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Monoclonal antibodies (mAb) 

Monoclonal antibodies are understood to be immunologically active proteins which act highly specifically against a certain epitope. They arise from a single B lymphocyte cell line (cell clone), which is why they are called monoclonal. Physiologically occurring antibodies are always polyclonal, ie they are directed against many different epitopes and are thus less specific. 

In the control of diseases, however, precisely this high specificity is often necessary in order to have therapeutic success without too great side effects. For this certain molecules have to be identified, which have a decisive role in a disease course. These are often cell membrane receptors or transport and signal proteins. They are then used as an antigenic structure, ie epitope for the monoclonal antibodies. The hybridoma technique, for which César Milstein and Georges Köhler received the Nobel Prize in 1984, are used to produce these. 

 

Monoclonal antibodies HPLC columns 

In order to adequately examine biological samples, a qualitative separation of the components is necessary. Here, too, basic techniques such as chromatography are used. 

 

Thermo Scientific MAbPac columns 

Thermo Fisher Scientific operates a series of HPLC columns for the processing of biological samples: In addition to protein and peptide HPLC columns, monoclonal antibody HPLC columns are also offered. They enable the characterization and quantification of monoclonal antibodies, their titer analysis and the separation of antibody fragments, aggregates and related biologicals. They also serve to monitor the stability and to control the efficiency and safety of antibodies. 

 

Thermo Scientific distributes five different product lines of its MAbPac ™ LC columns: 

  • Reversed phase monoclonal antibody columns 
    • Now you can use reversed-phase conditions for higher resolution separation of intact mAbs and fragments by HPLC and LC-MS. 
    • Products: MAbPac™ RP LC columns 
  • Hydrophobic interaction chromatography (HIC) mAb columns 
    • Use these columns for mAbmAb fragment, antibody-drug aggregate, and related biologic separations. 
    • Products: MAbPac™ HIC LC columns 
  • Affinity monoclonal antibody columns 
    • Use these columns for fast and accurate titer analysis of monoclonal antibodies in harvest cell cultures. 
    • Products: MAbPac™ Protein A LC columns 
  • Strong cation exchange monoclonal antibody columns 
    • Use these columns for high-resolution, high-efficiency analysis of mAbs and associated variants. 
    • Products: MAbPac™ SCX LC columns 
  • Size exclusion monoclonal antibody columns 
    • Choose these columns for optimal mAb analysis, including the high-resolution separation of monomers, aggregates and fragments. 
    • Products: MAbPac™ SEC LC Säulen 

 

The following table should help you find the right MAbPac ™ LC column for your mAb analysis. If you still have a question, our team will gladly advise you.

 

Analysis

Description

Columns & Buffers

 mAb Capture & titer analysis

  • mAb titer determination (concentration) & screening 
  • mAb capture for analysis workflows

 Aggregate analysis 

  • Routing screening for mAb aggregates and fragments

 Charge variant analysis

  • Routine charge variant profiling/screening, including lysine truncation, deamidation & acylation

 Methionine & tryptophan  oxidation

  • Targeted analysis of methionine and tryptophan oxidation 

 Antibody drug conjugate (ADC) analysis

  • ADC DAR analysis

 Intact mAb & fragment analysis

  • Light chain (LC) and heavy chain (HC) analysis
  • Fab and Fc analysis 
  • scFc and F(ab’)2 analysis 

 Sequence & structural analysis 

  • Primary sequence analysis 
  • peptide mapping 
  • peptide & glycopeptides structural & linkage analysis

 Glycan profiling

  • Profiling of released glycans

 

Hybridoma technique 

In this technique, antibody-producing B-lymphocytes are fused with myeloma cells to unite characteristics of both cell types. First, mice are infected with an antigen to which the monoclonal antibodies are directed (for example, receptor or signal proteins). In the mouse, B lymphocytes, which produce antibodies directed against this antigen, develop in the course of the immune response. Then they accumulate as immune complexes in the spleen. From this, the B lymphocytes can be isolated and fused with plasma cells from a myeloma. These tumor cells have the property of multiplying extremely rapidly and are therefore regarded as "immortal". Thus, this ability is combined with that of the B lymphocytes to produce highly stable, long-lived cells which continuously produce monoclonal antibodies. 

 

Medical use 

The extensive use of monoclonal antibodies in diagnostics and therapy has made them ever more indispensable since the first discoveries in the 1970s. 

In the laboratory, they are used for immunohistological procedures or ELISA and enable better reproducibility than polyclonal antibodies. They are also used in in vivo diagnostic procedures to identify different tumors. 

However, they are increasingly being used in the therapy of diseases in which key molecules have been identified and against which monoclonal antibodies have been developed. 

These therapeutically used antibodies are generally named after their origin: In murine antibodies, both the constant and the variable region consist of mouse proteins. These antibodies could only be used very little, since they were recognized and combated by the human immune system as an antigenic structure. The development to fully human antibodies by chimeric antibodies (containing only about 30% murine proteins) and humanized antibodies (90% from human proteins) nowadays allows the promising use in many different indications without an increased risk of immune defenses. 

Examples include Infliximab as an antibody to TNF-alpha in psoriasis, Rituximab as an antibody to CD20 for the treatment of non-Hodgkin's lymphoma or Omalizumab, which is used in severe bronchial asthma and binds to IgE to reduce anaphylactic reactions.