COMMUNICATION TO THE EDITOR
Multiple cell line sources are used as hosts to produce recombinant
prophylactic and therapeutic proteins for human use. Recombinant CHO
cell lines (Puck et al., 1958) remain a preferred host due to the
reliability, robustness and maturity of the technology in generating
clonally-derived cell line. Despite the high level of production,
batch-to-batch consistency and robustness of a clonally-derived CHO cell
line, the approach is challenging due to the burden of time and
resources required for stable clone isolation, selection, and provision
of tested cell banks for cGMP manufacturing. To overcome this challenge,
we wanted to evaluate the possibility of using non-clonal stable CHO
cell pools to expediate production of a monoclonal antibody (mAb)
against SARS-CoV2, CC6.35, particularly through cGMP manufacturing of a
single batch of material for a Phase 1 clinical trial. Since this
approach has not been rigorously tested for scaled-up cGMP manufacturing
and concerns remain that the cellular and genetic heterogeneity of
non-clonal stable CHO cell pools may result in production variability
and concomitant heterogeneous product qualities between batches, we
present a case study wherein we used non-clonal qualified cell banks and
platform processes to accelerate manufacturing of CC6.35 mAb. To do so,
we used the novel transposon-based Leap-In
Transposase® system (Rajendra et al., 2017; Rajendran
et al., 2021) for the development of stable CHO cell lines. The
codon-optimized DNA sequence encoding the amino acid sequence for the
Heavy chain(HC) and Light chain(LC) of CC6.35 mAb along with
corresponding signal peptide and the novel expression constructs based
on the Leap-In transposon® system were designed and synthesized. These
synthesized DNA constructs along with transposase mRNA (Rajendran et
al., 2021; Wilson et al., 2007) were used to co-transfect HD-BIOP3
glutamine synthase (GS) knock-out CHO-K1 host cells. Two promoter
components were used to generate two unique sets of CHO cell pools: one
using the EF1 promoter and another using the CMV promoter.
Post-transfection the recovery of these two CHO cell pools was
performed; after the initial recovery phase, the positive CHO cell pools
were selected by outgrowth in a glutamine-free formulation at 37°C in
5% CO2 and 70-80% relative humidity and expanded
further before cryopreservation and generation of Research Cell Banks
(RCBs). In order to estimate productivity of the CC6.35
antibody-expressing stable CHO cell pools, both cell pools were expanded
in a small-scale cell culture based on fed-batch process. After 14-days,
the harvested supernatant was measured for titer by binding to a
Protein-A biosensor on the Octet® System.Table 1 shows the percent (%) viability and expression levels
(in g/L) for both EF1-CC6.35 and CMV-CC6.35 stable CHO cell pools.
Based on the higher titer, the CMV-CC6.35 cryopreserved RCB vials were
advanced for use in scale-up production. This (CMV-CC6.35) RCB was
tested for safety and regulatory acceptance for producing the two
pivotal lots of material: one for preclinical safety studies and another
for Phase 1 clinical trial.
For producing material for pre-clinical safety study, the RCB vials were
thawed and expanded using a seed-train to support a 200 L bioreactor.
The key cell culture process indicators included cell growth, cell
viability, metabolic profiles (for ammonia and lactate), bioreactor
regulation profiles (for pH, glucose, osmolality and
pCO2) and antibody titer. After 14 days, the cell
culture supernatant was harvested, clarified, and subjected to a
3-column chromatography platform purification process (involving
Protein-A column chromatography, Anion-Exchange Column Chromatography
and a Cation-Exchange column chromatography) steps to generate purified
Drug Substance (DS), formulated at 20 mg/mL in an antibody platform
buffer as Drug Product (DP) for intra-venous administration.
For producing material for the Phase 1 clinical study, a similar
upstream (cell culture) process strategy was employed except that in
this case, the RCB vials were thawed and expanded using a seed-train to
support a 2000 L bioreactor. After 14 days, the supernatant from the
2000 L bioreactor was harvested, clarified, and purified via the same
3-column chromatography platform process to generate purified Drug
Substance (DS), formulated at 20 mg/mL in a histidine-based buffer as
Drug Product (DP) for intra-venous (i.v.) administration.
Both Drug Substances and Drug Products (200L and 2000 L scales) were
tested based on a proposed analytical panel and results compared to
agreed acceptance criteria. The analytical panel, comprising of
quantitative test (protein concentration and biological activity),
qualitative tests (physico-chemical and microbiological purity) and
compendial methods, was set based on prior knowledge on antibodies of
similar structure, and after confirmation of their suitability for the
purpose. In addition, a formal analytical comparability was performed to
ensure that the two products, from 200L and 2000L scales, are
”essentially similar” despite the changes in their production scale and
minor change in processes. The results of the analytical comparability
study are reported in Table 2 .
In addition to results in Table 2 , Circular Dichroism (CD)
analysis showed that both DS materials are comparable in terms of
secondary and tertiary structures (data not shown). Thermal analysis (by
Differential Scanning Fluorimetry) showed that both DS samples have
similar thermal denaturation profiles with a temperature of onset
(Tonset) at 60-61 °C with two inflection points (IP) for
both samples, one at 67 °C and another at 78 °C (data not shown).
Extensive characterization of glycans (glycan mapping) of the two DS
materials showed that the various detected N-linked glycan species are
comparable, except for some minor differences in relative distribution
of the galactosylated, fucosylated and sialylated species (data not
shown). Additionally, LC-MS analysis was performed on the two DS
materials for intact mass and deglycosylated/reduced mass. Intact mass
LC-MS analysis showed that the main proteoform for both non-cGMP DS
batch and the cGMP DS materials was the intact molecule PyroQ-LC +
HC[-K] coupled with FA2-FA2 glycans (data not shown). With respect
to the expected mass of 150622.60 Dalton, the non-cGMP DS had an
experimental mass of 150622.35 Dalton, whereas the cGMP DS had a mass of
150623.80 Dalton. Some other fragments were detected and identified in
both samples, amongst which the most abundant was the LC-LC dimer (data
not shown). After deglycosylation/reduction, the LC-MS analysis revealed
that the two DS materials were overall comparable in terms of intact
molecule, except for slightly higher levels of HC C-terminal truncation
in the cGMP batch (data not shown). Finally, non-reducing peptide
mapping by LC-MS/MS showed that the (nine) detected disulphide bridges
were aligned in line with expected (canonical) ones.
In summary, we demonstrate that using non-clonal stable CHO cell pools
and use of platform processes can expediate early clinical development
of monoclonal antibodies during pandemic outbreaks of emerging
infectious such as COVID-19. We show that the antibodies produced from
these stable CHO pools at two large scales bioreactors were comparable
using platform upstream and downstream processes, and through robust
analytical tested they were deemed suitable for clinical use. Since
clinical development timeline continue to thwart rapid evaluation of
therapeutic and prophylactic interventions during pandemics and
improvements in development span essential (Kelley, 2020), we believe
that this approach of using non-clonal stable CHO cell pools, which
enabled manufacturing of early clinical trial material within 4.5
months, is a feasible alternative for rapid cGMP manufacturing, and a
means that can accelerate the pace of therapeutic and prophylactic
protein evaluation in clinic.