Engineering Conferences International

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Lee, Jae Seong

Min, Honggi

Kim, Dongwoo

Lee, Sumin

Lee, Solhwi

Kim, Seul Mi

Poster Number 60 
HYBRID CELL LINE DEVELOPMENT SYSTEM UTILIZING SITE-SPECIFIC INTEGRATION AND 
METHOTREXATE-MEDIATED GENE AMPLIFICATION IN CHINESE HAMSTER OVARY CELLS 
 
Jae Seong Lee, Dep. of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea 
jaeseonglee@ajou.ac.kr 
Honggi Min, Dep. of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea 
Seul Mi Kim, Dep. of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea 
Dongwoo Kim, Dep. of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea 
Solhwi Lee, Dep. of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea 
Sumin Lee, Dep. of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea 
 
 
Key Words: CHO cells, CRISPR/Cas9, DHFR knockout, gene amplification, targeted integration 
 
Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster 
ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits 
their practical applications. In this study, we developed a hybrid CLD platform combining site-specific integration 
of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate 
high-yield recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette 
exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a 
CHO genomic hot spot, the C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to 
various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in 
EGFP expression in the presence of 200 nM MTX, accompanied by an increase in DHFR and EGFP copy 
number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity 
(qmAb) of recombinant mAb-producing targeted integrants by 2.8-fold, reaching a qmAb of 9.1–11.0 pg/cell/day. 
Fluorescence in situ hybridization analysis showed colocalization of the DHFR and mAb sequences at the 
intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted 
integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting 
stable gene expression in amplified cell lines. Our results suggest that the present CLD platform can increase 
the productivity of targeted integrants by amplifying transgene copies. 
 
Given the wide use of the CHO cells and potentials of site-specific integration-based CLD, accelerated 
generation of highly producing targeted integrants is promising. Increasing the transgene copy number from 
established and well-characterized targeted integrants can minimize clonal variation and increase productivity 
without the need for multicopy targeted integration. The present method could provide a new CLD platform for 
the efficient production of biopharmaceuticals and will be of broad interests to both medical and biotechnological 
research communities. In this presentation, I will discuss pros and cons of the present method by comparing it 
with the classical CLD method. Furthermore, I will discuss the perspectives of this new platform for product-
specific CHO CLD and cell engineering.  
 
 
 


English

Hybrid cell line development system utilizing site-specific integration and methotrexate- mediated gene amplification in Chinese Hamster Ovary cells

Liu, Ren

 Poster Number 33 
HIGH ER CONTENT AND UPREGULATED UPR PATHWAY RENDER A UNIQUE CHO HOST WITH HIGH 
ANTIBODY PRODUCTIVITY AND PRODUCT QUALITY 
 
Ren Liu, Bioprocess R&D, MSD 
Ren.liu@merck.com 
 
 
Key Words: CHO, ER, UPR, productivity, product quality. 
 
Chinese hamster ovary (CHO) cells are the work horse for the manufacturing of biological therapeutics. The 
productivity of CHO cell culture has improved significantly in the past decades. However, the specific 
productivity of CHO cells still pale in comparison to professional secretory cells.  We have developed a CHO 
host (CHO-A) that demonstrated specific productivity markedly 
higher than other CHO hosts, reaching as high as 148 
pg/cell/day, similar to the levels observed in professional 
secretory cells. This CHO host cell also produced complex 
molecules such as multi-specific antibodies with higher quality 
compared to other CHO host cells. We discovered that this host 
has unusually high endoplasmic reticulum (ER) content (Figure 
1). It also had significantly increased levels of critical genes in 
unfolded protein response (UPR) pathway such as ATF6, XBP1, 
Sec61, BiP, and EDEM1/3 (Figure 2). In addition, high 
productivity recombinant clones derived from this host 
demonstrated stability for at least 75 generations of continuous 
culturing. These results suggest that selecting / engineering 
CHO host with larger ER content is a promising way to achieve 
superior productivity and product quality for biologics 
manufacturing.  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. Upregulated UPR pathway in CHO-A cell (1) compared to another CHO host (2) 
Figure 1. Higher ER content in CHO-A 
  


High ER content and upregulated UPR pathway render a unique CHO host with high antibody productivity and product quality

Sen, Saurabh

Thursday, April 27, 2023        Session 6: Production of Viral Vectors and Other Emerging Therapeutic Modalities 
DEVELOPMENT OF A SUBCLONAL HOST CELL LINE FOR AAV PRODUCTION 
 
Saurabh Sen, Cell Line & Cell Bank Development, Sanofi 
Saurabh.Sen@sanofi.com 
 
 
Key Words: Viral Vectors, Gene Therapy, AAV, Producer Cell Line (PCL), HeLaS3 host 
 
The Genomic Medicine Unit (GMU) CMC group at Sanofi is dedicated to the establishment of best-in-class 
manufacturing platforms to support development of life-changing advanced cell and gene therapy products. 
Recombinant adeno-associated virus (rAAV) has emerged as one of the leading viral vectors for gene therapy. 
Sanofi’s AAV producer cell line (PCL) platform represents a leading technology for rAAV vector production due 
to its scalability and the capacity of the platform to produce viral vector of high and consistent quality. PCLs 
supporting projects within the Sanofi GMU portfolio are typically generated from a HeLaS3 host cell line.  
There are a few reasons for creating a new host cell line: first, reducing the heterogeneity that is characteristic of 
the HeLa cells; second, selecting a new host that has improved critical attributes compared to our current host, 
such as improved Population Doubling Time (PDT), growth kinetics, transfection efficiencies, vector yield, etc.; 
and finally, selecting a subclone capable of single-cell growth in a serum-free media, enabling process efficiency 
and reducing potential viral risk.  
 
The current abstract focusses upon the technologies available to produce recombinant adeno-associated virus 
(AAV)-based viral vectors towards treatment of various diseases. Efforts to develop a new HeLaS3 host cell line 
have been initiated and the current content will highlight upon Sanofi’s PCL based platform for production of 
gene therapy vectors and will zoom in on the development of a sublconal host cell line. 
 
 
 
 


Development of a Subclonal host cell line for AAV production

Du, Zhimei

Chang, Meiping

Nelson, Luke

Tang, Xiaoyan

Nyberg, Gregg

Wednesday, April 26, 2023                                                                     Session 5: CCE for Cell-based Therapies 
DIFFERENTIAL EFFECTS ON NATURAL KILLER CELL PRODUCTION BY MEMBRANE‐BOUND 
CYTOKINE STIMULATIONS 
 
Zhimei Du, Landmark Bio 
zhimeidu@gmail.com 
Meiping Chang, Merck Research Lab 
Xiaoyan Tang, Merck Research Lab 
Luke Nelson, Merck Research Lab 
Gregg Nyberg, Landmark 
 
 
Key Words: Allogeneic Cell Therapy, NK cell, Production process design 
 
Robust manufacturing production of natural killer (NK) cells has been challenging in allogeneic NK cell‐based 
therapy. Here, we compared the impact of cytokines on NK cell expansion by developing recombinant K562 
feeder cell lines expressing membrane‐bound cytokines, mIL15, mIL21, and 41BBL, individually or in 
combination. We found that 41BBL played a dominant role in promoting up to 500,000‐fold of NK cell expansion 
after a 21‐day culture process without inducing exhaustion. However, 41BBL stimulation reduced the overall 
cytotoxic activity of NK cells when combined with mIL15 and/or mIL21. Additionally, long‐term stimulation with 
mIL15 and/or mIL21, but not 41BBL, increased CD56 expression and the CD56bright population, which is 
unexpectedly correlated with NK cell cytotoxicity. By conducting single‐cell sequencing, we identified distinct 
subpopulations of NK cells induced by different cytokines, including an adaptive‐like CD56brightCD16‐CD49a+ 
subset induced by mIL15. Through gene expression analysis, we found that different cytokines modulated 
signaling pathways and target genes involved in cell cycle, senescence, self‐renewal, migration, and maturation 
in different ways. Together, our study demonstrates that cytokine signaling pathways play distinct roles in NK 
cell expansion and differentiation, which sheds light on NK cell process designs to improve productivity and 
product quality. 
 
 
 
 
(a) NK cell expansion fold induction (y‐
axis in log10 scale) from three donors with 
indicated process conditions.  
 
(b)  Single cell analysis, TotalSeqC panel 
measured RNA expression and surface 
protein expression, reveals 10 NK cell 
clusters. Different process conditions drive 
NK cells to follow distinct differentiation 
trajectories, leading to divergence in cellular 
composition that affect the bioactivities of the 
cellular products.  
 
 
 
 
 
 
Figure 1: Developing robust NK cell 
manufacturing processes with high yield 
and desired differentiation subsets 


Differential effects on natural killer cell production by membrane‐bound cytokine stimulations

Aifuwa, Ivie

Wednesday, April 26, 2023                                                                     Session 5: CCE for Cell-based Therapies 
ADAPTIVE T CELL PROCESSING THROUGH INTEGRATED PROCESS ANALYTICAL TECHNOLOGIES 
 
Ivie Aifuwa, Bristol Myers Squibb 
Ivie.Aifuwa@BMS.com 
 
 
Key Words: Process Analytical Technologies, Adaptive Control, Cell Therapy, Process Technologies 
 
Cell therapy continues to be a novel therapeutic modality for the treatment of hematological malignancies, solid 
tumors and autoimmune diseases. Within the past decade, cell therapy has evolved dramatically from academic 
to closed, GMP processes, with a number of approved commercial products. Further, cell therapy processing 
and production strategies continue to evolve to enable autologous, as well as allogeneic products with healthy 
donor derived and iPSCs applications. This rapidly evolving therapeutic space therefore requires significant 
technological advancements to accelerate development and enable affordable and robust products. This 
requires technology manufacturers to rapidly provide novel and innovative tools to support this growing field. 
Process Analytical Technologies (PAT) represent the primary methodology to enable adaptive T cell 
manufacturing and accelerate the evolution of cell therapy processing. Within cell therapy, PAT has primarily 
been considered for cell expansion and cultivation steps, however, the direct integration of PAT systems with 
end-to-end processing and production technologies provide a unique means of guiding cellular phenotypic state 
at the unit operation level and ensuring alignment with the desired target product profile. Here, we will share a 
framework for end-to-end adaptive T cell manufacturing, as well as consider the benefits, challenges, and 
requirements for tools utilized to support adaptive processing.  
 
 
 
 
 
 
 


Adaptive T cell processing through integrated process analytical technologies

Segatori, Laura

Tuesday, April 25, 2023           Session 3: Systems and Synthetic Biology for Improved Cell Culture Performance 
FEEDBACK-RESPONSIVE CELL FACTORIES FOR BIOMANUFACTURING 
 
Laura Segatori, Departments of Bioengineering, Chemical & Biomolecular Engineering, Biosciences. Rice 
University 
segatori@rice.edu 
 
 
The increasing demand for therapeutic proteins has resulted in a significant amount of global manufacturing 
capacity devoted to protein production and substantial effort to improve methods for protein manufacturing. 
Despite these efforts, the production of soluble and functional recombinant proteins remains one of the main 
challenges in the field of biotechnology. 
 
Manufacturing of recombinant therapeutic proteins depends both on cell productivity and growth as protein 
overexpression causes accumulation of misfolded proteins and proteotoxic stress, which leads to activation of a 
global response aimed at restoring homeostasis that may ultimately inhibit protein synthesis and reduce cell 
viability. Because most therapeutic proteins are secretory proteins, protein folding and quality control occur 
within the ER. Accumulation of misfolded proteins in the ER triggers activation of the unfolded protein response 
(UPR), which manifests through activation of three interconnected signaling cascades controlling a 
transcriptional and translational regulatory program for ER stress attenuation through upregulation of protein 
quality control. Upon sustained ER stress, the UPR executes apoptosis.  
 
The outcome of UPR induction, which ranges from stress attenuation to cell death, depends on the relative 
kinetics of activation of the three signaling responses, which, in turn, is shaped by the intensity and duration of 
ER stress induced by the expression of recombinant proteins. This highly dynamic system requires 
sophisticated tools for both detection and manipulation. To address the limitation of currently available tools 
mainly based on exogenous reporters and de-regulated alterations of key UPR genes, we developed a synthetic 
biology platform technology for (i) monitoring the UPR with high sensitivity and dynamic resolution of the stress 
stimulus and (ii) manipulating the UPR in response to feedback signals generated by ER stress, with the 
ultimate goal to enhance the innate cellular capacity to buffer proteotoxic stress.  
 
We have pioneered a platform technology for engineering sense-and-respond cellular devices that detect 
specific transcriptional signatures, and, in response, activate a biomolecular output, ranging from the expression 
of diagnostic signals to the actuation of therapeutic programs.  Our approach is based on the use of the 
NanoDeg (ACS Synth Biol. 2018 Feb 16;7(2):540-552)—a bifunctional system that mediates proteasomal 
degradation of a target protein with high specificity and exquisite control over the rate of decay. We leveraged 
this technology to design a platform technology for quantitative, multiplexed profiling of gene expression 
signatures of the stress response with high sensitivity and dynamic resolution of the stimulus causing 
proteotoxic stress (Nat Chem Biol. 2020 May;16(5):520-528). 
 
Current work is focused on deploying this technology for manipulating stress signaling pathways in response to 
feedback signals generated at different stages of protein production. The goal is to engineer cells for enhanced 
and sustained expression of proteins. This talk will describe strategies for engineering cells with feedback 
systems that enhance the stress attenuation mechanisms in response to proteotoxic stress and delay apoptosis 
with respect to activation of the stress attenuation response. Finally, we will describe the design of next-
generation cell factories for biomanufacturing that tune protein expression to the UPR capacity to cope with 
proteotoxic stress. 
 
 
 


Feedback-responsive cell factories for biomanufacturing

Omasa, Takeshi

Yamano-Adachi, Noriko

Horiuchi, Takayuki

Monday, April 24, 2023                             Session 2: Cell Line Development: Current State and Future Directions 
NEWLY-ESTABLISHED CHINESE HAMSTER-DERIVED CELL LINE FOR PROTEIN PRODUCTION 
 
Takeshi Omasa, Osaka University/ Manufacturing Technology Association of Biologics (MAB) 
omasa@bio.eng.osaka-u.ac.jp 
Noriko Yamano-Adachi, Osaka University / MAB 
Takayuki Horiuchi, Chitose Laboratory / MAB 
 
 
Key Words: Chinese hamster, lung, ovary, cell line establishment, serum-free adaptation 
 
Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to 
their capacity for correct folding, assembly, and post-translational modification. In particular, Chinese hamster 
ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins.  
We newly established cell lines from the Chinese hamster (Cricetulus griseus) from lung and ovary tissues.  For 
lung tissue, according to Puck et al. (1958), minced lung tissues from female Chinese hamsters were placed in 
IMDM (20% FBS). Primary cultured cells mainly showed a fibroblast-like morphology. We continuously 
maintaining cell culture for a long time. Cells were immortalized by spontaneous transformation and could be 
cultured more than about 500 days. Chromosome number analysis showed that the ratio of aneuploid cells 
increased in the process of acquiring immortalization. Some translocations with the same pattern as CHO-DG44 
and CHO-K1 derived cells were observed in Chinese hamster lung derived cells. Constructed cell pools were 
adapted to the serum-free medium by gradual decreases in the serum concentration. The serum-free-adapted 
cell pool was named Chinese hamster lung (CHL)-YN. For ovary tissue, we also established cell line using the 
same procedure and named it Chinese hamster ovary (CHO)-MK. 
 
The time courses of CHL-YN and CHO-K1 cells 
using serum-free medium were shown in Fig.1. As 
shown in this figure, the doubling time of CHL-YN 
was 10.5 hours in animal component-free chemically 
defined medium. From the cell cycle assay by Cell-
Clock Cell Cycle Assay Kit (Biocolor), it was 
suggested that the CHL-YN cells have shorter G0/G1 
cell cycle phase, because there are lower 
percentage of G0/G1 cells compared to CHO-K1 
cells. The CHO-MK cells also showed a similar 
doubling time. In the presentation, we would like to 
show the therapeutic antibody production using 
CHO-MK cells attaining 7g/L at 7 days in 2L-fed-
batch cultivation and the metabolites analysis of 
CHL-YN cell line. 
 
 
 
Figure 1 – Time course of CHL-YN and CHO-K1 cells1) 
 
                  Closed circle: CHL-YN cells, Open Circle: CHO-K1 cells 
 
 
1.Yamano-Adachi et al., Scientific Reports 10:17612 (2020) DOI: 10.1038/s41598-020-74735-0 
 
 
 


Newly-established Chinese hamster-derived cell line for protein production

Agrawal, Neeraj

Monday, April 24, 2023          Session 1: Multispecific Modalities: Commercialization Successes & Future Trends 
TRANSFORMING MOLECULE SELECTION AND PROCESS DEVELOPMENT THROUGH ATTRIBUTE 
FOCUS AND THE DEPLOYMENT OF HIGH-PERFORMANCE COMPUTING TOOLS 
 
Neeraj J Agrawal, Amgen, USA 
agrawaln@amgen.com 
 
 
Key Words: attributes, in silico modeling, molecule assessment. 
 
A deep understanding of the critical quality attributes (CQAs) of a molecule is essential both for the selection of 
an optimal therapeutic candidate and for developing robust processes that will consistently deliver product of the 
highest quality. These CQAs are impacted by process conditions (e.g. cell line and bioreactor process 
parameters, raw material quality), molecule sequence, and analytical method variability. Understanding the 
contribution from each of these factors as a CQA is necessary both in molecule and manufacturing process 
selection of a biotherapeutic. To enable this, we have coupled state of the art analytical measurements with 
digital [1] and high-performance computing capabilities [2] that allows us to probe the impact of the process and 
method variability, and protein sequence/structure on quality attributes. For example, this approach has helped 
us to quickly de-risk the chemical modification liabilities in an antibody sequence [1] and to understand the 
impact of sequence features such as the contribution of surface exposure and protein backbone flexibility on the 
rate of methionine residue oxidation [2]. Similarly, this approach enhanced our understanding of how sequence 
features, buffer and media conditions or stress conditions impact the degradation rate [3, 4]. For example, our 
studies have demonstrated that the presence of bicarbonate has a highly stimulatory effect on most lysine 
residues leading to higher total glycation levels [3]. 
 
Overall, we believe that advanced computational approaches are an essential complement to high-resolution 
analytical approaches and their joint deployment is necessary for the selection and commercialization of a 
biotherapeutic that comprehensively satisfies an unmet medical need. 
  
 
References: 
1. Jacobitz, AW; Rodezno, W; Agrawal, NJ “Utilizing cross-product prior knowledge to rapidly de-risk 
chemical liabilities in therapeutic antibody candidates.” AAPS Open 2022. 
2. Agrawal, NJ; Dykstra, A; Yang, J; Yue, H; Nguyen, X; Kolvenbach, C; Angell, N “Prediction of the 
Hydrogen Peroxide–Induced Methionine Oxidation Propensity in Monoclonal Antibodies.” J. Pharm. Sci. 2018. 
3. Jacobitz, AW; Dykstra, A; Spahr, C; Agrawal, NJ “Effects of Buffer Composition on Site-Specific 
Glycation of Lysine Residues in Monoclonal Antibodies.” J. Pharm. Sci. 2019. 
4. Jacobitz, AW; Liu, Q; Suravajjala, S; Agrawal, NJ “Tryptophan oxidation of a monoclonal antibody under 
diverse oxidative stress conditions: distinct oxidative pathways favor specific tryptophan residues.” J. Pharm. 
Sci. 2021. 
 
 
 


Transforming molecule selection and process development through attribute focus and the deployment of high-performance computing tools

Rask Poulsen, Bjarne

Heitmann, Martin

Marx, Nicolas

Nymand Nilsson, Claes

Poster Number 241 
 
DEVELOPMENT OF SCALE-DOWN SYSTEM FOR PERFUSION PROCESSES 
 
Bjarne Rask Poulsen, Novo Nordisk A/S, Denmark 
bjrp@novonordisk.com 
Martin Heitmann, Sanofi-Aventis GmbH, Germany 
Nicolas Marx, University of Natural Resources and Life Sciences, Vienna, Austria 
Claes Nymand Nilsson, Novo Nordisk A/S, Denmark 
 
 
Key Words: Perfusion, scale-down, chemostat, exponential increasing in-flow, discontinuous out-flow. 
 
As a consequence of recent initiatives towards continuous bioprocessing, and since it often involves perfusion 
processes, there is an increasing need to scale these down and enable experiments with a decreased resource 
usage for each culture. In this poster, strategies are presented to enable screening of multiple process 
parameters, while focus is on simple, yet predictive, scale-down systems for perfusion processes. Due to the 
difficulty of implementing cell retention in small scale, it was decided to develop a chemostat scale-down 
system. The conception of a chemostat with exponential increasing in-flow and discontinuous out-flow (EIDO) 
yields a constant dilution rate (Figure 1). Its implementation in 10-15 mL scale ambrTM cultures is presented. The 
scale-down system is benchmarked towards bench top ATF perfusion cultures, illustrating the predictability of 
the scale-down system. The power of using chemostats to scale down perfusion is shown: the cells experience 
the same metabolite concentrations, and the same product concentration is observed, in the two systems, when 
the dilution rate in the chemostat is equal to the bleed rate in the perfusion. Therefore, this scale-down system 
can also predict product quality attributes. 
 
 
 
Volume Dilution rate Flow
Figure 1. Scaled-down chemostat culture 
with Exponential increasing In-flow and 
Discontinuous Out-flow (EIDO) 


Development of Scale-down system for perfusion precesses

Gong, Weiliang

Xu, Hui

. Pegg, Ian L

CLASS C FLY ASH ACTIVATED BY LOW ALKALINITY ACTIVATOR WITH CONTROLLED SETTING 
  
Weiliang Gong, the Vitreous State Laboratory, Catholic University of America, USA 
gongw@vsl.cua.edu 
Hui Xu, the Vitreous State Laboratory, Catholic University of America, USA 
Ian L. Pegg, the Vitreous State Laboratory, Catholic University of America, USA 
 
 
Key Words: Class C fly ash; Low alkalinity activation; Controlled setting; Ultralow carbon materials 
 
The American Society for Testing and Materials (ASTM) standard C618 recognizes two major classes of fly 
ashes, Class C and Class F.  The lower limit of (SiO2 + Al2O3 + Fe2O3) for Class F fly ash (FFA) is 70 wt.% and 
for Class C fly ash (CFA) 50 wt.%. Typically, CFA has a higher calcium oxide content (20 to 30 wt.%) than FFA. 
CFA usually displays self-cementing behavior by reacting with water to produce calcium silicate and calcium 
aluminate hydrates. Hydration of CFA exhibits rapid stiffening and fast setting, yielding lower compressive 
strength. Recently, there has been an increased interest in alkali activated high-Ca fly ashes. Alkali silicate 
activated high-Ca fly ash-based geopolymers exhibit good strength and durability. The time of setting of a 
geopolymer paste decreases exponentially with increasing CaO content in the fly ash. Above 10 wt.% CaO, the 
time of setting is typically less than 30 min. 
 
In this paper, we report on Class C fly ash activated by low alkalinity activator with controlled setting. The fly ash 
contained about 83.0% glass, 6.7% tricalcium aluminate (C3A), 5.3% quartz, 3.3% periclase, and 1.7% lime. 
w/CFA ratio was fixed at 0.32. Tests were conducted with the following compositions (by weight of CFA): 1. 
Water only; 2. 2% NaOH; 3. 2% NaOH and 2% Na3PO4; 4. 2% NaOH and 4% Na3PO4; 5. 2% NaOH and 6% 
Na3PO4; and 6. 2% Na3PO4 only. 2% NaOH (~1.6 M) was employed as the alkali activator and Na3PO4 as the 
sole set retarder. Mortar and paste samples were characterized for set times by AutoVicat, heat of hydration by 
isothermal calorimetry (IC), compressive strength, and reaction products by XRD and SEM/EDS. In addition, the 
set retarding mechanism was explored by a set of leaching experiments (compositions #1, #2 and #3) with 
w/CFA at 40.  
 
Both water and 2% NaOH activated CFAs exhibited flash setting (<14 min). Initial set time increased with 
increasing Na3PO4 while NaOH was fixed at 2%. The initial set was as long as 177 min while Na3PO4 increased 
to 6% (Composition #5).  With 2% Na3PO4 alone, the initial set was 322 min (Composition #6). Our studies 
reveal strong set retarding effect of Na3PO4 is related to instant removal of calcium cations by precipitating 
apatite during early stages of hydration or alkali activation.  
 
XRD and SEM/EDS results show stratlingite is the primary secondary phase and it occurs in clusters embedded 
in the (C,N)ASH gel in the 2% NaOH activated CFAs.  
IC results for hydration of CFA in water show an initial heat flow peak related to rapid hydration of C3A present in 
CFA at ~55 min with acceleration starting from ~10 min and a main heat flow peak related to stratlingite 
formation occurring at ~300 min.  The initial peak shifted to 10 min in the 2% NaOH activated CFA while the 
main peak was missing. When Na3PO4 was introduced in the 2% NaOH activated CFA compositions (#3, #4, 
and #5), heat flows for both the initial and main peaks were greatly reduced. Apparently, a greater extension of 
set time by Na3PO4 is related to a greater reduction of the rate of katoite. 
 
We have observed a strong synergistic effect of NaOH and Na3PO4 on compressive strength development. The 
2% NaOH activated CFA exhibited flashing setting with a low compressive strength of 13.9 MPa (56 days), 
whereas compressive strength was as high as 65 MPa (56 days) with a set time extended to 177 min while 
Na3PO4 was amended. This synergistic effect can be explained by precipitation of apatite to removed calcium 
cations by Na3PO4 during the earliest curing stages, resulting in a great extension of set time and an increased 
concentration of NaOH in the pore solution that lead to accelerated alkali activation of CFA during later curing 
times.  
 
The carbon dioxide emission was estimated to be ~45kg CO2e/kg binder as compared to 820kg CO2e/kg for 
Portland cement. Low alkalinity activated CFA represents a low cost, ultralow carbon, high strength alkali 
activated fly ash material.  
 


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