*4.4. Purification of Antibodies*

Ab417 and Ab612 were purified using a Protein A affinity column. Harvested Cell Culture Fluid (HCCF) containing a monoclonal antibody was loaded on to pre-packed protein A column (HiTrap MabSelectTM Sure, Cytiva, Marlborough, MA, USA) equilibrated with binding buffer (20 mM sodium citrate, pH 6.0). The column was then re-equilibrated with binding buffer followed by a wash step at pH 6.0 and finally elution with sodium citrate buffer at pH 6.0 and 2.5 (gradient elution). The gradient elution experiments were carried out using a 30 column volume (CV) linear gradient from 0% to 100% buffer B (20 mM sodium citrate, pH 6.0). For virus inactivation, the elution fraction was incubated at 4 ◦C for 1 h and then neutralized with 1 M Tris-HCl (pH 9.0) to minimize the effect on the structure of the antibody under low pH conditions. The column was then regenerated and sanitized using 0.5 N NaOH.

Desalting chromatography (HiPrep™ 26/10 Desalting, Cytiva, Marlborough, MA, USA) was performed to replace the buffer in the primary purified product (affinity eluate) suitable for the loading condition (50 mM Na-Acetate, pH 5.0). Further purification of Ab417 and Ab612 was performed using cation-exchange chromatography. The sample was applied to a HiTrap Capto SP column (Cytiva, Marlborough, MA, USA) and eluted with a linear gradient of 0 to 500 mM NaCl, 30 CV.

Pooled fractions were further concentrated using Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane (Merck Millipore, Burlington, MA, USA). A concentrated protein sample was loaded onto the HiLoad Superdex 200 pg column (Cytiva, Marlborough, MA, USA) equilibrated with Phosphate-buffered saline (PBS) buffer at the rate of 1.0 mL/min. The elution profile was analyzed by the absorbance at 280 nm.

The purity and aggregation of purified protein were determined using high-performance size exclusion chromatography (SE-HPLC). A Waters HPLC (Alliance 2695) system was used with a Bio SEC-3 column (3 μm, 300 Å, 4.6 × 300 mm, Agilent, Santa Clara, CA, USA) at 0.3 mL/min flow rate (isocratic) using a mobile phase buffer of 20 mM Na-phosphate w/150 mM NaCl, pH 6.8.

### *4.5. Dynamic Light Scattering (DLS)*

DLS was performed using Zetasizer (Malvern, Herrenberg, Germany). After the Z-average of antibody sample was measured at 25 ◦C, 50 μL of the sample was added to disposable cuvettes (ZEN0040, Malvern, Herrenberg, Germany) and gradually the temperature was increased from 30 ◦C to 80 ◦C with 3 ◦C of temperature interval and a fixed angle of θ = 173◦. The Z-average and intensity were calculated using Zetasizer Software version 7.02 (Malvern, Herrenberg, Germany).

#### *4.6. Affinity Determination of Antibodies Using Octet red384 System*

The affinity of the antibody was determined using the Octet Red384 system (Sartorius, Goettingen, Germany). Anti-human IgG sensor AHC (ForteBio, Fremont, CA, USA) was firstly soaked in 0.1% PBA for 20 min. The antibody (0.2 mL of 0.5 μg/mL) was captured for 10 min followed by washing with 0.1% PBA for 2 min. hL1-s1 or mL-s1 (25, 12.5, 6.25, 3.125, or 1.5625 nM in 0.1% PBA) was then incubated with the antibody captured on the sensor. Association and dissociation rates were measured for 10 min and 30 min, respectively. For correction of baseline drift, a control sensor was designated as an antibody-captured AHC sensor exposed to running buffer only. All analytes were recalculated by subtraction of the rate of a control sensor. The operating temperature was maintained at 30 ◦C and agitated at 1000 rpm. Data were analyzed using a 1:1 interaction model (fitting global, Rmax unlinked by a sensor) with analysis software (ForteBio, ver. 8. 2).

### *4.7. Capillary Isoelectric Focusing (cIEF) Analysis*

cIEF was conducted according to the SCIEX application protocol. All cIEF experiments were performed on SCIEX PA800 plus instrument with a 50 μm i.d. neutral coated capillary (SCIEX P/N 477441) at the length of 30.2 cm. The UV detector was used to detect absorbance at 280 nm wavelength. A cIEF master mix solution was composed of 3 M urea-cIEF gel solution, pharmalyte 3–10 carrier ampholytes, cathodic stabilizer (500 mM arginine), anodic stabilizer (200 mM iminodiacetic acid), and five pI markers (10.0, 9.5, 7.0, 5.5, 4.1). Analytes (5 mg/mL) were also mixed with 10 μL of the master mix solution. All experiments were performed in triplicate. The pI values of the sample were calculated using qualitative analysis of a 32 Karat software.

#### *4.8. In Vivo Antitumor Activities of Ab417 or Ab612*

All the animals were housed under a 12/12 h light/dark cycle (light phase, 8:00 A.M. to 8:00 P.M.) with a standard laboratory diet and water ad libitum. All animal handling and experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of preclinical CRO Biotoxtech (180678). Nude mice (BALB/cSlc-nu, 5 weeks old) were obtained from Japan SLC, Inc (Shizuoka, Japan). Choi-CK cells (1 × <sup>10</sup>6) were inoculated into the right flank of each mouse. Constructed Choi-CK tumor tissue (3 × <sup>3</sup> × 3 mm3) was subcutaneously inoculated into the back of mice. After tumor volume reached a mean of 100 mm<sup>3</sup> (*n* = 8 per group), the antibody at a dose of 10 mg/kg was i.v. injected 3 times per week for 3 weeks. Tumor growth was monitored by measuring the length and width of the tumor with a caliper and calculating tumor volume based on the following formula; TV (mm3) = L (mm) × W2 (mm2) × 1/2, where L is length and W is width. Body weight was measured twice a week, and tumor tissues were taken out and weighed at the end of the experiment. Tumor growth inhibition rate (IR) was calculated as the following formula; IR (%) = (1 − T/C) × 100. T is the mean tumor weight of the antibody treated group, and C is the mean tumor weight of the mock control group.

Data were validated using SAS (Version 9.3, SAS Institute Inc., Cary, NC, USA). Each point represents the mean ± SD. Statistical comparison between groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett's t-test.

#### *4.9. Production of Ab612 from Research Cell Bank (RCB)*

#### 4.9.1. Construction of RCB

RCB was generated by Shanghai OPM Biosciences Co., Ltd. (Shanghai, China). CHO-K1 cells were grown in CD CHO Medium (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) containing 6 mM L-glutamine (Sigma, St. Louis, MO, USA). Cells were incubated under the condition of 120 rpm, 37 ◦C, and 8% CO2. Freshly prepared linearized plasmid was transfected into CHO-K1 cells by electroporation using a Bio-Rad system. For each sample, 1 × 107 cells were transfected with a total of 40 <sup>μ</sup>g of the linear plasmid. At 24 h later, the transfected cells were plated into a 96-well plate with 4000 cells/well. The culture medium was CD CHO Medium containing 50 μM L-Methionine sulfoximine (MSX) (Sigma, St. Louis, MO, USA) and 1 × GS-Supplement (Sigma, St. Louis, MO, USA). Cells were statically cultured in an incubator with 8% CO2 and 37 ◦C. The plasmid was transfected into cells twice by electroporation, and then the cells were plated into 20 pieces of a 96-well plate. Mini pool fed-batch assay medium was OPM-CHO CD07 Medium (OPM, China) with 1:200 anti-clumping Agent (ACA, OPM, China). The CDF18 (OPM, China) at 3%, 5%, 6%, 6% and 5% concentration and CDF26 (OPM, China) at 0.3%, 0.5%, 0.6%, 0.6% and 0.5% were fed on day 3, 5, 7, 9, and 11, respectively. The glucose was fed as needed to maintain at 2–6 g/L. The fed-batch was stopped when the viability is about 60% or on day 12.

#### 4.9.2. Stability Study of Research Cell Bank (RCB)

In suspension culture from RCB stock, cells were passaged once every 3 days for 69 days with 0.4 × 106 cells/mL seeding density in OPM-CHO CD07 Medium with <sup>50</sup> <sup>μ</sup><sup>M</sup> MSX and 1:200 Anti-Clumping Agent. In a fed-batch assay, cells were incubated at the density of 0.8 ± 0.1 × <sup>10</sup><sup>6</sup> cells/mL with 30 mL culture volume. The medium was OPM-CHO CD07 Medium with 1:200 ACA. The CDF18 was fed on day 3, 5, 7, 9 and 11 at 3%, 5%, 6%, 6% and 5%, respectively, and CDF26 was fed at 0.3%, 0.5%, 0.6%, 0.6% and 0.5%. The glucose was fed as needed to maintain it at 2–8 g/L. The fed-batch was stopped when the viability is about 60% or on 12 days. The culture supernatants were harvested by centrifuging at 3500 rpm for 30 min under 4 ◦C.

#### 4.9.3. Production and Purification of Antibodies

The HCCF obtained from RCB production was purified using protein A (Repligen, Waltham, MA, USA) packed in XK26/20 column (Cytiva, Marlborough, MA, USA) affinity chromatography. The purified antibody was stored in 10 mM sodium phosphate buffer containing 5% sorbitol and 0.01% tween 20 and dialyzed using HiPrepTM 26/10 Desalting Column (Cytiva, Marlborough, MA, USA). The concentration of purified antibody was determined with a Nanodrop (Thermo fisher scientific, Nanodrop 2000) based on the molar extinction coefficient. The produced Ab612 from RCB was used for analyzing purification by SEC-HPLC and antigen-binding activity by ELISA.

#### **5. Conclusions**

In conclusion, we successfully improved the biophysical properties and affinity of Ab417 through antibody engineering based on computational methods, which generated an optimized antibody, Ab612. This antibody exhibited enhanced in vivo anti-tumor efficacy compared to Ab417. Additionally, we successfully generated a highly productive and stable RCB and confirmed the scalability of the production process to a pilot scale. Ab612 is considered a promising candidate antibody for preclinical development.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms22136696/s1, Figure S1: Variant combinations of Ab417, Figure S2: pI value of Ab417 and Ab612 using cIEF.

**Author Contributions:** Conceptualization, H.H. and S.Y.; methodology, H.C., S.C., M.J., K.K., D.C., J.L. (Jaeyoung Lee), J.H. and J.L. (Jiwoo Lee); formal analysis, D.C.; investigation, H.C., S.C., M.J., K.K., D.C., J.L. (Jaeyoung Lee), W.N. and S.Y.; writing—original draft preparation, H.C., D.C., W.N. and H.H.; writing—review and editing, H.H. and S.Y.; Funding acquisition, H.H. and S.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Korea Drug Development Fund (KDDF) grant (KDDF-201212-12) and the Basic Science Research Program grant (NRF-2018R1D1A1A09084274) to H.J.H. and by the BIG3 Innovative startup package support Program (No. 10449069) & the Technology development Program (No. S3029721) supported by the Ministry of SMEs and Startups to S.Y.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We thank Lonza Biologics plc (UK) and Shanghai OPM Biosciences Co. (China) for the excellent services of computational methods and RCB generation, respectively.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

