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Antibody Expression Advantage

ATUM has utilized Design of Experiment (DoE) methodologies and machine learning to create more than 30 novel protein expression vectors optimized for transient protein production in HEK or CHO cells, ideal for the expression of antibodies, antibody-like, cytoplasmically expressed, secreted and integral membrane proteins. These unique vectors are able to increase protein expression more than 5-fold relative to commercial alternatives.

ATUM combines gene synthesis and protein expression services to test proteins in our suite of vectors, maximizing protein production in any expression host. The Vectorology suite of vectors combined with GeneGPS® codon optimization multiplies potential functional protein production.

Herceptin_vectorGPSv2
Trastuzamab_vectorGPS
Vectibix_vectorGPSv2
Rituxan_vectorGPS
MM121V_vectorGPS
23LN_vectorGPS
Expression data of six antibodies in different ATUM vector backbones, showing significant protein specific differences in expression. Proteins were expressed from HEK293 cells in suspension culture (96-well format) and purified on protein A resin. Protein quality was confirmed by SDS-PAGE, OD 280nm and HPLC analysis.


ATUM can screen an unlimited number of variables to boost protein productions. Test antibody expression was increased by screening 35 unique signal sequences. Gram levels of expression are possible in batch culture in just 5 days.

Signal_sequence
Optimization of signal sequence can significantly alter protein expression, approximate 8-fold increase over wild type (wt).

Specialized high-throughput and large-scale expression services for antibodies.

State-of-the-art antibody expression platform for small and large scale production with accelerated delivery times and rigorous QC.


Mel and Robot

Fully automated, bar-coded, time-stamped process for screening of large numbers of antibody constructs.

Robot2

1 mL to 10 L culture formats available for expression in HEK and CHO suspension cell lines.


Available Options

Cell Types HEK and CHO suspension cell lines
Culture Formats 1mL, 10mL, 30 mL, 100mL, 1L, 2L, 5L & 10L cultures
Deliverables
  • Filter sterilized, purified protein
  • Gel image
  • OD280 Quantification
  • HPLC Size Exclusion Chromatography
  • Endotoxin Tested


Virtual Sequence to Purified Protein in as little as 4 weeks

Lg_small_scale_process

References :
          “Virtual Sequence” image derivative of work by surfupvector.
          Licensed under Adobe Stock

ATUM offers multiple QC options

           Aggregation Analysis

Analytical size exclusion chromatography is performed on microbore silica columns (Sepax Zenix-C) which are ideal for rapid determination of monodispersity, multimerization or percentage of aggregation present in protein samples.

Detection of eluted material is by absorbance at 280mn and 220nm, which enables coverage of a large range of sample concentrations, including very low levels of aggregated material. See sample data report for typical size exclusion profiles.

Second column purification, such as IEX or preparative size exclusion chromatography is available to separate species and buffer screening to deaggregate samples is also available.

           Endotoxin

Lipopolysaccharides from the outermembranes of Gram-negative bacteria can confound some downstream assays or cell based experiments, so as standard we assay and report the level of endotoxin in final protein samples.

In the unlikely event of endotoxin contamination, endotoxin can be reduced by further treatment of protein samples if necessary.

           N-Terminal Sequencing

We can N terminal sequence customer proteins to determine the amino acid sequence of the mature processed protein.

Different numbers of N terminal amino acids reads are possible by Edman degradation depending on the service requested.

Turn around time is typically between 7-10 days and success is dependent on an unblocked N-terminal anion group being present

           Mass Spectrometry

MALDI-TOF mass spectrometric detection of intact mass of proteins (reduced/ non reduced) pre or post deglycosylation is available.

This service enables unambiguous conformation of protein identity or post translational modifications.

See sample data for an antibody.

              Sample Data Package

Inquire here

           Inquire for other assays

Antibody Humanization

Humanized antibodies are antibodies made from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. Antibody humanization is used for reducing the immunogenicity of animal (mostly murine) monoclonal antibodies (mAbs) and for improving their activities in the human immune system.


Our antibody humanization platform combines advantages of both rational and empirical antibody humanization approaches.

Humanized antibodies are designed using our proprietary humanization and optimization algorithm and made by gene synthesis and fusion of humanized antibody variable regions with human antibody backbones.

Full antibody

Hybridoma/Antibody Sequencing

Hybridoma sequencing or antibody sequencing is the sequencing of the cDNA encoding the variable heavy (VH) and variable light (VL) domains of your hybridoma cell line. Antibody sequence information is essential for monoclonal antibodies (mAbs) engineering, function optimization, database banking and patent application.



Sequencing_Flowchart

ATUM provides:

  • Determination of variable heavy and light chain sequences
  • Species and IgG sub-type confirmation
  • Follow on services
    • Combine antibody sequencing with our antibody engineering, humanization and antibody expression services



References :
         “Sequencing Infographic” image, derivative of icons by Rashad Ashurov and Blumer1979
         Licensed under Adobe Stock

Antibody Purification

Affinity purification is typically the first purification step for antibodies and antibody like molecules. We utilize Kaneka KanCap A resin to capture molecules with suitable Fc regions, and after high and low salt washes, the proteins of interest are eluted at pH 3.5, which minimizes low pH destabilization.

For proteins that interact poorly with Protein A, we utilize other resins :

  • Protein G
  • Protein L
  • Alternate resins that bind to specific light chains

Proteins are desalted or dialysed into PBS or customer specified buffer immediately after elution from affinity resin.

Other proteins may be purified by alternate affinity tags :

  • His tags
  • Strep tags
  • FLAG tags
  • Specific affinity resins for fusions such as HSA

Please contact sales for discussion of purification strategies tailored for your proteins, particularly for low expressing proteins or cytoplasmic expression.

Second column purification by ion exchange chromatography or polishing steps via size exclusion chromatography are often required (e.g. to remove aggregated material or co-purifying molecules) as is protein concentration or buffer optimization.


Chemical Modifications

Purified proteins can be chemically modified to aid in downstream processes. As standard, we offer Biotinylation, Fluorescent protein labeling, cross linking, protease digestion or PEGylation.