Stable cell lines for monoclonal antibody production

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    Are low antibody yields stopping you from reaching the market? Improve the odds of monoclonal antibody development success by choosing a stable cell line development platform focused on developability. Drawing from 30 years of experience in protein production and over 100 stable cell lines generated, our pipeline offers high and stable yields tied to the market’s strongest guarantees. Monoclonal antibody production made simple with our CMO-transferrable FTO cell lines with high-yields (>7g/L) at competitive prices.

    Why choose ProteoGenix' antibody
    phage display services?

    FTO cell lines for stable antibody production
    FTO cell lines

    Benefit from our royalty-free approach to speed up clinical development

    Ease of transference of stable cells and protocols
    Ease of transference

    Fast cell line and protocol transference to your CMO partners for cGMP bioproduction

    Emphasis on monoclonal antibody developability
    Emphasis on developability

    Minimize production risks thanks to our extensive early testing of antibody candidates

    Cell line diversity for stable antibody production
    Cell line diversity

    Proprietary CHO, CHO-S, CHO-DG44, HEK293 or even your preferred cell line… Get a service adapted to your requirements!

    Record monoclonal antibody productivity
    Record productivity

    Ensure your cell line generates the highest possible yields thanks to our proven amplification protocols

    Stable and high yields guaranteed
    Yields guaranteed

    Plan for the long-term and secure your investments by choosing a stable cell line development service with strong guarantees

    Protein production and stable cell line generation experts
    Protein production experts

    500+ monoclonal antibodies developed, 100+ stable cell lines generated, 28+ years of experience in protein production. Choose a leading bioproduction company.

    Full control over your stable cell line generation process
    Full process control

    Stay in complete control of your project thanks to our milestone-oriented approach for better flexibility

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    Our process of stable cell line generation
    for monoclonal antibody production

    Stable expression vector construction for monoclonal antibody production
    Expression vector construction
    • Gene synthesis optimized for the target expression system.
    • Gene subcloning in a high-performing expression vector.
    Antibody developability study
    Developability study
    • Production tests in transient expression systems.
    • Evaluation of yield, stability, solubility, and purity.
    • Full report including guarantees, estimated lead times, and time.
    GO/NO GO
    Host transfection and selection of positive stable pools
    Host transfection and selection of positive pools
    • Co-transfection with the expression and selection vectors.
    • Growth in selective media for the selection of positive pools.
    • Characterization of the most productive stable pools.
    GO/NO GO
    Isolation of the best monoclones for stable antibody production
    Isolation of the best monoclones
    • Isolation of monoclones by limiting dilution or VIPS™.
    • Screening of clones by ELISA, WB, FACS, IP or Octet Red96.
    Characterization of the best monoclones for stable production
    Characterization of the best monoclones
    • Productivity evaluation in batch and/or fed-batch culture.
    • RCB preparation and stability study.
    GO/NO GO
    Stable production process optimization
    Process optimization
    • Test of several culture media, feed strategies, culture conditions, among others
    Stable cell line and protocol transference for large-scale antibody production
    Stable cell line and protocol transference

    Stable cell line and protocol transference to the customer

    Customer

    Stable cell line and protocol transference to a CMO

    CMO

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    Our recombinant antibody production
    service content

    Step Content Timeline Deliverables
    Gene synthesis
    • Gene design including codon optimization
    • Gene synthesis
    • Subcloning in expression vector
    		 3 to 4 weeks
    • Gene optimized for the required expression system
    • Gene cloned in pUC57 plasmid
    Developability study
    • Production tests in transient systems
    • Evaluation of yield, stability, solubility, and purity
    		 3 to 4 weeks
    • Full report including project feasibility, custom protocol, guarantees, lead times, and price
    Stable pool generation
    • Stable transfection
    • Pool selection and amplification
    		 9 to 10 weeks
    • 50mL of the supernatant from stable pool
    • Stable pool vials
    • Detailed report
    Single cell clones screening and subcloning
    • Subcloning of the top producing clones
    • Characterization of clones
    • Stability study
    • RCB preparation
    		 ~4 months
    • Research cell bank -tested for Mycoplasma
    • Certificate of Analysis
    • Detailed report
    Process optimization
    • Test of several media/feed conditions
    		 4 to 5 weeks
    • Detailed report
    • Optimized protocol transfer

    Optional services:

    Before starting:

    • Antibody engineering: humanization, affinity maturation, antibody reformatting, bispecific antibody development
    • Screening of multiple antibody leads to choose the best for further development
    • Extensive antibody characterization: KD determination, thermostability, aggregation, glycosylation profile, epitope mapping

    After stable cell line development:

    • Research Cell Bank stability study
    • Additional antibody characterization
    • Master and Working Cell Bank development
    • Scale-up production (up to Biorector)
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    Stable cell lines and selection systems used for monoclonal antibody production

    Chinese hamster ovary cell lines for antibody production

    Chinese hamster ovary (CHO) cell lines

    CHO-S, CHO-DG44, CHO-K1, proprietary GS-CHO cell line (upcoming) among others

    Human embryonic kidney cell lines for antibody production

    Human embryonic kidney (HEK) cell lines

    HEK293F, HEK293E, among others

    Customer-provided cell lines for antibody production

    Customer-provided cell lines

    Baby hamster kidney (BHK), mouse myeloma (NS0) cells, Per.C6, among others

    Most mammalian cell lines can either be selected using metabolic or antibiotic markers. The most widely used markers for recombinant monoclonal antibody production are metabolic, including:

    • Metabolic markers
      • MTX/DHFR system
        Dihydrofolate reductase (DHFR) is a key enzyme in the de novo synthetic pathways of purines and certain amino acids, while methotrexate (MTX) is a known DHFR-inhibitor. Using DHFR- deficient cells (e.g. CHO-DG44) or growing in the presence of MTX allows the selection and amplification of positive clones in medium devoid of hypoxanthine and thymidine (HT).
      • MSX/GS system
        Glutamine synthetase (GS) catalyzes the condensation of glutamate with ammonia to produce glutamine thus controlling the nitrogen cycle and preventing toxic ammonia accumulation inside the cells. Methionine Sulfoximine (MSX) is a known GS inhibitor. Using GS-deficient cells (e.g. NS0) or growing cell cultures in the presence of MSX allows the selection and amplification of positive clones.
    • Antibiotic markers
      • G418/Geneticin
      • Blasticidin
      • Hygromycin B
      • Puromycin
      • Zeocin
      • …

    Metabolic selection markers have remained the gold standard for stable cell line generation. This preference can be explained by the versatility of these systems. Particularly in CHO cells, the presence of increasing concentrations of MTX/MSX is unknown to result in the amplification of antibody encoding genes. Consequently, this leads to a significant increase in production yield in comparison to other recombinant expression systems.

    However, our focus remains on flexibility. For this reason we offer a vast choice of selection systems and cell lines allowing us to adapt the process of stable cell line generation to your unique requirements and needs.

    Discuss your project with our PhD account managers to receive a tailor-made proposal:

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    Case study: stable expression of an IgG1 monoclonal antibody

    I. GENE SYNTHESIS, SUBCLONING, AND EARLY TESTING

    Antibody genes were optimized for expression in CHO cell lines, synthesized, and subcloned into our proprietary transient expression system.

    CHO-K1 cells (30 ml) were transiently transfected, transient pools were grown in 30 ml, and antibody purification was performed using protein G resin.

    Yield: 18.2 mg/L | Quantity produced: 0.18 mg | Purity >90%
    A final QC was performed by reduced and non-reduced PAGE analysis
    confirming the integrity of the antibody

    II. DEVELOPMENT OF STABLE-TRANSFECTED POOLS

    • Construction of stable expression vectors containing:
      • Heavy and light antibody chains
      • CMV (human cytomegalovirus) promoters
      • Polyadenylation signals for enhanced transgene expression
      • Glutamine Synthetase (GS) – the selection marker
      • Determination of natural resistance to Methionine Sulfoximine (MSX)
    • Determination of natural resistance to Methionine Sulfoximine (MSX)
    • Vector linearization
    • Transfection of CHO-K1 suspension cultures
    • Selection in the presence of MSX
    Case study – development of stable pools

    3 positive stable pools were further characterized in fed-batch experiments and antibodies characterized by SEC-HPLC to confirm purity and detect potential aggregation

    Pool ID Quantity (mg) Yield (mg/L) Purity
    1 37.3 1243 >90%
    2 39.4 1313.3 >90%
    3 43.6 1453.3 >90%

    III. PRODUCTION AND PURIFICATION SCALE-UP

    1L fed-batch production was carried out in 3L flasks and purification
    was performed using protein A resin
    Final yield: 2015.5 mg/L | Final purity: >92%

    VI.ISOLATION AND SCREENING OF STABLE MONOCLONES

    Monoclones were isolated using the limiting dilution method in 96-well plates and
    expression evaluation was performed by ELISA with anti-Fc antibodies
    2 Rounds of limiting dilution
    29 Best performing clones
    Best monoclones were evaluated by SDS-PAGE and ELISA (Fc-antibody).
    ELISA data (below) revealed 10 monoclones with high expression levels.
    Case study – screening of stable monoclones
    Bar length represents intensity obtained in ELISA, numbers represent the clone ID.

    V. EVALUATION OF STABLE MONOCLONES

    Clone ID 1 3 4 7 10 15 17 19 30 31
    Yield (g/L) 2.05 2.86 1.07 1.68 4.34 2.57 3.21 2.60 1.45 1.25
    Monoclones 3, 10, 17, and 19 maintained good stability and viability in
    subsequent tests and were selected for further development

    VI. PRODUCTION SCALE-UP AND OPTIMIZATION

    Monoclone 10 was used for initial scale-up with excellent results.
    Final yield: 7.91 g/L | Final purity: >95%

    Why focus on monoclonal antibody developability?

    95% of biologics under development fail to reach the market or clinic due to undetected developability issues. Early testing or screening has thus become crucial to overcome low success rates and minimize development risks. Producing monoclonal antibodies in vitro via transient systems is still the best way to produce small amounts of antibodies for measuring:

    • Affinity/avidity
    • Specificity/selectivity
    • Aggregation profile
    • Stability
    • Ease of purification
    • Ease of scalability

    Detectable problems at this stage can be corrected by additional engineering of antibody leads. These engineering efforts, although laborious, can save considerable costs and time on the long run to license your therapeutic antibodies.

    To learn more about stable cell line development, visit our frequently asked questions (FAQs) page. On this page, we cover all seminal principles and knowledge regarding monoclonal antibody production in stable cells and provide detailed insights into ProteoGenix’s unique platform for stable cell line development.

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