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.

Our process of stable cell line generation
for monoclonal antibody production

Expression vector construction

  • Gene synthesis optimized for the target expression system.
  • Gene subcloning in a high-performing expression vector.

Developability study

  • Production tests in transient expression systems.
  • Evaluation of yield, stability, solubility, and purity.
  • Full report including guarantees, estimated lead times, and time.

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.

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

  • Productivity evaluation in batch and/or fed-batch culture.
  • RCB preparation and stability study.

Process optimization

  • Test of several culture media, feed strategies, culture conditions, among others

Stable cell line and protocol transference



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)

Stable cell lines and selection systems used for monoclonal 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 (HEK) cell lines

HEK293F, HEK293E, among others

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 systemGlutamine 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:

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
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%
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%

IV.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

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.

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.

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.