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Desmond Schofield

Desmond Schofield

Director of Business Development at evitria

Recombinant antibodies are mono-specific antibodies produced with recombinant DNA technology, which means they are generated in vitro using synthetic genes introduced into mammalian cell lines rather than through hybridoma cell culture or animal immunization.

Immunization of an animal or cultivation of hybridomas is not necessary. As recombinant antibodies are produced from a known DNA sequence, they have enhanced quality and reproducibility compared to polyclonal and traditional hybridoma-based monoclonal antibodies (Basu, Koli et al. 2019). Recombinant antibodies are used in medicine as well as in life science. They are therapeutic treatments for many diseases, including cancer or autoimmune disorders.

At evitria, we focus on delivering a recombinant antibody expression service for our international customers. Our experience and expertise in recombinant antibody production is based on over 120,000 CHO cell culture transfections and 20,000 antibodies purified. In this article, we would like to share 7 facts about recombinant antibodies. From the application in medicine and science to recombinant antibody production and the advantages of recombinant technology, this article will teach you all you need to know about recombinant antibodies.

1. What is a recombinant antibody?

Recombinant antibodies show mono-specific binding to a single epitope, just like a monoclonal antibody. Their production begins at the genomic level and is entirely in vitro. Using molecular biology techniques, synthetic genes coding for the antibody of interest are designed and introduced into cell lines.

Recombinant antibodies have been around since 1984, when Morisson SL. and Neuberger MS. cloned Ig genes from hybridomas to modify them in vitro. They expressed the first chimeric antibody, the first version of recombinant antibodies.

Advances in recombinant technology facilitated several breakthroughs, leading to the advent of recombinant antibody production on a large scale. Recombinant DNA technology allows the design and cloning of custom tailored genes into mammalian cells, which in turn produce antibodies in unprecedented quality and reproducibility. evitria is a leading service provider for recombinant antibody expression and custom recombinant antibodies – therefore, we rely on CHO cell expression.

Transient antibody expression in CHO (in vitro) even circumvents the questionable use of laboratory animals in contrast to in vivo antibody generation. If you make the Abs using a synthetic or human Ab library, then that eliminates the use of animals.

recombinant antibody

2. Recombinant antibody production

Recombinant antibody production commences with the isolation of promising genetic material (nucleic acids) coding for antibody candidates or the use of a library of genes with randomized antigen binding site sequences for antibody engineering.

Promising genes are introduced into expression vectors that display the associated antibodies on their surface (antibody phage display library technique). In the so-called panning technique, such a bacteriophage library is exposed to an immobilized antigen, the weak binders can be washed away from strong binders, which adhere to the antigen unless special reagents are used.

Repetition of this selection process with increasingly stringent conditions leaves only the strongest and most specific antibodies in the antibody library.

This in vitro process allows the manipulation of the genes to create new antibodies, reduce immunogenicity or even only select for antibody fragments (e.g., scfv or fab fragments).

Next, the most promising antibody genes are cloned into suitable cell lines that function as expression platforms, such as HEK or CHO cells: Since antibodies are rather complex proteins that undergo modifications after expression, higher cells (eukaryotic cells) are usually needed. Bacterial cells such as Escherichia coli would not yield the desired antibody products as they cannot perform post-translational modifications, such as the assembly of disulfide bridges, that are required for correct antibody production.

Most commonly, antibodies are produced with CHO cells (chinese hamster ovary cells) and HEK cells (human embryonic kidney). As a result of methodology research and ongoing mammalian cell culture optimizations, the yields of human antibodies in these expression systems could be improved to over 12 g / liter​​.​1​ CHO cells are the most widely used host for recombinant antibodies. About 70% of all recombinant proteins are produced in CHO cells stated by Feng Li et al.

Recombinant antibody production

3. Advantages of recombinant antibodies

Advantages of recombinant antibodies at a glance:

  • Scalability
  • In vitro & animal-free
  • Customizable
  • Best quality
  • Consistency and batch-to-batch reproducibility

Find out more about the advantages:

Scalability

Once a recombinant antibody is established and the optimal genetic sequence is known, the antibody can be produced at different scales with more predictable behaviour.

Further, the use of well-established expression platforms facilitates the upscaling of the necessary manufacturing processes, which are comparatively lean.

What is an antibody sequencing service?

In vitro process – animal-free

Being an in vitro process entirely, recombinant antibody production is an agile technology that can switch between individual assignments on short notice. In contrast, the immunization of animals or production through hybridoma cell lines is more time consuming.

Additionally, in vitro processes are more economical and ethical since they do not require as many resources,produce as much waste, or use animals in the production process.

Customizable

Once the amino acid sequence of the antibody is known, it can be edited and customized easily, allowing the properties of the recombinant antibody to changed for new applications. This is not possible when working with polyclonal or hybridoma-derived antibodies.

Best quality

Since the underlying genetic material is easily optimized through the phage display method, recombinant antibodies can be designed to exhibit superior high affinity, sensitivity and specificity over traditional monoclonal antibodies.

Consistency and batch-to-batch reproducibility 

Hybridoma cell lines are prone to spontaneous mutations, thus leading to potential consistency issues between batches. Polyclonal antibodies are known for lower reproducibility, since each batch is derived from individual animals.

Production of recombinant antibodies relies on entirely defined and well-controlled genetic sequences and thus yields highly consistent antibody products. This process leads to very good reproducibility and validation between batches.

4. Applications of recombinant antibodies

Recombinant antibodies are used in numerous applications in medicine and life sciences, such as:

  • Biologic therapeutics against cancer, autoimmune diseases, infections
  • Tracers in imaging methods (e.g., PET scans)
  • Diagnostic tools in lateral flow test kits, ELISA, and other immunoassays
  • Basic science tools in molecular biology (e.g., Western blots, immunofluorescence, flow cytometry, immunohistochemistry)
Application of recombinant proteins – evitria

5. Customization of antibodies

Recombinant antibodies offer remarkable versatility due to their ability to be customized and reformatted for specific applications. Let’s explore key insights into this exciting field and explore the customization methods we employ at evitria.

Chimerization: Creating Hybrid Antibodies

Chimerization involves combining the variable domains of an antibody (usually from a mouse, rat, or rabbit) with the constant domains from a different species (e.g., human). This results in a chimeric antibody that retains specificity while minimizing immunogenicity.

Chimeric antibodies bridge the gap between species, allowing us to harness the best features of each. For example, a humanized chimeric antibody can be used for therapeutic purposes without triggering adverse immune responses.

Humanization: Making Antibodies More Human-Like

Traditional antibodies derived from animals can provoke immune reactions when administered to humans. By grafting the complementarity-determining regions (CDRs) of an antibody onto a human antibody framework, we create humanized recombinant antibodies. These retain specificity while minimizing immunogenicity, making them ideal for therapeutic use.

Isotype Switching: Tailoring Antibody Functionality

Isotype switching allows altering the isotype or subtype of an antibody. For instance:

  • Convert an IgG to an IgM for increased avidity.
  • Overcome aggregation issues associated with certain subtypes.

Different isotypes have varying effector functions and stabilities. Isotype switching enables fine-tuning of an antibody’s properties.

Antibody Fragments: Compact and Functional

In some situations, full-length antibodies may not be optimal. Recombinant expression platforms allow us to convert any antibody into fragments, followed by purification. Antibody fragments offer advantages:

  • Single Chain Fv (scFv): Consists of a single variable domain, useful for research and diagnostics.
  • Fab (Fragment antigen-binding): Retains antigen-binding capacity.
  • Fab2: Contains two Fab fragments, useful for bispecific antibodies.

Species and Format Flexibility

  • Species: Beyond human and mouse, we can engineer antibodies for various animals, including rat, rabbit, hamster, and more.
  • Formats: Our proprietary cloning system enables reformatting into diverse antibody formats:
    • IgG, IgA, IgE, and IgM
    • Fc domains
    • Fabs and Fab2
    • Fc fusions
    • Bispecifics

Custom Engineering

If you need a unique format, we’re ready to guide you through the design choices.

The Future of Recombinant Antibodies

As technology evolves, so do recombinant antibodies. From personalized medicine to targeted therapies, recombinant antibodies continue to shape the future of healthcare. Scientists, clinicians, and industry experts collaborate to unlock the full potential of these customizable molecules.

In summary, the customization and reformatting of recombinant antibodies empower us to tailor their properties for specific applications. Whether it’s humanization, chimerization, or creating antibody fragments, recombinant Abs offer a dynamic canvas for scientific innovation.

6. Recombinant antibody importance

The scope of recombinant antibody applications is tremendous. The recent pandemic outbreak of SARS-CoV-2 virus had a big impact on everybody’s life. The strategies to fight the pandemic rely heavily on recombinant antibodies. 

One example is the development of therapeutic antibodies that neutralize virus particles in infected patients. Recombinant antibodies that bind to surface proteins of SARS-CoV-2 are used in lateral flow test kits (“antigen tests”) to detect acute infections.

A recent publication of Canadian scientists in the Journal of Molecular Biology​​2​ highlights the possibility for the synthesis of highly sensitive and specific reporter assays against SARS-CoV-2 virus particles using recombinant antibodies.

They created a conjugate reporter system consisting of two halves of a luminescence enzyme. Each half is attached to an antibody via a peptide linker. The antibodies were developed to have affinity to regions of viral surface proteins using recombinant and phage display technologies.

In the presence of the viral surface proteins, the antibodies bind to them, thus enabling the attached enzyme halves to reconstitute to a functional enzyme. The reporter system then emits light. This publication illuminates how rAbs can be rapidly developed into tools for important applications.​

7. Recombinant antibodies history

When it comes to the history of antibodies, the name Paul Ehrlich is inevitable, as it was him to discover a cure against syphilis and to coin the German term “Antikörper” in 1891. As a matter of fact, though, it was referenced already a year earlier by Emil von Behringand and Shibasabura Kitasato, as they found that animals infected with diphtheria can be cured by the transfer of serum from other, already immunized animals.

In 1975 Georges Köhler and César Milstein invent the monoclonal antibodies. This was the start of the modern era of antibodies. The first version of a recombinant antibody called “chimeric antibody” at that time was created by independently by Morrison SL. and Neuberger MS in 1984.

However, there is to know a lot more about the history of antibodies and the several milestones in this field that proved their immense relevance for life sciences and medicine, revolutionizing both areas significantly.

history of antibodies

Recombinant antibody review

When it comes to reviewing which recombinant antibody is to selected, there are several factors to consider. They can be produced with different methods and tailor-made for specific applications. Nevertheless, there is no one-size-fits-all solution.This and more has to be considered when choosing which antibody is to be used.

Recombinant antibodies from evitria

At evitria, we use our CHO cells antibody production and expression platform for the generation of recombinant monoclonal antibodies. We can produce custom recombinant antibodies in large-scale within 5 weeks. Antibody production in CHO guarantees highest quality of recombinant antibodies and fast expression. You want to know more about our services? We would be pleased if you contact us.

Advances with afucosylated recombinant antibodies

Natural antibodies not only bind their respective antigen, they highlight it to the immune system as harmful and mark it for neutralization. This effect is called antibody dependent cellular cytotoxicity (ADCC) and is of high significance in the development of therapeutic antibodies in oncology.

Read more: Therapeutic afucosylated antibodies with enhanced ADCC

The underlying signalling process involves the recognition of a chain of carbohydrates on the antibodies. Research has shown that the nature of the carbohydrates influences the severity of the ADCC and that afucosylated antibodies (i.e., lacking fucose in the carbohydrate chain) have a particularly positive effect on ADCC.

Therefore, the ability to effect afucosylation of recombinant antibodies adds a whole new dimension to the scope of antibody technology. In addition to being able to develop highly specific antibodies to virtually any antigen through recombinant antibody technology, afucosylation allows the fine tuning of the strength of the immune system’s response to the antigen.

This fact opens the door to developing more potent and more tolerable therapeutic antibodies.

Poster Download: antibody development and quality control by mass spectometry

The production of high quality, reproducible material is critical for the development of antibody-based therapeutics. The evitria-Genovis workflow combines rapid, high quality, antibody production with high-throughput mass spectrometry for greater insight and control of key quality attributes.

Download the poster to learn more.

FAQs – Recombinant Antibodies

Antibodies (Abs) are proteins that belong to the class of immunoglobulins. They are naturally produced by certain types of blood cells of vertebrate animals and are part of the adaptive immune system. Humans produce several types of antibodies, with the so-called immunoglobulins G (IgG) and their various subtypes (such as IgG1) being the most common among them.

Antibodies are generally Y-shaped molecules consisting of heavy chains and light chains that bind to their target, or antigen, with a variable region. The antigen binding sites are located at the two tips of the “Y” and they mediate a very strong and specific binding interaction between antibody and antigen, similar to a receptor. The base of the “Y” is termed constant region (in contrast to the variable domain), although it’s sequence varies between species.

Antigens are usually parts of bacteria, microorganisms, or surface constituents of viruses. Antibodies bind to such pathogens, covering their surfaces, thus inactivating them and marking them as foreign to the immune system.

The high specificity of the antibody-antigen binding process led to the development of antibodies as valuable tools for diagnostic and therapeutic medicine and biological research. Nowadays, polyclonal, monoclonal and recombinant antibodies are available, and they come with their individual profile of pros and cons, depending on the specific application.

To trigger polyclonal antibody production, animals are injected with the antigen of interest. Further, repeat injections after initial animal immunization may be beneficial in order to increase the Ab titer in the blood.

Next, the polyclonal antibodies are harvested as solution in the serum by bleeding the animal and removing all blood cells. The polyclonal antibodies may be subjected to further purification by removing serum proteins. The choice of animal in antibody production depends on the desired amounts of Abs, their isotypes and immune response. Common animals for Ab production are rabbits, mice, horses, goats and llamas.

Key advantages of this process are the relatively low capital investment to obtain antibodies, but the use of animals poses ethical questions. Another disadvantage is the batch-to-batch variability, since they are obtained from different individual animals. Polyclonal antibodies are often used for immunoprecipitation, co-IP and ChIP applications.

Monoclonal antibodies (mAbs) are produced by single cells, unlike polyclonals which stem from a population of diverse B cells. The isolation of such a B cell for antibody production on large-scale is next to impossible and in addition, B cells have a relatively short expected life time.

A major breakthrough was the development of the hybridoma technology: B cells are harvested from immunized animals and then fused with myeloma cells. The resulting hybridoma cells carry the ability for antigen production of the B cell and the immortality of the myeloma cell. Hybridoma cells are then selected for specificity, and individual cells isolated.

Recombinant antibodies or recombinant monoclonal antibodies are antibodies that are produced by the means of recombinant antibody coding genes. They are monoclonal antibodies that are created in vitro, which means that hybridomas and animals are not needed. Find a full article on: What is a recombinant monoclonal antibody?

Recombinant antibodies are produced in a process called recombinant antibody expression, which is performed in vitro. By the means of genetic manipulation, new antibodies are created and then cloned into suitable cell lines. Read the full article about how are recombinant antibodies made.

One method for producing large numbers of identical antibodies is the hybridoma technology, which starts by injecting a mouse with an antigen to provoke an immune response. In recombinant antibody production, no animals are needed. When using a synthetic or human Ab library, there is no need for an immunization of an animal. No mice or hamsters are used in the recombinant antibody production! It does not require any animals in the production process as cell lines cultured in a lab are used as the base material.

While native antigens are produced in vivo, recombinant antigens are created artificially. A vector can be used to initiate their production, followed by their purification.

In biology, “recombinant” means that genetic material from different sources is combined. Read more: What is recombinant?

Recombinant DNA is produced by the means of different laboratory procedures in order to combine, split or rearrange DNA segments, resulting in a modification of the original DNA.

When an antibody is recombinant, this means that the antibody has been produced in vitro rather than by infecting living organisms. Instead, recombinant DNA is inserted into host cells by the means of a vector. This provokes the host cells to produce (recombinant) antibodies accordingly, which can then be harvested.

One prominent (and early) example of a recombinant is insulin; the genetic code for human insulin is inserted in bacteria, which are then “programmed” to produce insulin accordingly.

Source and studies:

  1. 1.
    Frenzel A, Hust M, Schirrmann T. Expression of Recombinant Antibodies. Front Immunol. Published online 2013. doi:10.3389/fimmu.2013.00217
  2. 2.
    Fellouse FA, Miersch S, Chen C, Michnick SW. Structure-based Design of a Specific, Homogeneous Luminescence Enzyme Reporter Assay for SARS-CoV-2. Journal of Molecular Biology. Published online June 2021:166983. doi:10.1016/j.jmb.2021.166983
Previous article Enhanced purification services for diverse projects

February 23, 2024 duration: 3 min

Enhanced purification services for diverse projects

Desmond Schofield

Desmond Schofield

Director of Business Development at evitria

From simple proteins to complex multi-specifics, our methods evolve with you.

In the dynamic landscape of antibody research and therapeutics, having a diverse array of purification methods is essential. At Evitria AG, we recognize the need for flexibility to meet various project requirements. Let’s delve into our updated purification services:

1. Protein Capture

1.1. Protein A and Alternative Resins

Protein A Affinity Chromatography: Our gold standard method efficiently captures antibodies via their Fc regions, ensuring high purity (typically >95%) and yields. Protein A can be cleaned and regenerated thousands of times, ensuring production costs and wastes are minimised.

However, we understand that different antibodies may have varying affinities for Protein A. To address this, we offer alternative resins:

Protein G: Suitable for capturing a broad range of antibody subclasses, Protein G can be used in instances where protein A does not bind, for instance with rat IgG2b productions.

Protein L: Specifically binds to the variable domain of kappa light chain of antibodies, another alternative when Protein A cannot be used.
For even more versatility, we also have an extensive range of specialized columns

1.2 Specialized Columns

Anti-CH1 Purification: Target the CH1 domain specifically for efficient purification of Fab domains and related formats.

KappaSelect and LambdaSelect Columns: Selectively capture either kappa or lambda light chains, streamlining purification.

IgM-Specific Purification: Tailored methods for IgM antibodies, addressing their unique properties.

IMAC (Immobilized Metal Affinity Chromatography): Ideal for purifying His-tagged proteins, IMAC yields high-quality results.

Antibody production services – a comparison

2. Protein Polishing

After an efficient and high-yielding capture step has been performed, polishing may be required to remove aggregates, multimers, and other impurities. Here we use two methods:

2.1 Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC), also known as gel filtration, is a powerful technique for separating biomolecules based on their hydrodynamic size. Here’s how it works:

Principle: SEC separates molecules by differences in size as they pass through a resin packed in a column. Unlike other chromatography techniques, such as ion exchange or affinity chromatography, SEC does not involve specific binding interactions. Instead, it relies on the exclusion of molecules from porous beads based on their size.

Matrix: SEC resins consist of spherical particles (beads) with a porous structure. Larger molecules cannot diffuse into the beads and elute first, while molecules of intermediate size penetrate the pores to varying degrees. Smaller molecules that can enter the total pore volume elute last.

Fractionation Ranges: SEC resins are designed with specific fractionation ranges, allowing separation of peptides, small biomolecules, or large proteins (e.g., antibodies). The exclusion limit defines the size of molecules excluded from the pores and eluting in the void volume.

2.2 Ion Exchange Chromatograph

Ion Exchange Chromatography is another workhorse in our purification arsenal. Here are the key principles:

Principle: IEX separates compounds based on their net charge. The stationary phase contains charged functional groups covalently bound to a solid support, creating either a cation or anion exchanger. Charged analytes are adsorbed by the opposite-charged exchanger, while neutral or similarly charged compounds pass through. Careful design and control of the pH and salt concentration allows for selective binding and elution of molecules.

Reversible Binding: The binding of charged compounds is reversible, allowing elution with a salt or pH gradient. IEX is versatile for various applications, such as bispecific antibodies

Media Selection: We offer a range of IEX media based on factors like the isoelectric point (pI) of the compounds and the ionic form, porosity, and particle size of the media.

Ready to optimize your antibody production? Contact our experts today! Whether you need custom antibodies, afucosylated variants, or Fc-silenced antibodies, evitria has you covered.

Previous article Dr. Desmond Schofield new CBO of evitria
Next article An Introduction to Recombinant Antibodies: 7 Key Insights

February 19, 2024 duration: < 1 min

Dr. Desmond Schofield new CBO of evitria

ZURICH, Switzerland – evitria AG, a leading preclinical service provider focused on transient antibody and protein production, announces that former Head of Business Development EMEA, Dr. Desmond Schofield, has been appointed Chief Business Officer as of 1st January 2024.

“evitria pioneered the use of transient CHO cells for antibody production, and now as the market evolves with new and exciting modalities, we are reshaping our business development organization, to closer align with our clients” said Dr. Stefan Schmidt, Chief Executive Officer.

Dr. Schofield added, “at evitria we have a long-established reputation for high quality protein production services, and I look forward to building on this to meet new market demands and further support our clients.”

Dr. Desmond Schofield, CBO evitria AG

Since 2010, evitria, based in Fahrweid near Zurich, has specialized in the production of antibodies and other proteins. Today, evitria, part of a group consisting of Atlas Antibodies and Histocyte Lab, is a sought-after partner worldwide for research, development, and production of antibodies.

Previous article Therapeutic antibodies: afucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity (ADCC)

February 13, 2024 duration: 5 min

Therapeutic antibodies: afucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity (ADCC)

Desmond Schofield

Desmond Schofield

Director of Business Development at evitria

Therapeutic antibodies stand at the forefront of modern biomedicine. These remarkable molecules, designed to target specific antigens, hold immense promise in the treatment of various diseases. In this article, we discover the mechanisms of antibody-dependent cellular cytotoxicity (ADCC) and explore the technique of afucosylation. Additionally, we’ll highlight the role of companies like evitria in spearheading the production of afucosylated antibodies, contributing to the evolution of therapeutic treatments.

Therapeutic antibodies

Therapeutic antibodies are the fastest-growing class of biological drugs. Their ability to interact with specific targets means they can be employed for a range of diseases, including cancer, autoimmune and infectious diseases. According to the Umabs Antibody Therapies Database, 162 antibody therapies had gained approval by at least one global regulatory body at the end of June 2022​1​.

Antibody-dependent cellular cytotoxicity (ADCC)

One critical mechanism underlying the clinical efficacy of therapeutic antibodies is antibody-dependent cellular cytotoxicity (ADCC). Although several classes of antibodies can mediate ADCC, most therapeutic antibodies belong to the IgG subclass. ADCC relies on the bifunctional structure of antibodies, where the antigen-binding fragment (Fab) portion recognises an antigen, and the fragment crystallisable (Fc) portion bridges the antibody to effector cells possessing Fc receptors (FcγR).

FcγRs are glycoproteins expressed on effector cells, like natural killer cells (NK), neutrophils and eosinophils. The typical ADCC involves the activation of NK cells following the binding of its FcγRIIIa (CD16a) to the Fc portion of IgG antibodies. This causes polarisation of cytotoxic granules and releases perforins and granzymes, which work in concert to induce cell death. In targeted cancer therapy, antibodies such as rituximab, trastuzumab and cetuximab increase tumour-cell killing via ADCC.

Glycoengineering of antibodies with enhanced ADCC properties

Recently, antibody engineering techniques have focused on enhancing ADCC to provide more efficacious antibody treatment. Glycoengineering involves removing the fucose residues on the Fc region of the antibody. This strategy targets the N-linked glycosylation site at residue 297 (N297) in the Fc region, which consists of a heptasaccharide core structure that can undergo further extension with glycan moieties, such as fucose.

Removing fucose from the Fc N-glycans of human IgG1 increases the affinity to human FcγRIIIa on NK effector cells by 10-100 fold compared with non-fucosylated counterparts​2–4​. Structural studies have explained that this increased affinity is due to the strengthening of the carbohydrate-carbohydrate interactions between the antibody Fc region and the FcγRIIIa on effector cells. As FcγRIIIa is a major activating receptor on NK mediating ADCC, this leads to a 2-40 fold increase in ADCC​3–5​. Moreover, afucosylation does not change other properties like antibody conformation and stability​6​.

Afucosylated Antibodies

Clinically approved afucosylated antibodies with enhanced ADCC functions

Afucosylated antibodies have a clinical advantage because of their enhanced immune activation of effector cells. Examples of clinically approved afucosylated antibodies include:

  • Obinutuzumab — which has a reduced fucose content (~30%) — was the first glycoengineered antibody developed in 2013 and displayed greater ADCC and direct cell death than rituximab​7​. Obinutuzumab is approved to treat previously untreated chronic lymphocytic leukaemia (CLL), a cancer of white blood cells (B lymphocytes).
  • First approved in 2018 by the Food and Drug Administration (FDA) for the treatment of Cutaneous T-cell lymphoma (CTCL) patients, mogamulizumab targets CC chemokine receptor 4 (CCR4). This afucosylated antibody has enhanced ADCC activity due to high-affinity binding with the FcγRs on effector cells​8​.

Benefits of low fucose Fc engineering

Afucosylated antibodies with either a complete absence or reduced levels of fucose can be generated using genetically engineered cell lines with defects in enzymes involved in the fucose biosynthesis pathway. Alternative strategies include recombinant fucosidases and post-manufacturing enzymatic treatment. Interestingly, afucosylation has overtaken other strategies — such as Fc mutations with five substitutions (L235V/F243L/R292P/Y300L/P396L) — to become the most popular strategy for enhancing antibody effector functions (Table 1).

Table 1​9​

Fc enhancing variantsNumber of INNs
Reduced levels of fucose (achieved by host cell engineering)10
E430G3
L235V/F243L/R292P/Y300L/P396L2
S239D/K274Q/Y296F/Y300F/L309V/I332E/A339T/V397M2
V215A2
G236A/A330L/I332E1
G236A/S239D/A330L/I332E1
N325S/L328F1
P247I/A339Q1
S239D/A330L/I332E1
S239D/I332E1
S267E1
S267E/L328F1
T393A1
V215A/E269R/K322A1
Table 1. Number of International non-proprietary names (INNs) filed with the World Health Organisation (WHO) that utilise Fc variants that enhance antibody effector functions​9​

evitria’s antibody expression service for producing afucosylated therapeutic antibodies

evitria is a global antibody expression service provider for afucosylated antibodies. evitria efficiently expresses afucosylated antibodies lacking the core fucose moiety by utilising ProBioGen’s GlymaxX® technology. For this, the heterologous expression of the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) deflects an intermediate in the de novo synthesis of fucose to a dead-end product​10​, resulting in the secretion of afucosylated antibodies. As part of the service, evitria can validate each antibody by assessing the ratio of G0/G0F using mass spectrometry (Figure 1) to calculate the percentage of afucosylation.

evitria excels in the transient expression of native and afucosylated antibody variants in Chinese hamster ovary (CHO) cells using the GlymaxX® technology. Afucosylated antibodies are produced at the same yields and possess the same stability as native antibodies, thus providing the perfect negative control. Unlike strategies using Fc mutations, evitria’s use of GlymaxX® avoids the risk of immunogenicity. Additional benefits of evitria’s afucosylated antibody service compared with alternative strategies are avoiding inefficiencies caused by steric hindrances of recombinant fucosidases or expensive post-manufacturing enzymatic treatment.

Figure 1. Evitria’s afucosylated antibody expression service validates antibodies using mass spectrometry. Source: evitria
Percentage afucosylation is calculated by comparing fucosylated (G0F) and afucosylated (G0) antibody forms.

evitria’s afucosylated antibodies for T-cell acute lymphoblastic leukaemia treatment

evitria has aided in the preclinical development of afucosylated antibodies for treating T-cell acute lymphoblastic leukaemia (T-ALL). Classed as an orphan disease (lacking immunotherapeutic options), T-ALL is a rare, aggressive form of leukaemia caused by the lack of proper development of T-cells, leaving them unable to fight infections properly. CD43 is highly expressed in T-cells. In a study published in the Journal for Immunotherapy of Cancer, researchers investigated whether an afucosylated humanised antibody targeting a unique epitope of CD43 (UMG1) — produced by evitria — could treat T-ALL.

The afucosylated UMG1 antibodies proved effective with significant ADCC against T-cells in vitro. In a leukaemia cancer mouse model, the afucosylated antibodies also exhibited treatment benefits with improved survival rates​11​.

References

  1. 1.
    Lyu X, Zhao Q, Hui J, et al. The global landscape of approved antibody therapies. Antibody Therapeutics. Published online September 6, 2022:233-257. doi:10.1093/abt/tbac021
  2. 2.
    Golay J, Da R, Bologna L, et al. Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab. Blood. 2013;122(20):3482-3491. doi:10.1182/blood-2013-05-504043
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    Umaña P, Jean–Mairet J, Moudry R, Amstutz H, Bailey JE. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol. Published online February 1999:176-180. doi:10.1038/6179
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    Shields RL, Lai J, Keck R, et al. Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity. Journal of Biological Chemistry. Published online July 2002:26733-26740. doi:10.1074/jbc.m202069200
  5. 5.
    Shinkawa T, Nakamura K, Yamane N, et al. The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity. Journal of Biological Chemistry. Published online January 2003:3466-3473. doi:10.1074/jbc.m210665200
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    Houde D, Peng Y, Berkowitz S, Engen J. Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol Cell Proteomics. 2010;9(8):1716-1728. doi:10.1074/mcp.M900540-MCP200
  7. 7.
    Chu Y, Awasthi A, Lee S, et al. Obinutuzumab (GA101) vs. rituximab significantly enhances cell death, antibody-dependent cytotoxicity and improves overall survival against CD20+ primary mediastinal B-cell lymphoma (PMBL) in a xenograft NOD-scid IL2Rgnull (NSG) mouse model: a potential targeted agent in the treatment of PMBL. Oncotarget. 2020;11(32):3035-3047. doi:10.18632/oncotarget.27691
  8. 8.
    Ishida T, Iida S, Akatsuka Y, et al. The CC Chemokine Receptor 4 as a Novel Specific Molecular Target for Immunotherapy in Adult T-Cell Leukemia/Lymphoma. Clinical Cancer Research. Published online November 15, 2004:7529-7539. doi:10.1158/1078-0432.ccr-04-0983
  9. 9.
    Wilkinson I, Hale G. Systematic analysis of the varied designs of 819 therapeutic antibodies and Fc fusion proteins assigned international nonproprietary names. mAbs. Published online September 15, 2022. doi:10.1080/19420862.2022.2123299
  10. 10.
    von Horsten HH, Ogorek C, Blanchard V, et al. Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase. Glycobiology. Published online July 15, 2010:1607-1618. doi:10.1093/glycob/cwq109
  11. 11.
    Caracciolo D, Riillo C, Ballerini A, et al. Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia. J Immunother Cancer. Published online February 2021:e002026. doi:10.1136/jitc-2020-002026
Previous article Scalability & Reproducibility – getting it right first time, then again and again…

February 6, 2024 duration: 2 min

Scalability & Reproducibility – getting it right first time, then again and again…

Desmond Schofield

Desmond Schofield

Director of Business Development at evitria

“Start with the end in mind” is the mantra for new product development activities; if you do not have a clear end-goal for your development, the temptation to chase bad results or over-invest in interesting (but non-essential) data can be too much to resist!

At evitria, we always advocate for careful forward planning and the design of production plans to fit your needs. Whether this be in the design of a bispecific panel for an initial feasibility, or the production of large quantities of antibodies for in vivo studies. However, all this planning is irrelevant if you do not have consistency in your material across time and scale. This is what evitria was founded to provide to researchers.

More than 90% of the time, antibody-related formats will be produced in CHO cells for clinical and commercial material supply. This is due to the many benefits of CHO cells, shown below.

HEK293 cells vs. CHO cells

At evitria, we pioneered the development of transient CHO services, to enable our clients to “start with the end in mind”, and work with their desired host system from day 1. However, this was not enough – in order to be a reliable partner for biotherapeutic R&D, we have to provide consistent material quality across time and scale.

We can demonstrate this when looking at our production history. Below is an example of a series of productions undertaken for one client. First, a small-scale production to support proof of concept work was delivered in summer 2020. Then a >2 year development gap followed while initial data and funding was gathered. After this, the client returned for several larger productions to support their later development and in vivo studies.

Production dateYield (% of 1st production)Purity (%)Scale (L transfected)
May-2010098.70.5
Nov-2210695.61
Jan-2311396.15
Apr-239696.22
May-2311296.24

Delivering material with the same quality at every scale, and with several years in between campaigns, enabled our client to rapidly move through development stages without having to undertake difficult troubleshooting activities or worry about their material quality.

Previous article Antibody reformatting and engineering – where will your variable domains take you?
Next article Enhanced purification services for diverse projects

January 22, 2024 duration: 2 min

Antibody reformatting and engineering – where will your variable domains take you?

Desmond Schofield

Desmond Schofield

Director of Business Development at evitria

The design of your antibody sequence is the single most important step in your therapeutic development journey. Weak binding, off-target effects, and insufficient activity can all improve via sequence engineering. If you want to explore novel and innovative formats (e.g. bispecifics), then further sequence engineering is necessary to introduce new mutations, build new structures, and combine new binding domains.

Throughout 14 years of operations, we have performed >130,000 successful production runs of different antibodies and antibody related formats, covering different species, subtypes, and isotypes, as well as a range of novel and engineered assemblies. This depth of experience is available for all our clients to benefit from, on a royalty-free, fee-for-service basis. Whether you need to create a murine analogue of your human IgG1 therapeutic for in vivo work, or want to stitch together different variable domains with a novel backbone, evitria’s scientists are ready and willing to guide you through the design choices and share lessons learnt.  

At evitria, the process always begins with the variable domains – once we establish these sequences, our modular cloning techniques enable rapid reassembly of your construct, whether a simple process (choosing from our library of >600 different constant domains) or a more bespoke design (such as the design of bi- and multi-specific antibodies).

Antibody reformatting

Frequently, our clients need to enhance immune effector functions, enhancing ADCC or CDC. Here, we have experience producing all commercially-approved ADCC enhancing mutation sets, as well as generating afucosylated antibodies with the GlymaxX® technology, an unique approach to enhancing ADCC activity. For clients working within anti-inflammatory therapeutics, or with many bispecifics, abolishing immune effector functions may be needed. Here, we can provide silenced Fc domains, and we can guide clients through the pros and cons of working with each mutation set, including the fully silent STR technology.

As soon as you begin a project with evitria, you can count on our team’s dedication and experience to guide the way – if you are thinking about where your variable domains can take you, why not just tell us where you want to go?

Previous article Transient antibody expression in CHO cells: Our approach at evitria
Next article Scalability & Reproducibility – getting it right first time, then again and again…

December 6, 2023 duration: 4 min

Transient antibody expression in CHO cells: Our approach at evitria

Richard Park

Richard Park

Global Head, Client Relations and Business Development at evitria

The transient expression of recombinant antibodies holds immense significance for scientific breakthroughs and therapeutic advancements. Recombinant antibodies serve as critical tools across various industries, from advancing medical treatments to facilitating groundbreaking research.

Among the methodologies employed in antibody expression, evitria has developed a distinctive approach known for its efficacy in transient antibody expression using CHO cells. You’ll learn more about it in this article.

Why recombinant antibodies?

Recombinant antibodies, synthesized through genetic engineering techniques, are pivotal tools in various scientific and medical applications.

These antibodies are crafted by introducing specific DNA sequences into host cells, enabling the production of antibodies with tailored characteristics.

Their significance lies in their versatility and precision. Unlike traditional antibodies derived from animals, recombinant antibodies offer several advantages:

  • Tailored characteristics: Genetic manipulation allows precise modification of antibody properties, such as enhancing specificity or altering functionality, catering to diverse research or therapeutic needs.
  • Consistency and reproducibility: Through controlled production methods, recombinant antibodies ensure uniform quality and reproducibility, crucial for reliable experimental outcomes and therapeutic consistency.
  • Reduced immunogenicity: By designing antibodies with minimized immunogenicity, potential adverse immune reactions in patients can be mitigated, a crucial aspect in therapeutic applications.
  • Diverse applications: These antibodies find utility in various applications, including disease diagnosis, targeted therapy, and probing intricate cellular mechanisms, fostering advancements across multiple scientific disciplines.

Recombinant antibodies, owing to their tailored properties and consistent quality, serve as indispensable tools in scientific research and medical advancements, driving innovation and precision in various fields. This is why evitria has specialized in their production.

Why CHO cells?

Within the spectrum of expression systems employed for recombinant antibody production, Chinese Hamster Ovary (CHO) cells stand out for various reasons.

CHO cells offer a unique blend of characteristics crucial for efficient antibody production. One notable advantage is their ability to perform complex post-translational modifications, mirroring human-like glycosylation patterns. This similarity in glycosylation ensures the production of antibodies with structures more closely resembling those found in humans, enhancing their efficacy and reducing immunogenicity.

Moreover, CHO cells demonstrate robust growth characteristics, allowing for scalability in culture, a pivotal aspect when aiming for large-scale antibody production. Their adaptability to suspension cultures simplifies the scalability process, enabling efficient handling and increased yields, essential for industrial-scale production demands.

Additionally, CHO cells (the use of which does not involve live animals today) are well-documented for their stability in long-term culture, maintaining desirable genetic traits over multiple passages. This stability contributes to consistent antibody production and minimizes the risk of genetic drift, ensuring reproducibility and reliability in the manufacturing process.

Transient transfection – which cell lines are the best option?

Why transient transfection?

Transient and stable transfection methods play distinct roles in recombinant antibody production. While stable transfection offers sustained antibody expression through integration into the host cell’s genome, evitria favors the transient method for its unique advantages.

Stable transfection involves integrating the foreign DNA permanently into the host cell’s genome, ensuring a continual production of antibodies. However, this method often demands extensive screening to select high-producing cell lines and requires more time and resources for development.

On the contrary, transient transfection offers flexibility and speed. This method doesn’t necessitate the integration of foreign DNA into the host cell’s genome, allowing for a rapid antibody production turnaround. Furthermore, transient transfection enables the swift expression of antibodies without the need for clonal selection or the establishment of stable cell lines.

evitria’s preference for transient transfection is driven by the accelerated production timelines and the ability to quickly adapt to diverse customer demands.

Read more: Benefits of transient transfection

Custom recombinant antibody production

We are a leading service provider for custom recombinant antibody production. Get in contact!

Custom recombinant antibody production

Antibody production workflow at evitria

At evitria, the antibody production workflow is meticulously designed to ensure efficiency, quality, and timely delivery of recombinant antibodies. The process is streamlined to meet stringent standards while maintaining a rapid timeline – typically five weeks from start to finish.

The workflow typically begins with the initiation of a project, which swiftly progresses through key stages:

Project initiation

  • Rapid commencement of the project, often within one day of customer approval
  • Process kicks off with a pilot study, advancing from the electronic sequence to quantified pilot supernatant within 15 to 20 business days

Larger-scale expression

  • Transition to larger-scale expression occurs efficiently within 5 to 10 business days
  • Scale-up facilitates increased antibody production while maintaining quality and consistency

Purification

  • Purified antibody isolation phase takes approximately 2 to 3 business days
  • Stringent purification protocols to ensure high-quality, reproducible antibodies

Final analytics

  • Comprehensive final analytics, including quality control measures, are completed within 2 to 3 business days
  • Thorough analysis to guarantee integrity and functionality of the produced antibodies

Shipment

  • The final stage involves the efficient delivery of the produced antibodies, typically within 1 to 2 business days.

The antibodies resulting from evitria’s workflow embody several key advantages. They are consistently high-quality, meeting stringent industry standards. With low levels of endotoxins (<1 EU/mg), these antibodies minimize potential adverse effects, ensuring suitability for a wide array of applications. Additionally, the workflow’s adaptability allows for tailored production, accommodating specific customer requirements.

Transient expression of rAbs in CHO cells – evitria’s service portfolio

evitria’s expertise in transient expression of recombinant antibodies (rAbs) using CHO cells stands as a cornerstone of its service portfolio. We have the technology and knowledge to deliver unparalleled solutions for rapid and high-quality antibody production.

The transient expression approach adopted by evitria leverages the advantages of CHO cells, ensuring a swift and efficient process that surpasses traditional techniques. This methodology allows for custom antibody production in large scales within remarkably short timelines – typically as brief as 5 weeks – while maintaining exceptionally low levels of endotoxins (<1 EU/mg).

The service portfolio offered by evitria spans a wide spectrum, encompassing custom development and manufacturing of various rAbs, including afucosylated antibodies, Fc-silenced antibodies, and other recombinant proteins. This allows us to cater to diverse customer needs across industrial, research, and therapeutic domains.

The key capabilities within evitria’s service portfolio include isotype switching, chimerization, glyco-engineering, and the development of bispecific or trispecific antibodies. These capabilities, coupled with the utilization of CHO cells, empower evitria to deliver tailored solutions meeting specific requirements while ensuring high quality and rapid turnaround times.

Previous article Transient transfection vs. stable transfection
Next article Antibody reformatting and engineering – where will your variable domains take you?

duration: 4 min

Transient transfection vs. stable transfection

Richard Park

Richard Park

Global Head, Client Relations and Business Development at evitria

Stable transfection establishes a lasting genetic change in cells, integrating foreign DNA into the host genome, ensuring consistent gene expression over time. Conversely, transient transfection introduces genetic material without permanent integration, resulting in short-term gene expression.

Despite this being the main gap between stable and transient transfection, it implies several further differences between these two approaches that are also essential in the expression of antibodies for various purposes.

Therefore, we will discuss the manifold differences between stable and transient expression – along with their implications – in this article.

What is transfection?

Transfection stands as a fundamental technique in genetic manipulation, allowing scientists to intentionally introduce external genetic material into cells. This process, facilitated by various methods like electroporation, chemical agents, or viral vectors, enables the incorporation of foreign DNA or RNA into the cell’s internal machinery.

It serves as a cornerstone in molecular biology, permitting the exploration of gene function, modulation of protein expression, and examination of intricate cellular mechanisms. By manipulating genetic material within cells, researchers can study diseases, engineer genetically modified organisms, or produce therapeutic proteins for medical purposes.

Transfection techniques are vital across diverse scientific disciplines, from pharmaceutical development to agriculture, paving the way for innovations in genetic engineering, disease research, and biotechnological advancements.

Stable transfection – process, benefits, and applications

Stable transfection involves the intentional introduction of foreign genetic material, typically through plasmids, into host cells. This process aims to establish a permanent alteration in the cell’s genetic makeup by integrating the foreign DNA into the host genome. It requires a meticulous selection process to identify and culture cells that successfully incorporate the desired genetic material.

The primary benefit of stable transfection lies in its ability to sustain consistent gene expression over extended periods. Once established, these genetically modified cells reliably produce proteins or exhibit specific traits dictated by the introduced genetic material. This reliability makes stable transfection crucial in various industries, particularly pharmaceuticals, where it serves as the foundation for continuous production of therapeutic proteins.

Moreover, stable transfection finds extensive application in disease modeling, allowing researchers to replicate specific genetic conditions for in-depth studies. It also plays a significant role in functional genomics, facilitating a deeper understanding of gene function and cellular pathways. Its long-term nature makes it suitable for conducting extended studies or maintaining continuous production of specific proteins for various scientific and industrial purposes.

Transient transfection – process, benefits, and applications

Transient transfection involves the temporary introduction of foreign genetic material, such as DNA or RNA, into host cells for short-term expression without permanent integration into the cell’s genome. This process usually lasts for a few days, allowing researchers to study immediate and short-term effects of introduced genes or proteins.

One of the significant benefits of transient transfection is its rapidity and simplicity. It allows for quick experimentation and doesn’t require the establishment of stable cell lines, making it a preferred choice for short-term studies or rapid production of proteins for research purposes.

Applications of transient transfection span various fields. It’s commonly used in protein expression studies, where researchers need to produce specific proteins quickly for analysis. Transient transfection is also vital in reporter assays, allowing the observation of gene expression and cellular processes in real-time. Moreover, it plays a crucial role in functional genomics research, enabling scientists to assess gene function and cellular responses promptly.

Despite its temporary nature, transient transfection serves as a valuable tool in scientific exploration, offering a swift and efficient means for studying gene expression and cell responses as well as protein production, without the need for establishing stable cell lines.

Custom recombinant antibody production

We are a leading service provider for custom recombinant antibody production. Get in contact!

Custom recombinant antibody production

Differences in an overview: Stable transfection vs. transient transfection

Stable transfection involves the integration of foreign genetic material, typically DNA, into the host cell’s genome, resulting in a persistent genetic modification. This approach provides a reliable platform for continuous protein production or long-term studies, as the introduced gene is consistently expressed alongside the host genome.

On the other hand, transient transfection introduces foreign genetic material into the cell’s cytoplasm, operating independently from the host genome. The lack of integration ensures that the host cell’s genetic material remains unaltered, with the introduced DNA or RNA undergoing transient expression for a limited duration.

This has a major impact on several aspects of these two methods, pointed out in the table below.

FactorsStable transfectionTransient transfection
Genetic alterationPermanent integration into the host cell’s genomeNo integration into the host genome; transient expression
Duration of expressionLong-term, sustained gene expressionShort-term, transient gene expression (typically a few days)
Cell line establishmentRequires establishment of stable cell linesNo need for stable cell line establishment
Experiment durationUseful for extended studies or experimentsPreferred for short-term studies or quick experimentation
Ease and speedGenerally more complex and time-consumingSimpler and quicker compared to stable transfection
ScalabilityOften less scalable due to the need for stable cell line selectionPotentially more scalable due to quicker experimental turnover
Risk of genomic integrationPossibility of unintended genetic changes due to integrationReduced risk of unintentional genomic changes as integration doesn’t occur
AdaptabilityUseful for consistent and ongoing studies with stable alterationsPreferred for experiments where temporary genetic modifications suffice

Stable transfection or transient transfection – what to choose when?

When deciding between stable and transient transfection methods, the choice hinges on the specific demands of the research goals. Stable transfection is often the preferred route for studies requiring prolonged and consistent gene expression or continuous protein production. It’s particularly valuable in disease modeling or when studying the effects of long-term genetic regulation.​1​

On the other hand, transient transfection is favored for shorter-term experiments or when swift protein production is needed without the requirement for lasting genetic alterations. This method offers flexibility and speed, minimizing the risk of unintended genomic integration and bypassing the need for establishing stable cell lines. It allows the quick examination of short-term protein expression and is, e.g., often used when studying gene silencing using inhibitory RNA or gene knockdown.​1​

When it comes to recombinant protein expression, stable transfection had long been the method of choice when aiming for larger-scale production. Recent advancements in the field of transient transfection, though, make use of cell lines like HEK293 or CHO cells for large-scale gene expression based on transient transfection, circumventing many laborious and time-consuming processes required for stable transfection.​1​

Read more: Transient transfection – which cell lines are the best option? | Transient antibody expression in CHO cells: Our approach at evitria

rAb production at evitria – with transient transfection

evitria specializes in services for the production of recombinant antibodies and other recombinant proteins, employing transient transfection of CHO cells as a core method. This approach allows for the rapid and efficient generation of antibodies without the need for establishing stable cell lines. Innovative strategies enable us to swiftly generate antibodies for various research and therapeutic purposes – efficiently, in high quality and in large amounts.

Furthermore, our specialization on transient recombinant antibody expression allows us to adapt quickly to changing demands, producing antibodies tailored to specific requirements without the constraints of long-term cell line establishment. This agility in antibody production enables us to cater to diverse research and medical needs.

  1. 1.
    Smith C. Stable vs. Transient Transfection of Eukaryotic Cells. Biocompare. Published January 2013. Accessed December 2023. https://www.biocompare.com/Editorial-Articles/126324-Transfection/
Previous article Benefits of transient transfection
Next article Transient antibody expression in CHO cells: Our approach at evitria

December 5, 2023 duration: 3 min

Benefits of transient transfection

Richard Park

Richard Park

Global Head, Client Relations and Business Development at evitria

The numerous benefits of transient transfection come from its ability to allow protein production without permanently altering the cellular blueprint. This method, unlike permanent genetic modifications, swiftly synthesizes proteins within host cells. The versatility and adaptability of transient transfection across various cell lines and its scalable nature redefine the boundaries of antibody expression.

In this article, we will explore the many benefits and real-world applications of transient transfection, shedding light on its substantial contribution to advancing biotechnological endeavors like the production of antibodies and other recombinant proteins.

Why is transient expression important?

Transient expression holds significance for its ability to rapidly produce proteins within cells without permanently altering their genetic structure. Its quick and adaptable nature distinguishes it from stable transfection methods, making it crucial for various practical applications.

This technique serves as a valuable tool in time-sensitive experiments, enabling researchers to swiftly study protein functionalities and conduct experiments without the permanence of genetic changes seen in stable methods. Moreover, transient expression is instrumental in producing therapeutic proteins, allowing for their generation without leaving a lasting genetic mark on the host cells.

5 advantages of transient transfection

Transient transfection presents numerous advantages that contribute to its significance in biotechnological applications. Its distinct characteristics redefine protein expression methods, offering:

  1. No permanent genome modification
  2. Fast protein expression
  3. Flexibility in cell line choice
  4. Scalable production processes
  5. Expression of various proteins

Now, let’s explore each of these advantages in more detail.

1. No permanent genome modification

Transient transfection is characterized by providing:

  • Temporary alterations – the ability to introduce genetic material for a limited duration without permanently integrating it into the host cell’s DNA
  • Preservation of cellular integrity – ensures the cell retains its original genetic blueprint once the transient expression period concludes
  • Safety in research and applications – minimization of potential risks associated with permanent genetic modifications, ensuring a safer approach for experimentation and application in therapeutic protein production

The absence of permanent genome modification in transient transfection offers a controlled and reversible means to conduct experiments and produce proteins, maintaining the integrity and safety of the host cells.

2. Fast protein expression

Transient transfection facilitates rapid protein expression by swiftly introducing genetic material into host cells, prompting immediate protein synthesis without the prolonged adjustments necessary in stable transfection methods. This rapidity is crucial for researchers needing quick access to proteins, particularly in time-sensitive experiments or when urgent protein production is required for therapeutic or research purposes.

The accelerated protein expression afforded by transient transfection benefits researchers and biotechnologists working on projects demanding swift protein synthesis. For instance, in drug development or therapeutic protein production, where timely access to proteins is vital for testing or treatment formulations, this rapid expression capability becomes invaluable.

Additionally, in research scenarios where quick experimentation turnover is essential, transient transfection’s ability to swiftly provide proteins enables faster iterations of experiments, accelerating the pace of discovery and innovation. This feature streamlines biotechnological processes, enhancing productivity and enabling quicker advancements in various fields reliant on protein synthesis.

Read more: Stable vs. transient transfection

Recombinant antibody expression – the process in detail

3. Flexibility in cell line choice

Transient transfection permits the use of varied cell lines, empowering researchers to select the most fitting for their experiments. This flexibility accommodates diverse research needs, allowing optimization based on scalability, protein type, and specific experimental requirements.

The choice also includes mammalian cell lines like HEK293 and CHO cells, enabling customization for optimal protein expression. This adaptability ensures tailored approaches, enhancing protein yields and overall efficiency in diverse biotechnological applications reliant on transient transfection.

Read more: Transient antibody expression in CHO cells: Our approach at evitria

4. Scalable production processes

Transient transfection streamlines scalability from small to large production scales without requiring extensive infrastructure. Enhanced transfection efficiency ensures consistency across scales, expediting the scaling process.

Its rapidity suits time-sensitive projects, offering adaptability and cost-efficiency. Moreover, its compatibility with various cell lines amplifies its versatility, catering to diverse protein production needs and enabling swift scalability for various applications.

5. Expression of various proteins

Transient transfection accommodates diverse protein expressions, spanning antibodies, enzymes, signaling molecules, and therapeutic proteins. This versatility is advantageous as it enables the synthesis of various protein types, including complex proteins with specific modifications or intricate folding.

Researchers benefit from this adaptability, using it to study protein functions, conduct assays, and produce therapeutic proteins. Additionally, it facilitates efficient screening and quick prototyping, expediting the assessment of protein characteristics and the development of novel proteins or formulations.

Custom recombinant antibody production

We are a leading service provider for custom recombinant antibody production. Get in contact!

Custom recombinant antibody production

Transient expression of recombinant antibodies at evitria

evitria specializes in leveraging transient expression systems for recombinant antibody production. Our focus on transient transfection technologies enables swift and efficient generation of high-quality antibodies. By optimizing expression and purification processes, evitria offers cost-effective and scalable solutions for biopharmaceutical manufacturing.

evitria’s expertise in transient expression empowers the rapid synthesis of antibodies, meeting the demands of therapeutic development, clinical and diagnostic applications. With our commitment to enhancing yield, quality, and speed of antibody production, we provide partners around the globe with high-quality antibody expression services.

Previous article How are afucosylated antibodies developed?
Next article Transient transfection vs. stable transfection

November 30, 2023 duration: 2 min

How are afucosylated antibodies developed?

Desmond Schofield

Desmond Schofield

Director of Business Development at evitria

The development of afucosylated antibodies implies a modification process altering the structure of antibodies, and involves the removal of a specific sugar molecule called fucose from the antibody’s carbohydrate chain. This modification significantly impacts the antibody’s functionality and interactions within the immune system. By eliminating fucose, afucosylated antibodies exhibit altered behavior, particularly in their ability to engage immune cells and potentially enhance their effectiveness in specific biological activities.

In this article, we will explore the methods and techniques used in afucosylation, shedding light on the science behind this crucial alteration in antibody structure.

Why are afucosylated antibodies developed?

Afucosylation of antibodies offers distinct advantages, primarily enhancing their ability to engage with the immune system. By removing fucose, afucosylated antibodies exhibit heightened antibody-dependent cellular cytotoxicity (ADCC).

This modification augments their capacity to bind more efficiently to immune cells, potentially leading to increased destruction of targeted cells or pathogens. Consequently, afucosylated antibodies hold the promise of significantly bolstering therapeutic efficacy, paving the way for more potent and targeted treatments against various diseases.

Developing afucosylated antibodies – basic principles

Creating afucosylated antibodies involves altering their structure by targeting the sugar components within the Fc region. This modification primarily focuses on reducing or eliminating fucose residues.

Read more: Applications of afucosylated antibodies

Scientists employ genetic engineering, enzymatic modification, or glycoengineering techniques to achieve this alteration. The goal is to manipulate the glycan structure to minimize fucose content, enhancing the antibodies’ functional properties.

Methods in afucosylated antibody development

There are different, very specific methods in afucosylated antibody development, some of which are listed below:

  • GDP-fucose control: Utilizing enzymes involved in the biosynthesis of GDP-fucose, a critical precursor for fucosylation, to modulate the fucosylation process.
  • FUT8: Manipulating the activity of the FUT8 gene, responsible for a specific enzyme adding fucose to antibodies
  • SLC35C1 modifications: Targeting the transporter gene SLC35C1 to regulate fucosylation in the Golgi apparatus.
  • Bisecting GlcNac generation: Introducing modifications in the glycan structures of antibodies by increasing bisecting GlcNAc formation, impacting fucosylation patterns.
  • Bacterial RMD expression in CHO cell cytosol: Disrupting the de novo pathway of GDP-fucose by expressing bacterial RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) in the cytosol of Chinese Hamster Ovary (CHO) cells.
  • Fucosylation inhibition with biohemicals: Employing specific inhibitors that hinder the fucosylation process within cells.
  • Plant cell expression platforms: Leveraging plant cells as alternative platforms for the production of antibodies with modified glycosylation patterns.
  • Chemoenzymatic remodeling: Using a combination of chemical and enzymatic methods to selectively modify glycan structures of antibodies, allowing controlled fucosylation alterations.​1​

Custom recombinant antibody production

We are a leading service provider for custom recombinant antibody production. Get in contact!

Custom recombinant antibody production

Afucosylated antibodies by evitria – afucosylation with GlymaxX® technology

evitria, in an exclusive partnership with ProBioGen’s GlymaxX® technology, offers high quality expression of afucosylated antibodies. Engineered for heightened Antibody-Dependent Cellular Cytotoxicity (ADCC), these antibodies demonstrate superior efficacy against tumorigenic and infected cells.

evitria’s approach, avoiding Fc mutations, ensures equivalent yields and stability to native antibodies with reduced immunogenic risks. Specializing in recombinant antibody production, evitria guarantees swift, high-quality results of afucosylated antibody expression. From sequence to antibody in just five weeks, evitria’s commitment is to timely support partners worldwide with high-quality afucosylated antibodies.

  1. 1.
    Pereira NA, Chan KF, Lin PC, Song Z. The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. mAbs. Published online May 7, 2018:693-711. doi:10.1080/19420862.2018.1466767
Previous article Applications of afucosylated antibodies
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