activity detection and other dimensions.
2. Comprehensive and strict quality management
The platform has established a comprehensive and strict quality management system to ensure compliance, authenticity, reliability and traceability of the results of the analysis.
3. International advanced instruments and equipment
The platform has international advanced instruments and equipment and standardized operating methods to ensure that the characterization and analysis results are accurate and stable.
4. Team members with rich experience
Our team has completed hundreds of related analysis projects and has rich experience in the characterization and analysis of different types of molecules such as monoclonal antibodies, multi antibodies and fusion proteins.
Service Features
1. Multidimensional physical and chemical characterization analysis with complete detection items
Multidimensional physical and chemical characterization analysis platform of Sanyou Bio has multiple detection items and detection methods, covering basic concentration, content, purity analysis, primary structure and advanced structure and residue detection.
2. Industry leading instrument platform
The characterization analysis platform of Sanyou Bio integrates a series of advanced detection equipment in the industry, which is committed to ensure accurate and stable characterization, detection and analysis results.
・ ProteinSimple Maurice
・ Waters ACQUITY UPLC-H-Class PLUS
・ Biacore T200
・ ProbeLife Gator
・ Agilent 1260 HPLC
・ ABI 7500 Fast RT-PCR、
・ MD SpectraMax iD3
・ MD AquaMax 4000
・ BioHandler NPS9100 workstation
Case Stastics
1. Concentration determination(Protein A-HPLC)
As shown in Fig. 1, the standard curve of control antibody IgG1 is drawn by protein A-HPLC method. The peak type is normally distributed, and the linear R2 between peak area and concentration is 0.995, which provides rapid data support for the determination of concentration of antibody protein.
Fig. 1 Standard curve of control antibody
Chromatographic column
MAbPac Protein A
Specifications
4×35 mm
Mobile phase A
50 mM phosphate buffer, 150 mM sodium chloride, pH 7.4
Mobile phase B
50 mM phosphate buffer, 150 mM sodium chloride, pH 2.5
Flow rate
2.0 mL/min
Injection volume
15 µL
Injection volume
25 ℃
Detection wavelength
280 nm
Instrument
Agilent HPLC 1260
2. Determination of protein content (UV280)
As shown in Table 1, confirm the system applicability of the instrument through commercial BSA, and then dilute the sample to be tested (Rituximab) to the absorbance range of the ultraviolet spectrophotometer for absorbance value determination (n=3). Finally, the concentration of the sample to be measured is calculated, which provides accurate data support for the determination of protein concentration through the relationship between concentration and absorbance, dilution multiple and extinction coefficient (s=A280×DF/ε).
Table 1 Record of absorbance measurement of sample to be tested
Sample ID
User Name
Date and Time
Comments
Triplicates 280 nm
Avg Abs 280 nm
Sample1
HMM1
3/25/2021 4:41:47 PM
BSA-b
0.675 0.676 0.676
0.676
Sample2
HMM1
3/25/2021 4:42:30 PM
BSA-c
0.695 0.695 0.695
0.695
Sample3
HMM1
3/25/2021 4:43:21 PM
BSA-d
0.677 0.678 0.679
0.678
Sample4
HMM1
3/25/2021 4:52:35 PM
Rituximab-B
0.661 0.661 0.661
0.661
Sample5
HMM1
3/25/2021 4:53:30 PM
Rituximab-C
0.651 0.650 0.650
0.650
Sample6
HMM1
3/25/2021 4:54:16 PM
Rituximab-D
0.658 0.658 0.658
0.658
3. Purity analysis
3.1. Purity analysis(SDS-PAGE)
As shown in Fig. 2, SDS-PAGE can accurately determine the molecular weight and the purity of the main band, so as to determine the purity of protein samples.
Fig. 2 SDS-PAGE
3.2. Purity identification(CE-SDS)
As shown in Fig. 3 and Fig. 4, CE-SDS can accurately quantify the molecular size distribution and the purity of the main peak, and has a higher resolution of antibody fragments, providing accurate data for the identification of antibody purity.
Fig. 3 Non-reduced CE-SDS
Fig. 4 Reduced CE-SDS
3.3. Purity analysis (SEC-HPLC)
As shown in Fig. 5, SEC can separate the protein based on the hydrodynamic size of the protein in the solution, and can accurately separate sample 1 with the polymer and monomer content of 4.21% and 95.79%, respectively. Achieving the purpose of polymer analysis and purity identification of antibody accurately.
Fig. 5 SEC
Chromatographic Column
ProPac WCX-10
Specifications
(4 × 250 mm) 10 μm
Mobile phase A
20 mM NaH2PO4,pH 4.5
Mobile phase B
20 mM K2HPO4,pH 9.5
Mobile phase C
Ultra-pure water
Mobile phase D
500 mM NaCl
Flow rate
1.0 mL/min
Injection volume
50 µg
Injection volume
20℃
Chromatographic column
ProPac WCX-10
Specifications
(4 × 250 mm)10 μm
4. Charge heterogeneity analysis (CEX)
As shown in Fig. 6, CEX pH gradient elution method can accurately isolate the acid variant content of Rituximab from the market, the principal component content is 60.56%, and the basic variant content is 26.271%, which is consistent with the literature report, providing accurate data for the analysis of antibody charge variant.
Fig. 6 CEX
Chromatographic Column
ProPac WCX-10
Specifications
(4 × 250 mm) 10 μm
Mobile phase A
20 mM NaH2PO4,pH 4.5
Mobile phase B
20 mM K2HPO4,pH 9.5
Mobile phase C
Ultra-pure water
Mobile phase D
500 mM NaCl
Flow rate
1.0 mL/min
Injection volume
50 µg
Injection volume
20℃
Chromatographic column
ProPac WCX-10
Specifications
(4×250 mm) 10 μm
5. Charge heterogeneity and isoelectric point analysis
As shown in Fig. 7, iCIEF can accurately determine the pI value of Trastuzumab, a listed antibody drug, is 8.97, which is consistent with the pI value provided by manufacturer instruction, indicating that this method is stable and reliable and provides accurate data for the analysis of pl value of antibody.
Fig. 7 pl measurement of Trastuzumab
6. High resolution molecular weight detection (LC-MS/MS)
As shown in Fig. 8, the precise molecular weight of protein polypeptide drug A can be obtained.
Fig. 8 mass spectrum of drug A
Glycoform
Theoretical Molecular Weight
M+G0F+G0F
148220.0
M+G0F+G1F
148383.0
M+G1F+G1F
148545.0
M+G1F+G2F
148707.0
M+G2F+G2F
148868.0
7. Peptide mass fingerprinting analysis (LC-MS/MS)
As shown in Fig. 9, the coverage rate of single enzyme digestion peptide spectrum of the protein sample has reached 98.4%. The coverage rate of three enzyme digestion peptide spectra reached 100%, which can accurately confirm the amino acid sequence of the test protein.
Fig. 9 peptide coverage
8. Peptide map
As shown in Table 2, the mass peptide map inspection method can obtain all the information of amino acid sequences such as peptide modification, peptide sequence, valence state, average molecular weight and response value of the protein sample.
Fig. 10 UV chromatographic labeling of trypsin hydrolysate
Table 2 Trypsin hydrolysate CDR peptide information (part)
CDR peptide
Peptide sequence
Retention time(min)
Batch 1
Batch 2
LCDR3(2)
TFGQGTK
14.37
14.35
LCDR1(1)
VTITCR
20.88
20.85
HCDR1(1)
LSCAASGFR
27.36
27.34
HCDR2(1)
APEWVSDINTR
42.93
42.89
9. Spectrum analysis of N sugar (HPLC/FLD)
The glycoform map of antibody drug a is shown in FIG. 11. The types and relative quantity of glycoform are clear and consistent with the glycoform spectrum reported in the literature, indicating that this method is stable and reliable and can be used for product consistency and stability evaluation.
Fig. 11 The N-sugar map detected by fluorescence detector (HPLC/FLD)
10. Sialic acid analysis(UPLC-FLD)
As shown in Fig. 12, this method can accurately detect the type and content of sialic acid (Neu5Ac, NANA) in the listed monoclonal antibody Adalimumab, and the retention time is consistent with the positive control, indicating that this method is stable and reliable.
Fig. 12 Analysis of sialic acid
Chromatographic Column
Waters BEH C18
Specifications
(2.1 mm×100 mm) 1.7 µm
Mobile phase
Methanol:acetonitrile: water (7:9:84)
Flow rate
0.25 mL/min
Injection volume
3 µL
Temperature
30℃
Detection wavelength
The excitation wavelength is 373 nm and the absorption wavelength is 448 nm
Instrument
Waters ACQUITY UPLC H-Class
11. Thermal stability analysis(DSF)
As shown in Fig. 13 and Fig. 14, DSF can accurately detect that the Tm1 value of antibody drug A on the market is 69.3°C and the Tm2 value is 81.6°C, The temperatures are consistent with the Tm value reported in the literatures, indicating that this method is stable and reliable.
Fig. 13 Fluorescence curve of drug A
Fig. 14 First derivative curve of drug A
12. Affinity kinetic analysis (BLI)
BLI technology can be used to analyze the binding ability between candidate antibodies and antigens, and then evaluate the druggability. Fig 15 shows a case of antibody antigen binding activity analysis. It can be seen from the figure that the affinity between antibody 1 (Ab 1) and antigen is one order of magnitude higher than antibody 2 (Ab 2).
Fig. 15 Binding ability between antibody and antigen (BLI)
Sample ID
KD (M)
Kon (1/Ms)
Koff (1/s)
R2
Ab 1
3.11x10-10
2.16x105
6.71x10-5
0.99
Ab 2
2.42x10-9
1.15x105
2.78x10-4
0.99
13. Protein level function evaluation(ELISA)
The better binding activity of antibodies can be screened for subsequent functional research based on ELISA technology.
Fig.16 shows a case of analyzing the binding activity of full-length antibody and antigen by ELISA. The binding activity of lead antibody 1 to antigen is 54.97% higher than that of the control antibody, and lead antibody 2 is inactive. Lead antibody 1 can be used for follow-up function research.
Fig.17 shows a case of analyzing the blocking ability of full-length antibody to ligand binding by ELISA. The blocking ability of lead antibody 1 is equivalent to that of the control antibody, and lead antibody 2 has no blocking ability, so antibody 1 can be selected for functional research.
Fig. 17 Binding activity analysis of full-length antibody (ELISA)
Fig. 18 Blocking ability analysis of full-length antibody (ELISA)
14. Host DNA residue detection
The residual DNA of CHO host cells in antibody drugs can be detected by fluorescence probe PCR. Fig. 18 shows a case of host DNA residue detection of an antibody. The results show that the CHO host DNA residue of the antibody is 5.9 pg/mg, and the extraction recovery rate is 83.53%, which meets the national standard (each bottle should not be higher than 100 pg (general rule 3407)).
Fig. 19 Host DNA residue detection
15. Host cell protein residue
Based on ELISA technology, the host cell protein residue detection kit can be used to detect the host cell protein residue in antibody drugs.
Fig. 19 shows a case of host protein residue detection of an antibody. The results show that the host protein residue of the antibody is 5.31 ng/mg, and the recovery rate is 90.18%, which meets the national standard (determined by ELISA (general rule 3429), which should not be higher than 0.01% of the total protein).
Fig. 20 Standard curve of host protein residue detection
16. Protein A residue detection
Based on ELISA technology, protein A residue detection kit can be used to detect protein A residual content in antibody drugs.
Fig. 20 shows a case of protein A residue detection of an antibody. The results show that the protein A residue of the antibody is 0.0001%, which meets the pharmacopoeia standard (determined by ELISA (general rule 3429), the protein A residue should not be higher than 0.001% of the total protein).
Fig. 21 Protein A residue detection