Supplementary Materials

Supplementary Figures

Figure S1: Growth characteristics of CHO Cell Line A in amino acid restricted HiPDOG fed-batch cultures employing pre-defined feeding strategy for controlling levels of ten amino acids in low concentration range. Viable cell densities (A), viability (B), titer (C) and ammonia (D) of Cell Line A cultivated in the Low 10AA () or Control () HiPDOG conditions. Control of ten amino acids at lower residual culture concentrations in Low 10AA condition of CHO Cell Line A led to lower accumulations of corresponding byproducts. This resulted in in higher viable cell densities and productivities in the Low 10AA condition when compared to Control condition (normal levels of all the ten amino acids). Ten amino acids controlled at low levels in Low 10AA: Leu, Ile, Val, Met, Phe, Tyr, Trp, Ser, Gly and Thr

Figure S2: Effect of nutrient limitation on accumulation of novel growth inhibitors in HiPDOG fed-batch cultures of CHO Cell Line A. HiPDOG cultures of CHO Cell Line A were carried out by maintaining the levels of ten amino acids (Low 10AA) in concentration range 0.5–1mM or at typical levels (Control) for first 7 days of the culture. Tyrosine (A) and phenylalanine (B) were controlled at low levels in the Low 10AA condition, which led to lower accumulations of 4-hydroxyphenyllactate (F) and 3-phenyllactate (G) in Low 10AA condition as compared to Control condition. Valine (C), isoleucine (D) and leucine (E) were controlled at low levels in the Low 10AA, which resulted in lower levels of isobutyrate (H), 2-methylbutyrate (I) and isovalerate (J) in Low 10AA condition as compared to the Control condition. Ten amino acids controlled at low levels in Low 10AA: Leu, Ile, Val, Met, Phe, Tyr, Trp, Ser, Gly and Thr; Low 10AA (), Control ().

Figure S3: Growth characteristics of CHO Cell Line C in amino acid restricted HiPDOG fed-batch cultures employing pre-defined feeding strategy for controlling levels of ten amino acids in low concentration range. Viable cell densities (A), viability (B), titer (C) and ammonia (D) of Cell Line C cultivated in the Low 10AA () or Control () HiPDOG conditions. Control of ten amino acids at lower residual culture concentrations in Low 10AA condition of CHO Cell Line C led to lower accumulations of corresponding byproducts. This resulted in in slightly higher viable cell densities and higher viabilities in the Low 10AA condition when compared to Control condition (normal levels of all the ten amino acids). Ten amino acids controlled at low levels in Low 10AA: Leu, Ile, Val, Met, Phe, Tyr, Trp, Ser, Gly and Thr.

Figure S4: Effect of nutrient limitation on accumulation of novel growth inhibitors in HiPDOG fed-batch cultures of CHO Cell Line C. HiPDOG cultures of CHO Cell Line C were carried out by maintaining the levels of ten amino acids (Low 10AA) in concentration range 0.5–1mM or at typical levels (Control) for first 7 days of the culture. Tyrosine (A) and phenylalanine (B) were controlled at low levels in the Low 10AA condition, which led to lower accumulations of 4-hydroxyphenyllactate (F) and 3-phenyllactate (G) in Low 10AA condition as compared to Control condition. Valine (C), isoleucine (D) and leucine (E) were controlled at low levels in the Low 10AA, which resulted in lower levels of isobutyrate (H), 2-methylbutyrate (I) and isovalerate (J) in Low 10AA condition as compared to the Control condition. Ten amino acids controlled at low levels in Low 10AA: Leu, Ile, Val, Met, Phe, Tyr, Trp, Ser, Gly and Thr; Low 10AA (), Control ().

Figure S5: Biochemical pathway for catabolism of phenylalanine-tyrosine and BCAAs. (A) Phenylalanine-tyrosine catabolic pathway. (B) BCAA catabolic pathway.

Figure S6: Growth and metabolic characteristics of 2x-tfxn, 2x-control-tfxn, 4x-tfxn and 4x-control-tfxn cell pools derived from CHO Cell Line D in HiPDOG cultures. 2x-tfxn and 4x-tfxn cell pools were expanded in tyrosine-free environment and evaluated in tyrosine-free HiPDOG cultures. 2x-control-tfxn and 4x-control-tfxn cell pools were expanded in tyrosine-supplemented environment and evaluated in tyrosine-supplemented HiPDOG cultures. (A) Viable cell density in HiPDOG cultures, (B) viability, (C) residual tyrosine concentrations, (D) concentration of 4-hydroxyphenyllactate and (E) concentration of 3-phenyllactate. 2x-control-tfxn (◊), 2x-tfxn (□),4x-control-tfxn (♦) and 4x-tfxn (■).

Figure S7: Correlation analysis of relative intracellular levels of metabolic intermediates or byproducts of phenylalanine-tyrosine catabolic pathway in CHO cells cultivated in fed-batch cultures. (A, C) The plot shows correlation between relative intracellular levels of 4-hydroxyphyenylpyruvate and 4-hydroxyphenyllactate in CHO cell fed-batch cultures. (B, D) The plot shows correlation between relative intracellular levels of tyrosine and 4-hydroxyphyenylpyruvate in CHO cell fed-batch cultures. (A, B) Intracellular metabolite data (time course) obtained from a published study where the authors cultivated CHO K1 cells in two different fed-batch processes (Sumit et al., 2019). The intracellular metabolite levels were assessed using LC-MS/GC-MS method (for details on the LC-MS/GC-MS method, see (Mulukutla et al., 2017)). One of two processes employed HiPDOG control strategy whereas the other was a typical fed-batch process. The two processes also differed in the basal and the feed medium used and, few other fed-batch process parameters. (C, D) Intracellular metabolite data (time course) obtained from the control HiPDOG culture condition of CHO Cell Line C described in Fig. S3. The intracellular metabolite levels were assayed using the same LC-MS/GC-MS method as above. Across all the plots, data points on day 0 were excluded from the analysis as they don’t necessarily represent (pseudo)steady state values. Data points that were imputed were also excluded. Imputed data points are those conditions where a metabolite was not detected and, therefore, the lowest measured value for the metabolite (among other conditions in which the metabolite was detected) was assigned to the said condition. a.u.: arbitrary unit.

Figure S8: Growth and metabolic characteristics of Cell Line A derived BCAT1 knockdown and negative (control) pools in HiPDOG cultures. (A) Viable cell density, (B) ratio of BCAT1 transcript levels between knockdown pool and negative (control) pool, residual culture levels of (C) isoleucine, (D) leucine, (E) valine, (F) 2-methylbutyrate, (G) isovalerate and (H) isobutyrate, amino acid consumption and corresponding byproduct production rates for (I) isoleucine and 2-methylbutyrate, (J) leucine and isovalerate, (K) valine and isobutyrate. BCAT1 knockdown pool and negative pool were derived from Cell Line A by performing knockdown using miRNA (Seq. 4-1) targeted towards BCAT1 or a scrambled miRNA, respectively. Both the cell pools were evaluated in HiPDOG cultures for growth and metabolic phenotypes. BCAT1 knockdown pool grew to slightly higher viable cell densities compared to control culture but produced similar titers. However, the knockdown pool had consistently lower BCAT1 transcript levels throughout the HiPDOG culture. Potentially due to lower BCAT1 enzyme levels, BCAT1 knockdown pools consumed BCAAs at lower rates and produced BCFAs at lower rates. This resulted in lower culture concentrations of BCAAs and higher concentrations of BCFAs in the BCAT1 knockdown pool when compared to the control pools.

Figure S9: Concentration of amino acids and metabolic byproducts in non-optimized HiPDOG cultures of BCAT1 KO clones, negative control clone and the wild type cell line . Concentration of (A) cysteine and (B) tyrosine in the HiPDOG cultures of WT, BCAT1 KO Clone 83, BCAT1 KO Clone 86 and BCAT1 Clone 90 in the experiment described in Figure 6. Concentration of (C) 3-phenyllactate and (D) 4-hydroxyphenyllactate in the HiPDOG cultures of WT, BCAT1 KO Clone 83, BCAT1 KO Clone 86 and BCAT1 Clone 90 in the experiment described in Figure 7. WT (), BCAT1 KO Clone 83 (), BCAT1 KO Clone 86 () and BCAT1 Clone 90 ().

Supplementary Tables

Table S1: “Top Strand” oligonucleotide sequence for miRNA expression constructs used to knock-down BCAT1 expression in CHO Cell Line A.

Name “Top Strand” Oligonucleotide SequenceSeq. 1.1 5’ TGCTGTTTAATGTGAGGCTTCTCCCAGTTTTGGCCACTGACTGACTGGGAGAACTCACATTAAA 3’Seq. 2.1 5’ TGCTGCAAAGTGGTCCTCACAGCAGAGTTTTGGCCACTGACTGACTCTGCTGTGGACCACTTTG 3’Seq. 3.1 5’ TGCTGAACAGATTCATCGTGCCCACTGTTTTGGCCACTGACTGACAGTGGGCAATGAATCTGTT 3’Seq. 4.1 5’ TGCTGTAGAGGAACAGATTCATCGTGGTTTTGGCCACTGACTGACCACGATGACTGTTCCTCTA 3’Seq. 5.1 5’ TGCTGATATCAGTCAGTTTGCCCAAGGTTTTGGCCACTGACTGACCTTGGGCACTGACTGATAT 3’

Scrambled miRNA 5’ GAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT 3’

Table S2: BCAT1 genomic sequence targeted with guide RNAs (gRNAs) for CRISPR Knockout

Serial # BCAT1 Genomic Sequence

1 TGCGGTGCGGCGGCGACACG

2 CTGGCGACACCTCCGCTAGA

3 AAAGCCAGACCCCGATACCCTGG

Table S3: Plasmid vectors used for the generation of transgenic CHO cell pools expressing mouse HPD, HGD, PAH, and PCBD1 genes.

Plasmid Vector Manufacturer Gene Expressed Antibiotic Selection TypepcDNA3.2/V5/GW/D-TOPO Thermo Fisher,

Waltham, MAPAH Neomycin (G418) Expression

pMONO-hygro-mcs InvivoGen, San Diego, CA

HPD Hygromycin Expression

pcDNA6.2/V5/GW/D-TOPO Thermo Fisher, Waltham, MA

HGD Blasticidin Expression

pSELECT-puro-mcs InvivoGen, San Diego, CA

PCBD1 Puromycin Expression

pcDNA3.2/V5/GW/CAT Thermo Fisher, Waltham, MA

Choramphenicol acetyl transferase (CAT)

Neomycin (G418) Control

pMONO-hygro-mcs InvivoGen, San Diego, CA

none Hygromycin Control

pcDNA 6.2/V5/GW-CAT Thermo Fisher, Waltham, MA

Choramphenicol acetyl transferase (CAT)

Blasticidin Control

pSELECT-puro-mcs InvivoGen, San Diego, CA

none Puromycin Control

Table S4: Oligonucleotides used for RT qPCR analysis of genes involved in CHO BCAA and Phe-Tyr catabolic pathways.

Gene 5’ Primer 3’ PrimerB-ACTIN 5’-CCT CTA TGC CAA CAC AGT GC-3’ 5’-CCT GCT TGC TGA TCC ACA TC-3’SLC2A1 5’-GCA TCC TTA TTG CCC AGG TG-3’ 5’-TCG TTG CGA TTG ATG AGC AG-3’

DLD 5’-TGG GTG GAA TTG GAA TTG AT-3’ 5’-GGC TTT ACC ACC AGA AGC AG-3’MCCC2 5’-TAT CTC GGT GAT GGG AGG AG-3’ 5’-CAC AGC CTT GCA CTG GAG TA-3’

IVD 5’-AGG TAC CCA ACG CCT TTT CT-3’ 5’-GAG GCC AGC AAG AGA CAA TC-3’BCKDHB 5’-GAT GTT GCC TTT GGT GGA GT-3’ 5’-CTG ATC AAA GGC AGG GAA AA-3’ACADM 5’-GAG CTG CTT GGG AAG TTG AC-3’ 5’-CTT GGC ATC CCT CAT CAG TT-3’HMGCL 5’-GAA GCC ACC AGC TTT GTC TC-3’ 5’-TGC TTA CTT CCT TGG CAC CT-3’BCKDHA 5’-ACC ACG GCA GAG TTT GTA GA-3’ 5’-TTC GGT CCA TGG TGT TGA GT-3’MCCC1 5’-CAG TCT GAC CAG TGC CTG AA-3’ 5’-GAA CTT CCA CAT GCC TTG GT-3’

DBT 5’-CAT CAC CAG CCG TTA CGA TG-3’ 5’-GCT CCT GTT TGC CTT TCC AA-3’BCAT1 5’-GGA AGG AGA ACG TGG AAG GA-3’ 5’-CCA CTC TGG GCT ACT TGA CA-3’AUH 3 5’-CAG TGT TGA GGA GGC TCT GA-3’ 5’-GGC TGG AGA AGG AGT GAG TT-3’AUH 5 5’-TTT CAA AAG CCG TGG ATG CA-3’ 5’-GCA CAG GGA GGT TAG CAA TG-3’

MIF 5’-GCG TCG CAC TGT CCT CTA CT-3’ 5’-CGC TAG AGC CGC TAA AAG TC-3’GOT2 5’-TGT GTT TGC CCT CCC AGT AT-3’ 5’-AGT CAA GCA GAA CGG GAT GA-3’GOT1 5’-ACT CCA GAG GAG TGG AAG CA-3’ 5’-GAG AAG GAC TGG GCA CAG AG-3’FAH 5’-GGC TAG ACT GAT CCT CGT CC-3’ 5’-GAT CTG GTC ACC GAT AGC CA-3’

GSTZ1 5’-GAG GGC TAT CAC TTC TGG CT-3’ 5’-GCA GGG CTT TGT TGA TGT GA-3’PAH 5’-AGC CCA TGT ACA CAC CTG AA-3’ 5’-ACA GAA GCC CAG CAC CAT AT-3’HPD 5’-CGG GAG AAT CTC AAG ACA GC-3’ 5’-GGA GTT GAA GTT GCC TGC TC-3’HGD 5’-CCT CAA GTC TGC CCC TAC AA-3’ 5’-GGT TAG GAT CAG GGC CAA CT-3’

PCBD1 5’-CAT AGA TCC GCT CCT TTT CG-3’ 5’-AAC ATG GCA GAG CCC TAA TG-3’GCH1 5’-GCC ACC TTG TTA GTG GCA AT-3’ 5’-GGT GAA AAA CCT GGC ACA GT-3’QDPR 5’-GCA GCT CTT GAC AGT TGC AG-3’ 5’-CTG GTC ACC TAG GAG CTT GC-3’SPR 5’-CCT GAA TGC CTT CCC AGA TA-3’ 5’-CAT AGC TCA GCA CCC TCA CA-3’PTS 5’-TGA AGA CAA GCC AGA CAG GA-3’ 5’-ATT CGG ATT GTT GCA TTT CC-3’

Table S5: Oligonucleotides used for RT qPCR analysis of transgenic cell pools expressing mouse orthologs of HGD, HPD, PAH, and PCBD1 genes.

Target 5’ Primer 3’ Primer SpecificityBActin 5’-CCTCTATGCCAACACAGTGC-3’ 5’-CCTGCTTGCTGATCCACATC-3’ CHOPAH 5’-AGCCCATGTACACACCTGAA-3’ 5’-ACAGAAGCCCAGCACCATAT-3’ CHOHPD 5’-CGGGAGAATCTCAAGACAGC-3’ 5’-GGAGTTGAAGTTGCCTGCTC-3’ CHOHGD 5’-CCTCAAGTCTGCCCCTACAA-3’ 5’-GGTTAGGATCAGGGCCAACT-3’ CHOPCBD1 5’-CATAGATCCGCTCCTTTTCG-3’ 5’-AACATGGCAGAGCCCTAATG-3’ CHOPAH 5’-AGCTTTGCCCAGTTTTCTCA-3’ 5’-TACTCCTGGCAGGCTGTCTT-3’ MouseHPD 5’-CATTTCCACTCGGTGACCTT-3’ 5’-CTCGAATGCGATGTCTTTCA-3’ MouseHGD 5’-GGTGGGGAGTTGCAGATAAA-3’ 5’-CTGGCCTTTTCAAAGCAGTC-3’ MousePCBD1 5’–AAGTAGAAGGCCGAGATGCT-3’ 5’-TCATGGGTGCTCAAGGTGAT-3’ Mouse

Table S6: Antibodies used to determine relative protein expression in transgenic CHO cell pools

Type Target Host Species Manufacturer Catalog Dilution

10 Antibody Beta Actin Rabbit Polyclonal AbCam, Cambridge, MA ab8227 1:2500

10 Antibody PAH Mouse Monoclonal AbCam, Cambridge, MA ab191234 1:1000

10 Antibody HPD Rabbit Polyclonal AbCam, Cambridge, MA ab93138 1:500

10 Antibody HGD Mouse Monoclonal Santa Cruz Biotechnology, Dallas, TX sc-376276 1:500

10 Antibody PCBD1 Rabbit Monoclonal AbCam, Cambridge, MA ab138518 1:5000

10 Antibody BCAT1 Rabbit Polyclonal AbCam, Cambridge, MA ab110761 1:500

20 Antibody Rabbit IgG Goat anti rabbit AbCam, Cambridge, MA ab6721 1:10000

20 Antibody Mouse IgG Goat anti mouse Santa Cruz Biotechnology, Dallas, TX sc-2031 1:2000

Table S7: Spent medium levels of 3-phenyllactate and 4-hydroxyphenyllactate in tyrosine-supplemented fed-batch cultures of 3x-tfxn, 3x-control-tfxn, 4x-tfxn and 4x-control-tfxn cell pools derived from CHO Cell Line A. 3x-tfxn and 3-control-tfxn cells pools were evaluated as part of one experiment whereas 4x-tfxn and 4x-control-tfxn cell pools were evaluated in a separate experiment. For each condition, cell pools were inoculated in fed-batch cultures, employing HiPDOG control strategy, at inoculation cell densities of 1E6 cells/mL. Cells pools within each experiment grew similarly and peaked at similar cell densities. The byproducts levels were measured on day 5 and day 7 for the 4x-tfxn and 4x-control-tfxn conditions. For 3x-tfxn and 3x-control-tfxn conditions, the byproducts levels were measured only on day 7. "-": not measured.

3-phenyllactate (M) 4-hydroxyphenyllactate (M)

Day 5 Day 7 Day 5 Day 7

3x-tfxn - 112 - 107

3x-control-tfxn - 116 - 215

4x-tfxn 56 78 45 138

4x-control-tfxn 76 160 81 187

Table S8: Spent medium levels of 4-hydroxyphenylpyruvate in fed-batch cultures of 2x-tfxn, 2x-control-tfxn, 4x-tfxn and 4x-control-tfxn cell pools derived from CHO Cell Line D. The 2x-tfxn and 4x-tfxn cell pools were grown in tyrosine-free fed-batch cultures whereas 2x-control-tfxn and 4x-control-tfxn were grown in tyrosine supplemented fed-batch cultures. All the fed-batch cultures employed HiPDOG strategy during the growth phase of the cultures. “-”: indicates no detection. Units: micro molar (M).

Conditions

2x-control-tfxn 2x-tfxn 4x-control-tfxn 4x-tfxn

Day

5 11.8 - - -

7 - - 13.5 -

9 9.9 16.1 - -

Table S9: Change in BCAT1 expression levels after transfection with miRNA knockdown constructs.

Treatment PlasmidCт Mean

B-ACTIN BCAT1 Δ Cт

Non-Transfected Pool NA 17.3 26.7 9.5

Transfected Pools

No Plasmid 17.3 26.3 9.0

Seq.1.1 16.0 26.3 10.2

Seq. 2.1 15.2 25.5 10.3

Seq. 3.1 15.9 25.4 9.5

Seq. 4.1 15.9 27.0 11.1

Seq. 5.1 16.2 27.0 10.8Scrambled

miRNA 15.3 24.8 9.6

References

Mulukutla, B. C., Kale, J., Kalomeris, T., Jacobs, M., Hiller, G. W., 2017. Identification and control of novelgrowth inhibitors in fed-batch cultures of Chinese hamster ovary cells. Biotechnology and bioengineering. 114, 1779-1790.

Sumit, M., Dolatshahi, S., Chu, A. A., Cote, K., Scarcelli, J. J., Marshall, J. K., Cornell, R. J., Weiss, R., Lauffenburger, D. A., Mulukutla, B. C., Figueroa, B., Jr., 2019. Dissecting N-Glycosylation Dynamics in Chinese Hamster Ovary Cells Fed-batch Cultures using Time Course Omics Analyses. iScience. 12 , 102-120.