Pharmacopoeial Standards for Therapeutic Monoclonal Antibodies: Rituximab A Case Study

  

    
S.No
    
Pharmacopoeia requirements/    Specifications/Monograph content
    
Pharmacopoeial    Specification
  
  

    
Rituximab (Drug Substance),(IP, 2022)
    
Rituximab injection (Drug    Product), (IP, 2022)
    
Infliximab    concentrated solution (Drug Substance), (Ph. Eur. 11.2) 
  
  

    
1
    
Host-cell-derived proteins  
    
NMT 100 ppm 
    
-
    
Limit as approved by the competent    authority 
  
  

    
2
    
Host-cell-    and vector-derived DNA
    
NMT 10 ng per dose 
    
-
  
  

    
3
    
Category 
    
Anticancer 
    
Anticancer
    
Monoclonal antibody (TNF alfa)
  
  

    
4
    
Description 
    
Clear colorless to pale yellow liquid free from particles that can be    observed by visual observation.
    
Clear to opalescent, colorless to    pale yellow liquid.
    
Opalescent or slightly opalescent, colorless or    light yellow liquid. 
  
  

    
5
    
Identification
    
Determine by    method B and any two methods from method A,C, D, E 
    
Determine by method A, B or D and    C
    
-
  
  

    
5.A
    
Bioassay
    
Complies with the biological    activity as described in assay
    
It complies with the limits of the assay    (potency) 
  
  

    
5.B
    
Method
    
Peptide mapping by HPLC
    
Capillary zone electrophoresis
    
Peptide mapping by HPLC
  
  

    
Specification
    
The retention time of    established marker peaks should be within ± 0.7 minutes of the reference    solution marker peaks 
    
Positive identity is confirmed if    the difference in migration time between the main peak of the reference    solution and test solution is equal or less than 0.1 minute
    
-the profile of the    chromatogram obtained with the test solution corresponds to that of the    chromatogram obtained with the reference solution; 

      -no additional peak in    the chromatogram obtained with the test solution has an area greater than    0.5 per cent of the sum of the areas of peaks 1 to 20 
  
  

    
5.C
    
Sodium dodecyl    sulphate–polyacrylamide gel electrophoresis
    
The principal band    in the electropherogram obtained with test solution is similar in position    and intensity to the principal band in the electropherogram obtained with    reference solution. 
    
-
  
  

    
5.D
    
Method
    
Capillary zone electrophoresis    (CZE)
    
Iso Electric Focusing using    capillary electrophoresis (IEF-CE)
    
-
  
  

    
Specification
    
Positive identity is    confirmed if the difference in migration time between the main peak of the    reference solution and test solution is less than or equal to 0.1 min. 
    
pI of principal band in reference    solution is 9.3±0.2. In the electropherogram obtained with the test solution, no band other than the    principal band is more intense than the principal band in the    electropherogram obtained with reference    solution.
  
  

    
5.E
    
IEF-CE
    
In the electropherogram obtained    with reference solution, the pI of principal is 9.3 ± 0.2. In the    electropherogram obtained with test solution no band other than the principal    band is more intense than the principal band in the electropherogram obtained    with reference solution. 
    
NA
    
-
  
  

    
6
    
Tests
  
  

    
6.1
    
pH
    
6.3 to 6.7
    
As approved by the competent    authority
  
  

    
6.2
    
Osmolality 
    
NA
    
Not less than 250mosmol per kg
    
NA
  
  

    
6.3
    
Impurities with molecular    masses differing from that of Rituximab
    
Method 1
    
Capillary Electrophoresis under    reducing and non-reducing conditions
    
Size-exclusion chromatography 
  
  

    
Specification
    
The corrected percentage area low molecular weight impurities are not more than 10.0 per cent. 
    
Under reducing conditions: The corrected percentage area of non-glycosylated heavy chain is Not more than 2.0 per    cent

      Under non-reducing conditions:  The corrected percentage area low molecular weight impurities is not more than 10.0 per cent 
    
sum of all peaks other    than the monomer peak: maximum 2 per cent 
  
  

    
Method 2
    
Sodium    dodecyl-sulfatepolyacrylamide gel electrophoresis
    
- 
  
  

    
Specification
    
The band(s) observed in the test    solution and reference solution match in position and intensity on visual    observation.
  
  

    
6.4
    
Related substances/related    proteins
    
Method
    
Size exclusion chromatography
    
Capillary electrophoresis (2.2.47) under both    reducing and non-reducing conditions
  
  

    
Specification
    
Complies with the limits approved    for the particular product
    
The sum of the peaks with    retention times less than that of the principal peak is NMT 2.0 per cent, the    sum of the peaks with retention times higher than that of the principal peak    is NMT 7.0 per cent and the sum of the peaks with retention times lesser and    higher than that of the principal peak is not more than 9.0 per cent.
    
Reducing conditions: 

      – sum of all    peaks other than heavy chain and light chain: maximum 2 per cent,    unless otherwise justified and authorised; 

      Non-reducing    conditions: 

      – sum of all    peaks other than the principal peak: maximum 8 per cent. 
  
  

    
6.5
    
Charged variants
    
Method
    
Ion-exchange liquid    chromatography
    
Isoelectric focusing (2.2.54): use suitable    agarose gels 
  
  

    
Specification
    
 
    
Acidic    variants = 45 %

      Main peak =    35%
    
– Electropherogram    obtained with the test solution is similar to the electropherogram obtained    with reference solution (b) and no additional bands obtained with test    solution. Isoelectric points of the principal components of the test solution    and reference solution (b) do not differ by more than    0.05 pI units; 
  
  

    
6.6
    
Glycan distribution
    
Method
    
Capillary electrophoresis with    fluorescence detection
    
Any suitable method as per general    chapter ‘Glycan analysis of glycoproteins’
  
  

    
Specification
    
The    corrected area percentage of each glycan should comply with the limits    approved by National Regulatory Authority (NRA).

      The percent    area of the peaks corresponding to galactosylated glycan should be between 35 and 65 per cent.
    
The    corrected area percentage of each glycan should comply with the limits    approved by National Regulatory Authority (NRA).The percent area of the peaks corresponding    to galactosylated glycan should be between 35 and    65 per cent.
    
percentage of    fucosylatedglycans: as authorized by the    competent authority; 

      percentage of    afucosylatedglycans: as authorized by the    competent authority; 

      percentage of    sialylatedglycans: as authorized by the competent authority. 
  
  

    
6.7
    
Bacterial endotoxins
    
NMT 1.0 EU per mg or equivalent    to EU per ml
    
NMT 1.0 EU per mg
    
NA
  
  

    
6.8
    
Protein A leachetes
    
Comply with the limits as    approved by NRA
    
-
    
As approved by the competent authority
  
  

    
6.8
    
Tests stated under Parenteral

      Preparations
    
NA
    
Complies 
    
NA
  
  

    
 
    
Residual    Protein A
    
NA
    
NA
    
Suitable immunochemical method based on an    enzyme-linked immunosorbent assay (ELISA). 
      
Limit: As approved by the competent authority 
  
  

    
6.9
    
Protein 
    
Method
    
Ultraviolet/visible    spectrophotometry
  
  

    
Specification
    
Not less than 90 per cent and Not    more than 110 per cent of the stated amount of protein.
    
NA
  
  

    
7
    
Assay

      Potency 
    
Method
    
Complement dependent cytotoxicity    assay by using WIL2-S cells
    
Suitable cell-based assay based    on the inhibitory action of infliximab on the biological activity of TNF-a 
  
  

    
Specification
    
Rituximab contains not    less than 80 per cent and not more than 125 per cent of the stated potency 
    
The estimated potency is not less than    80 per cent and not more than 120 per cent relative to the    reference solution. 
  
  

    
8
    
Storage
    
Store at temperature as approved    by NRA.
    
Store at 2 to 8° in an airtight    container.
    
In an airtight container, under    approved conditions
  
  

    
9
    
Labeling
    
NA
    
The label    states (1) Content of rituximab in mg per ml (2) name of product with generic    name (3) drug product (injection) in mg per ml (4) Potency; (4) storage    temperature.
    
The label states the content in    milligrams of protein per millilitre.
  

Table 4: Summary of pharmacopoeial specifications of Rituximab Monographs (IP, 2022) and Infliximab concentrated solution (Ph.Eur. 11.2.).