菜单
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 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

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
    1. 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
    1. 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
    1. 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
    1. 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 Studies
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