Antibody production: development and quality control

  • In-house antibody generation
  • State-of-the art manufacturing processes
  • Standardized Quality Control

Overview of recombinant antibody production

Research and Development

At Miltenyi Biotec, we are very proud of our large R&D department, which allows us to invest a lot of time into the generation of new antibody technologies. Already in 2012 we decided to focus on recombinant antibodies and therefore developed the REAfinity™ Recombinant Antibody Portfolio. These antibodies are specifically developed for flow cytometry applications to minimize background signal and increase lot-to-lot consistency. Today, we are extending our antibody development not only for flow cytometry, but also for other applications, such as imaging and microscopy. The majority of our antibody generation occurs in-house, making it possible for us to optimize each one for its intended application right from the development stage. In addition, clone selection, dye selection, and isotype selection are carefully managed by experts throughout the production process, including extensive testing to verify clones' specificity. This infrastructure allows us to react quickly to new trends and requirements within the scientific community as we select the antibody targets for future products.

Development and production of REAfinity Recombinant Antibodies consists of the following steps:

1. Determining the genetic sequence of the antibody of interest
2. Synthesis of the desired DNA and cloning into expression vectors 
3. Utilizing mammalian cells to express the requisite antibody fragments
4. Purifying and analzying the antibody

For recombinant antibody production, stable cell lines, such as CHO and HEK293, are often used.

During development, our antibodies are rigorously validated to ensure they are specific, sensitive, and reproducible. Specific validation steps are of course dependent on the intended use of the antibody; Hence, we develop tailored protocols for validation experiments, and verify that the results meet our pre-defined specifications. In addition, we routinely compare our products with comparable ones from other vendors to ensure at least equal, and often superior, antibody quality. To find out more about our validation efforts, please click here.

Antibody production

After successful development of a new antibody in our R&D department, it needs to be transferred into our production facility for up-scaling of the antibody production, monitoring of product quality, and to ensure product consistency over time. Read more about the antibody production steps in the next section.

Antibody production methods of hybridoma-derived monoclonal antibodies and recombinant monoclonal antibodies
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Recombinant antibody production consists of the following workflow:

We manufacture our REAfinity Recombinant Antibodies by cloning the antigen-binding region from a traditional hybridoma-derived monoclonal antibody (mouse IgG) with the human IgG1 Fc region. Mutating the Fc region prevents their binding to Fcγ receptors. A mammalian cell line cultured under standardized conditions is used to express this chimeric DNA and the same cell line is used to produce all REAfinity Antibodies in order to ensure lot-to-lot consistency. Our state-of-the art recombinant production method ensures that there are no light chain-, cell-, or serum-derived impurities. These advantages highlight how important it is to invest into reliable research by using reliable recombinant antibodies from the beginning. Doing so avoids the need for re-validation and saves time.

Conjugation of an antibody

After production of the pure antibody, it can be coupled to a fluorochrome. For this, we use robust and consistent state-of-the-art conjugation processes.

Conjugation of antibodies to fluorochromes with subsequent purification, functionality, and lot-to-lot consistency testing.
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Purification steps

Only quality-controlled conjugates are used for the conjugation process. After conjugation, it is important to ensure that the final product contains neither free fluorochromes, nor unconjugated antibodies or activation reagents, all of which could compromise antibody sensitivity in the final product. Hence, for different production steps, different state-of-the-art chromatographic techniques are used to purify the intermediate and final product.

Antibody quality control

We follow a certified quality management system in compliance with international standards. Every step during production of an antibody is rigorously monitored and controlled by adequate measures that follow predefined standard operating procedures (SOPs). This includes entry controls of all raw materials, in-process controls, and quality assays of pure antibody batches, as well as final products.

In-process control

During production of an antibody several in-process control steps are performed. Release testing for every lot includes an evaluation of the purity, F/P ratio, sensitivity, specificity, and lot-to-lot consistency. New lots are tested in a specific application on biological samples. If new batches do not meet defined criteria for mean fluorescence intensity (MFI), stain index (SI), target cell frequency ‒ or other factors, such as background signal and staining pattern ‒ in a given application, they are not released for sale.

Quality control of final filled product

After a product has passed all in-process control steps, it is finally filled into vials. At this stage, it goes through rigorous QC testing to guarantee high quality and consistent product performance from lot to lot. The final quality control is also performed to ensure that every product sent to customers has been tested in its final formulation and filling. This is performed for a given application on suitable tissue samples with internally defined specifications. Some of our antibodies can be used for several applications, although they will have been initially tested and released for only one of these. In such cases, not every application has been run through our final QC testing procedure, but has been instead reported or tested during development. You can easily find this information on every product page within the application section (reported, during development, or QC tested).

Lot-specific Certificates of Analysis (CoA)

You can download a CoA for every lot by entering the lot number into our CoA database.

Lot-to-lot consistency

Consistent performance among different antibody lots is of critical importance in both research and clinical settings. At Miltenyi Biotec, each lot is tested at different dilutions and compared to previous lots to ensure lot-to-lot consistency with respect to their performance. This means that when the recommended dilution is used by the end customer, new lots do not need to be titrated. 

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Technical update

Quality assured – from lot to lot

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