Recombinant Protein Chromatography: A Core Strategy from Capture to Polishing

Recombinant proteins occupy a core position in modern biomedicine (such as therapeutic antibodies, enzymes, vaccines, cytokines), diagnostic reagents and industrial enzyme preparations. Their production process usually involves expression in genetically engineered host cells (such as E. coli, CHO cells, yeast, insect cells), followed by isolation and purification of target proteins from complex cell cultures or lysates. Chromatography technology has become the cornerstone of recombinant protein purification technology because of its high resolution, high selectivity and good scalability. Developing a chromatographic purification process that is robust, efficient, cost-effective and compliant with regulatory requirements requires a systematic strategy.

Core Objectives of Process Development

• High purity: Effectively removes host cell proteins (HCP), nucleic acids (DNA/RNA), endotoxins, viruses (if applicable), media components, product-related impurities (aggregates, degraded fragments, misfolded forms, post-translationally modified variants, etc.).

• High recovery rate: maximize the yield of target protein and reduce production cost.

• Robustness: The process is tolerant to small changes in raw materials (e.g. buffers, resins), fluctuations in operating parameters.

• Scalability: Processes developed at laboratory scale can be successfully scaled up to pilot and production scale.

• Economics: Optimize process steps, reduce the number of chromatography steps, increase resin loading and service life, and reduce buffer consumption.

• Compliance: Meets Good Manufacturing Practice (GMP) requirements, has a good process characterization (PC) and process validation (PV) basis, and ensures product quality and safety (especially virus clearance ability).

• Speed: Shorten the purification cycle time and improve the production efficiency.

Chromatography Process Development Strategy Framework

Phase 1: Preliminary Research and Objective Definition

In-depth understanding of target proteins: 

• Physicochemical properties: molecular weight, isoelectric point (pI), hydrophobicity, stability (pH, temperature, redox, shear force), post-translational modifications (glycosylation, phosphorylation, etc.).

• Biological activity: methods of functional detection.

• Critical Quality Attributes (CQAs): Attributes that directly affect product safety and efficacy (purity, activity, aggregate content, charge heterogeneity, glycoform distribution, host impurity residues, endotoxins, viral particles, etc.).

• Characteristics of expression system and harvest solution: host cell type (determining the main impurity profile), expression form (intracellular, secretory, inclusion bodies), composition, viscosity, particulate matter content of harvest solution/lysate.

Clarify process objectives:

Set purity and yield targets, cost caps, time requirements, scale-up targets, regulatory requirements (e.g. virus clearance requirements).

Literature research and platform reference:

Refer to the purification strategy of similar proteins, and use existing platform technologies (such as the Protein A platform of monoclonal antibodies).

Phase 2: Capture Phase Development

Objective: Rapidly concentrate the target protein, remove most of the volume and major impurities (e.g. cell debris, nucleic acids, lipids, most of HCP), and stabilize the target protein.

Preferred technology:

Affinity chromatography is the gold standard for the capture stage (if available and economical)

• Monoclonal antibody/FC fusion protein: Protein A/G/L Affinity chromatography. Development focus: optimization of binding/elution conditions (pH, ionic strength, additives), loading optimization, washing/regeneration protocol, aggregate control, Protein A ligand shedding.

• Tag fusion proteins: His-Tag (IMAC), GST-Tag, FLAG-Tag, etc. Development focus: binding specificity, elution conditions (imidazole concentration, reducing agent, pH), tag removal strategy (e.g., enzymatic digestion), potential tag-related impurities.

  
  

Alternative techniques (when affinity chromatography is not applicable):

• Ion exchange chromatography (IEX): strong anion exchange (such as MaXtar® Q, Q Chromstar® FF) is often used to capture negatively charged impurities (nucleic acid, acidic HCP) and allow the target protein to flow through; Or selected according to the isoelectric point pI.

• Hydrophobic interaction chromatography (HIC): binding of target proteins or impurities at high salts. Sensitive to salt concentration.

• Precipitation/flocculation: Sometimes used for preliminary concentration and impurity removal (e.g. nucleic acid precipitation) as a pretreatment before chromatography.

Case 1: Recombinant subunit herpes zoster project uses MaXtar® Q-binding elution mode, 20% B elutes impurity and 35% elutes target protein.

• Chromatography column and resin: 2.6 cm in diameter and 20 cm in height

• Sample: Culture supernatant. The target protein concentration was 0.7 mg/mL

• Loading volume: 804 mL

• Solution A: 20 mM PB pH6.8

• Solution B: 20 mM PB 1M NaCl pH6.8

• Flow rate: 120 cm/h

• Gradient: 20% elution, 35% elution

Key points of capture phase development:

• High binding capacity and high flow rate: Choose resins with large particles and good tolerance to high flow rates.

• Pre-filtration/clarification: Optimize depth filtration, centrifugation or tangential flow filtration (TFF) to remove particulate matter and protect the column.

• Sample adjustment: Optimize pH, conductivity, additives (e.g. protease inhibitors, reducing agents) to promote binding and stability.

• Flow-through mode vs. binding-elution mode: selected according to impurity profile and target protein properties.

Phase 3: Intermediate Purification Stage Development

Objective: Remove critical impurities (e.g. HCP, aggregates, mismatched isomers, shed affinity ligands, host DNA) with similar properties to the target protein.

Core technologies: Ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) are the most commonly used intermediate purification steps, using differences in charge or hydrophobicity for separation. Usually orthogonal to the capture step (different separation principle).

IEX: Fine optimization of pH (operating near target protein pI to maximize charge difference), gradient or step elution conditions, buffer species, and ionic strength. Cation exchange (such as MaXtar® S, SP Chromstar® ) and anion exchange (such as MaXtar® Q, Q Chromstar® ) is selected depending on the pI and impurity profile of the target protein.

HIC: Optimize salt species (usually ammonium sulfate), starting salt concentration, gradient or step elution conditions, additives. Combine at high salt and elute with reduced salt concentration. It has a good effect on removing aggregates.

Other techniques:

• Multimode chromatography (MMC): combining multiple forces (e.g. ion exchange + hydrophobic/hydrogen bonding), the selectivity may be higher, but the mechanism is more complex and slightly more difficult to develop (e.g. MaXtar® MMC).

• Hydroxyapatite chromatography (HAP): It has a unique effect on certain difficult-to-separate impurities (such as shedded Protein A, DNA, aggregates).

Case 2: Phenyl Chromstar was used in the second step of the recombinant subunit shingles project Phenyl Chromstar® HP binding elution mode, 30% B elutes impurity and 70% B elutes protein of interest.

• Chromatography column and resin: Diameter 2.6 cm, height 13.8 cm

•  Sample: MaXtar® Q elution collection (ammonium sulfate added to 98-105 ms/cm conductance), sample concentration 1.86 mg/mL

• Loading volume: 218 mL

• Solution A: 0.6 M (NH4) 2SO4 · PBS

• Solution B: Water

• Flow rate: 120 cm/h

• Gradient: 30% elution, 70% elution

Phase 4: Polishing Phase Development

Objective: Removal of trace impurities (especially those extremely close to the properties of the target protein, such as trace HCP, product-related variants, dimers/oligomers), ultimately meeting the desired high purity standards. It is also often a critical virus clearance step (for mammalian cell expression products).

Core technology:

• Ion exchange chromatography (IEX): Final purification under finer conditions (shallower gradient, higher resolution resins), separating the charge isomers.

• Size exclusion chromatography (SEC): based on molecular weight differential separation, it is the gold standard for removing aggregates (especially high molecular weight aggregates) and degraded fragments, while exchanging the buffer. The disadvantages are slow processing speed, low binding capacity, and diluted samples. Often used in final steps or analysis.

• Anion exchange chromatography flow-through mode:  As a polishing step, the target protein is flowed through at high pH (> pI) and impurities are bound to the column, taking advantage of the negatively charged nature of most HCP and virus particles.

  
  

Case 3: The third step of the recombinant subunit herpes zoster project purified chromatography uses SP Chromstar® The FF flow-through mode further removes impurities.

• Chromatography column and resin: 2.6 cm in diameter and 10 cm in height

• Sample: Solution after ultrafiltration, concentration 0.9 mg/mL

• Loading volume: 178 mL

• Solution A: 20 mM PB pH5.8

• Solution B: 20 mM PB 1M NaCl

• Flow rate: 60 cm/h

• Gradient: flow-through mode

  
  

Key Success Factors and Challenges

In-depth understanding of target protein and impurity profile: The basis for choosing the correct separation method. Use analytical techniques (HPLC, CE, SDS-PAGE, Western Blot, MS, SEC-MALS, etc.) to monitor the whole process.

Principle of orthogonality: The combined use of chromatographic modes based on different separation principles is the key to achieving high purity.

Platform strategy: For similar categories of products (such as monoclonal antibodies), the adoption of standardized platform processes can greatly accelerate development.

Quality by Design (QbD): Incorporate QbD philosophy from the early stage of development, focus on CQAs, conduct risk assessments (e.g. FMEA), use DoE optimization, and define design space.

Viral safety: The process of mammalian cell products must contain and validate at least two potent virus clearance/inactivation steps with different mechanisms (typically low pH incubation + AEX flow-through + nanofiltration).

Cost control: Chromatography resins and consumables are the main costs. Optimizing biding capacity, flow rate, buffer consumption, resin life is critical. Consider new techniques such as continuous chromatography.

Data analysis and modeling: Accelerate development and optimization with advanced data analysis tools and mechanism/statistical models.

Conclusion

Recombinant protein chromatography process development is a complex, iterative, and goal-oriented system engineering. Successful strategies begin with a deep understanding of the target proteins and impurities and run through the careful selection and optimization of the capture, intermediate purification, and polishing steps. Always keep in mind the core objectives of purity, yield, robustness, scalability, economics, compliance and virus safety. Applying QbD concepts, DoE tools, platforming strategies and advanced technologies (such as continuous chromatography), combined with in-depth process characterization, can develop high-quality, efficient and sustainable chromatographic purification processes that meet the needs of commercial production.