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Overexpression of SERPINB1 in Chinese Hamster Ovary Cells Increases Recombinant IgG Productivity

By: Nan Lin, Jeanne Brooks, Natalie Sealover, Christopher Limmex, Henry J. George and Kevin J. Kayser, Cell Sciences and Development, SAFC/Sigma-Aldrich, 2909 Laclede Ave., Saint Louis, MO 63103 USA

Abstract

We report the discovery and validation of a novel CHO cell engineering target (Serpinb1) that has significant effects in enhancing recombinant IgG productivity. We performed transcriptomic studies using cDNA microarrays and compared cultures with IgG heavy and light chain transcription transiently repressed versus cultures with non-targeting siRNA. The transcription of serpin peptidase inhibitor, clade B, member 1 (Serpinb1), a member of the serine protease inhibitor (Serpin) superfamily, was up-regulated 2 fold post HC and LC siRNA transfection. A lentiviral vector was used to overexpress the Chinese Hamster Serpinb1 in a CHOZN® Glutamine Synthetase (-/-) recombinant IgG producing CHO cell line. The transduction led to a stable pool with 4.2-fold over-expressed SERPINB1 compared with the non-transduced control. The peak viable cell density and peak IgG volumetric productivity in fed-batch increased 1.3 and 2.0 fold, respectively, as a result of the over-expression. As verification, a plasmid expressing SERPINB1 was transfected to the CHOZN® GS (-/-) host cell line to create several stable pools. Over sixty single-cell clones were isolated from these stable pools and characterized for their SERPINB1 expression levels and exponential phase growth rate in fed-batch cultures. Paradoxically, the clone (SERPINB1 Clone 27) with the highest SERPINB1 expression isolated from the plasmid stable pools had decreased exponential phase growth rate. Selected clones with varied SERPINB1 over-expression levels were subsequently evaluated for their IgG expression capabilities using GS selection. Clone 27 has much reduced outgrowth using the GS expression system, but the “minipool” transfectants demonstrated higher expression. Two intermediate SERPINB1 OE clones (#42 and 47) demonstrated similar “minipool” productivity but reduced outgrowth under (-) glutamine selection, implying increased selection stringency. Clone #42 demonstrated increased productivity in “bulk” pool selection. We conclude that manipulating Serpinb1 expression level can lead to increased recombinant IgG productivity, but the effect in host cell lines may vary by clone. This work is among the ongoing effort in applying “-omics” findings to novel CHO host cell line engineering.

Background

Serpinb1 is up-regulated responding to hydrolysate feeds and may play a role in IgG production

serpinb1

Serpinb1 belongs to a superfamily of “suicide inhibitors”

Serpin peptidase inhibitor, clade B, member 1

  • Member of serine protease inhibitor (Serpin) superfamily
  • SERPINB1 is known to inhibit elastase and cathepsin G in neutrophils and is protective of cell damage at inflammation sites
serpinb1-gbc

Methods

Workflow

overexpression-workflow

Results

Serpinb1 is over-expressed in the lentiviral stable pool

lentiviral-stable-pool

SerpinB1 Lentiviral Stable Pool Demonstrated Increased IgG Volumetric Productivity

serpinb1-lentiviral-stable-pool

serpinb1-plasmid-stable-pools

bulk-pool-minipool

Conclusions

  • SerpinB1 overexpression may lead to increase in both growth and productivity in rIgG producing CHO cells as observed
    in the lentiviral stable pools derived
  • Confirmed OE in single-cell clones derived from plasmid stable pools generated using CHOZN GS host cell line
  • Clone-associated biological effects were observed in the five SERPINB1 OE clones studied
    - Highest SEPRINB1 OE (Clone #27) led to reduced growth rate
    - Clone #27 has much reduced outgrowth using the GS expression system, but the “minipool” transfectants
      demonstrated higher expression
    - Two intermediate level SERPINB1 OE clones (#42 and 47) appear to have similar productivity but reduced minipool outgrowth
      under (-) Gln selection, implying increased selection stringency; SERPINB1 OE Clone #42 demonstrated much increased
      productivity in “bulk” pool selection
    - Further investigation of more clones is necessary to clarify the clone-associated effect

Materials

     
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