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CMC Case Study

Rich CMC experience with> 90 CMC projects

30+ IND approvals

40% IND approvals from challenging molecules

Nowadays mAb CMC development is becoming more and more standardized. However, there is no standard CMC strategy for multispecific antibody and recombinant protein. Customized CMC strategy should be developed according to the molecular features. There are many challenges in the development of recombinant protein CMC, such as low titer, light and heavy chain mismatch, low stability, complex purification process and challenging analytical development, which raise high requirements for CDMOs.

ProBio has a complete antibody protein drug process development platform, and has undertaken > 90 CMC projects. 40% of the 30+ IND approvals are from proteins and muti-specific antibodies. The most advanced project has entered into the clinical phase III.

Recombinant Protein CMC Development

Cell Line Development

Upstream Process

Downstream Process

Analytical Development

Focus on product quality
Titer improvement

Optimize product quality based on molecule features
High throughput parameter screening
Ion additive
Harvesting time

Filter adsorption
Resin selection
S/D inactivation
PTM

Analytical method development capability
Special assay kit development

Cell Line Development—Focus on quality and titer

ProBio’s cell line development platform is based on the proprietary CHOK1-GenS system with high productivity and excellent stability. The average titer for mAb can reach 5.7g/L. CHOK1-GenS system is compatible with different molecular types, such as bispecific antibody and recombinant protein. It only takes 10 weeks from DNA to PCB.

For recombinant protein, ProBio pays attention to product quality and purity, and increase cell pool and clone selection scale to improve final titer.

Upstream Process Development

1. Through the use of high-throughput platforms such as Ambr15 for process development, ProBio can efficiently complete clone screening, media screening and parameter optimization (i.e. pH, temperature, feed strategy). ProBio optimizes product quality based on molecular features.

2. Suitable additives to improve product quality
In a recombinant protein case, ion additives help improve monomer purity.
3. Define the best harvesting time
By extending the cell culture time and analyzing the trend of activity and viability, ProBio can define the best harvesting time is D11.

Downstream Process Development

1.Attention to filter adsorption
Variant filter adsorption in recombinant protein cases.
C filter shows the lowest adsorption performance.
2. S/D substitutes low pH
Protein will fully precipitate under low pH, while S/D inactivation method can act as the substitution. S/D can also help improve the purity due to the aggregate precipitation.

3. AC resin selection
Many recombinant proteins are not suitable for protein A affinity chromatography. Multimodal resin can fulfill the purity and yield requirements.
4. Polish resin selection
Multimodal resin is suitable for recombinant protein polish purification and can remove aggregates.

Analytical Development

Recombinant protein has special molecular properties. Conventional analysis methods cannot meet the needs of the projects. So recombinant protein projects need strong analytical development platform, including structure, residuals and bioactivity. This table shows some challenges and solutions in the protein projects.

Challenge	Solution
Recombinant protein is unstable in mobile phase Standard mAb SEC is not applicable	Mobile phase optimization and selection
Recombinant protein may precipitate during pretreatment Standard mAb glycan analysis is not applicable	Optimize pretreatment buffer Increase sample volume
O-glycan site identification ESI-MS is not applicable	SialEXO&OpeRATOR LC-MSMS to identify the O-glycan site
O-glycan quantitative analysis β-elimination method is not stable	LC-MSMS measurement
No commercial Chaperon assay kit No established method	Develop Chaperon assay kit based on ELISA

Case Study: Recombinant Protein

Entered into clinical phase Ⅱ

Content		Customer Expectation	GenScript Delivered	Note
Titer		30 mg/L	500 mg/L	Titer improved 15 times Pilot scale only need 7L
Pilot Scale		100L	7L	Titer improved 15 times Pilot scale only need 7L
Purity	SDS-PAGE	≥95%	>98%	Purity >98% by USP&DSP
	SEC-HPLC	≥95%	>98%
	RP-HPLC	≥95%	>98%
Impurity	Aggregate	≤2.0%	<1.0%	Aggregate <1.0%
	P35 residue	≤1.0%	Undetectable	Remove p35, p40 and p80 residue effectively by DSP
	P40 residue	≤1.0%	Undetectable
	P80 residue/td>	≤1.0%	Undetectable
	HCP	≤0.05%	≤0.01%	Low level of host residual
	HCD	≤10ng/Dose	≤0.01ng/Dose	Low level of host residual
In vitro activity		80%-150%	90%-110%	Stable bioassay method Consistent batch quality

Multispecific Antibody CMC Development

Cell Line Development—Focus on quality and titer

ProBio’s cell line development platform is based on the proprietary CHOK1-GenS system with high productivity and excellent stability. The average titer for mAb can reach 5.7g/L , and the average titer for bsAb is 6.7g/L. It only takes 10 weeks from DNA to PCB.

Upstream Process Development

1. Suitable additives to improve product quality
In one bispecific antibody case, modified amino acids help improve titer to 150%
2. Process optimization to improve titer
In one bispecific antibody case, optimizing culture conditions such as pH and temperature to improve titer.

3. Process intensification
In addition to the traditional fed-batch process, ProBio also provides process intensification solutions such as high density inoculation to help improve titer.
4. Perfusion culture helps improve product quality
Perfusion culture helps increase full length ratio and purity

Downstream Process Development

1. High-throughput downstream process screening platform

2. S/D method in place of low pH can help improve purity

VI Method	Sample ID	Conc.(mg/ml)	SEC-HPLC(%)			CE-SDS-N
VI Method	Sample ID	Conc.(mg/ml)	HMW	Main	LMW	(%)
Control	AC Eluate	12.44	1.65	98.35	ND	99.61
S/D	AC Eluate	12.60	1.47	98.54	ND	99.54
Low pH	pH3.8, 1hr	10.73	3.33	96.42	0.24	99.11
Low pH	pH3.7, 1hr	10.39	14.61	85.40	ND	99.18

3. Aggregate removal
Multimodal resin is suitable for multispecific antibody purification and can remove aggregates.
In one trispecific antibody case, through downstream process optimization, purity was improved from 84.7% to 98.6%.

Analytical Development

Multispecific antibodies often have chain mismatches, so appropriate analysis methods need to be developed to monitor the occurrence of mismatches.

LC-MS method can sensitively monitor chain mismatch.

ProBio has strong analytical development capabilities, and can design suitable bioassay and functional activity methods based on molecule features and MOA

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