1. Introduction
With the iterative upgrading of biopharmaceutical technologies, complex structured biologics including bispecific antibodies and fusion proteins have evolved beyond laboratory research and been widely applied in clinical treatment of tumors, autoimmune diseases, and other severe disorders, emerging as a core innovative track in the biopharmaceutical industry. Compared with conventional monoclonal antibodies (mAbs), these complex molecules possess multiple subunits, complicated chain assembly mechanisms, and diverse post-translational modifications, resulting in distinctive technical bottlenecks in upstream cell culture processes.
During industrial production, complex biologics commonly suffer from high chain mismatch rates, insufficient protein titers, elevated aggregate and fragment impurities, significant charge heterogeneity, and poor controllability of glycosylation. Moreover, their yield and quality are highly sensitive to cellular microenvironments. Conventional mAb culture processes lack targeted adaptability to address the specific production defects of complex molecules, constituting a critical barrier to large-scale and stable manufacturing.
Based on industrial-scale process development and scale-up practices, this article systematically summarizes six core technical challenges in the upstream culture of complex biologics. Centering on three key objectives including yield improvement, metabolic regulation, and quality homogeneity control, it establishes systematic solutions covering cellular metabolic regulation, precise microenvironment control, nutrient system optimization, and targeted impurity inhibition. This study provides standardized, implementable technical references for manufacturability optimization and industrial scale-up of bispecific antibodies, fusion proteins, and other complex biologics.
2. Core Technical Challenges in Upstream Culture of Complex Biologics
The structural uniqueness of complex biologics determines the complexity of their cellular expression and culture processes. The upstream process challenges are highly coupled and interactive, ultimately leading to low product yield, pronounced quality heterogeneity, and insufficient production stability. The six core issues are elaborated as follows:
2.1 Disordered Antibody Chain Assembly and High Mismatch Impurity Levels
Complex multi-subunit molecules represented by bispecific antibodies require the co-expression of multiple heavy and light chains. The random intracellular chain assembly process frequently causes heavy/light chain mismatches and abnormal homodimer formation, generating a large number of structurally aberrant impurities. These impurities exhibit highly similar physicochemical properties to target products, making them difficult to remove via downstream purification. This dilemma substantially increases purification difficulty and production costs, while compromising final product purity and medication safety.
2.2 Insufficient Overall Expression Level and Limited Production Capacity
The co-expression of multiple subunits imposes significant transcriptional and translational metabolic burdens on host cells, resulting in unbalanced subunit expression and inhibited cell proliferation. Meanwhile, the accumulation of reactive oxygen species (ROS) and glycolytic metabolic disorders during culture further impair cell viability and protein synthesis efficiency, ultimately leading to low volumetric productivity that fails to meet the demands of large-scale industrial production.
2.3 Uncontrolled Lactate Metabolism and Instable Culture System
Abnormal rebound and massive accumulation of lactate frequently occur in the middle and late stages of cell culture. Excessive lactate causes continuous acidification of the culture system, inhibits cell proliferation and protein synthesis, and shortens the high-efficiency production window of cells. Furthermore, lactate accumulation exacerbates cellular metabolic disorders, further reducing product yield and quality stability, which serves as the most common inducement of process fluctuations during upstream scale-up.
2.4 Pronounced Charge Heterogeneity and High Acidic Variant Proportion
Complex biologics are prone to diverse post-translational modifications including deamidation, amino acid oxidation, non-enzymatic glycation, and aberrant sialylation during cell culture. Disordered disulfide bonds also trigger charge deviation, resulting in massive generation of acidic charge variants. Excessive charge variants directly alter the in vivo pharmacokinetic (PK) profiles and target binding activity of products, and elevate potential immunogenicity risks, which are key and difficult points in critical quality attribute (CQA) control.
2.5 Severe Protein Aggregation and Fragmentation with Excessive Impurities
Complex molecules expose more hydrophobic regions. Under the stress of microenvironmental changes, protease hydrolysis, and disulfide bond mismatching during culture, high-molecular-weight (HMW) aggregates and low-molecular-weight (LMW) fragments are readily formed. HMW and LMW impurities reduce the effective purity of products, and aggregates significantly increase the immunogenicity risk of drugs, directly affecting product safety and efficacy.
2.6 Fluctuant Glycosylation and Poor Controllability of Functional Attributes
Glycosylation is a core post-translational modification that determines the in vivo half-life, antibody-dependent cellular cytotoxicity (ADCC) activity, and immunogenicity of biologics. The glycosylation modifications of complex biologics (including fucosylation, sialylation, and galactosylation) are highly sensitive to culture conditions. Minor fluctuations in process parameters can lead to substantial deviations in modification ratios, causing batch-to-batch quality differences and restricting product quality consistency.
3. Systematic Optimization Solutions for Upstream Processes
Targeting the above six core pain points, this study constructs five targeted optimization technical systems based on industrial process development experience, with the core goals of quality improvement, yield enhancement, process stabilization, and impurity control. Multi-parameter collaborative regulation realizes stable, efficient, and controllable upstream culture of complex biologics.
3.1 Bidirectional Collaborative Regulation for High-Efficiency Product Expression
The final protein yield is jointly determined by the integral viable cell density (IVCD) and specific cellular productivity (qP). The traditional optimization strategy relying solely on cell density expansion has obvious limitations. This study adopts a combined strategy of intensified fed-batch (IFB) culture, precise nutrient matching, and microenvironment parameter control to simultaneously improve cell proliferation and single-cell productivity.
In terms of process mode, the IFB process is adopted with an optimized seeding density of 10×10⁶ cells/mL, and precise feeding is initiated on the day of inoculation to support rapid cell proliferation and prolong the effective production duration. Process verification shows that this strategy increases the system IVCD by 138% and raises the product titer from 575.5 mg/L to 1552.3 mg/L, achieving a significant capacity improvement.
In terms of nutrient regulation, metabolomics-driven precise amino acid optimization is performed. The targeted combination of valine, serine, cysteine, and tyrosine improves protein expression while reducing the accumulation of ammonia, lactate, and other metabolic by-products. Supplementary addition of TCA cycle-related amino acids activates cellular energy metabolism pathways and markedly elevates qP.
In terms of microenvironment control, pCO₂ is strictly maintained below 120 mmHg to avoid cell metabolic damage caused by hypercapnia. A moderately hyperosmotic system (460–500 mOsm/kg) is applied to activate protein synthesis and improve qP without significant growth inhibition. Meanwhile, staged temperature shift, precise pH regulation, trace element optimization, and antioxidant supplementation are integrated to comprehensively optimize cell physiological status and achieve steady yield improvement.
3.2 Remodeling Cellular Metabolic Pathways for Precise Lactate Control
Excessive lactate accumulation stems from overactivated intracellular glycolysis and insufficient mitochondrial aerobic oxidation, which prevents pyruvate from entering the TCA cycle for complete catabolism and results in massive lactate secretion. The core optimization principle is to inhibit invalid glycolysis and enhance aerobic oxidation, fundamentally eliminating lactate rebound.
A restricted glucose feeding strategy is applied to precisely regulate glucose concentration and feeding rate, weaken glycolytic flux, reduce excessive pyruvate generation, and cut off the substrate supply for lactate synthesis. Meanwhile, supplementary addition of TCA cycle intermediates and copper ions improves mitochondrial respiratory chain efficiency, enhances pyruvate aerobic oxidation, and directs carbon flux toward protein synthesis rather than by-product formation.
A glucose-responsive dynamic feeding system is established to realize on-demand precise feeding based on real-time glucose monitoring, completely avoiding lactate rebound caused by nutrient excess in the late culture stage. Auxiliary strategies including staged temperature reduction, steady-state pH control, and precise regulation of dissolved oxygen and pCO₂ stabilize cellular metabolism, maintain cell viability in late culture, and ensure consistent yield and process stability.
3.3 Multi-Dimensional Regulation to Reduce Acidic Charge Variants
Acidic charge variants are mainly induced by oxidative stress, deamidation, non-enzymatic glycation, and abnormal sialylation, serving as the primary cause of product charge heterogeneity. Multi-dimensional strategies including environmental regulation, antioxidant system construction, and amino acid ratio optimization can effectively inhibit negative charge modifications and reduce acidic variant proportions.
Precise pH adjustment of the culture system slows down the deamidation rate of asparagine residues and reduces deamidation-derived acidic variants. An antioxidant protection system is constructed by supplementing free tryptophan to inhibit protein oxidation and Mn²⁺ to eliminate intracellular ROS, alleviating oxidative stress-induced charge deviation.
Strict control of cysteine concentration is essential; excessive cysteine triggers disulfide bond reduction, thiolation, and subsequent oxidation, significantly increasing acidic variant levels. Precise cysteine reduction effectively mitigates charge heterogeneity. Meanwhile, appropriate supplementation of basic amino acids increases the net positive charge of protein molecules, dilutes the overall proportion of acidic variants, and optimizes product charge homogeneity.
3.4 Stabilization of Protein Spatial Structure to Inhibit Aggregation and Fragmentation
HMW aggregates and LMW fragments critically affect product purity, safety, and efficacy. Their core inducements include protein unfolding, disulfide bond mismatching, extracellular protease hydrolysis, and stress-induced structural instability. Efficient impurity control can be achieved via process optimization, redox homeostasis regulation, and dynamic medium replacement.
Staged temperature reduction reduces cell metabolic rate and extracellular protease secretion, alleviates protease-mediated protein hydrolysis, and decreases protein unfolding probability to inhibit hydrophobic intermolecular aggregation. Supplementary Cu²⁺ optimizes intracellular redox homeostasis, corrects abnormal disulfide bond pairing, and stabilizes protein spatial structure, increasing the SEC main peak purity by approximately 10% according to process validation data.
The introduction of continuous perfusion culture realizes real-time removal of metabolic wastes, proteases, and denatured proteins via continuous medium exchange, cutting off the intrinsic causes of protein degradation and aggregation. This process reduces protein fragmentation levels by 15.6% and significantly improves product main peak purity. Auxiliary measures including dissolved oxygen stabilization, reasonable culture cycle shortening, and chelator addition to eliminate metal ion stress further reduce aggregate and fragment formation.
3.5 Precise Glycosylation Regulation for Stable Product Functional Attributes
Glycosylation is a critical product CQA that directly determines the in vivo half-life, ADCC anti-tumor activity, and immunogenicity of complex biologics, requiring multi-factor coordinated regulation for precise modification control.
For sialylation optimization, a composite regulation system consisting of Mn²⁺, uridine, galactose, and dexamethasone is constructed to provide sufficient substrates and enzymatic reaction conditions, increasing the sialylation rate to 92.3%. Supplemental Cu²⁺ inhibits extracellular sialidase activity to prevent sialic acid degradation and maintain high-level sialylation throughout the culture process, ensuring the long-term in vivo efficacy of products.
For afucosylation optimization, a fucose analog competitive inhibition strategy is adopted to target fucosyltransferase and block the fucosylation pathway. After process optimization, the afucosylation ratio of products increases from 6.45% to 45.59%, which significantly enhances product ADCC activity and anti-tumor efficacy.
Furthermore, steady-state control of macroscopic parameters including temperature, pH, and osmotic pressure, combined with precise matching of metal ions, nucleotide sugar precursors, and hormones, realizes directional glycosylation regulation, minimizes batch-to-batch modification fluctuations, and guarantees product quality consistency.
4. Conclusion and Outlook
Different from the standardized manufacturing mode of conventional mAbs, the upstream culture processes of bispecific antibodies, fusion proteins, and other complex biologics are characterized by multi-factor coupling, high specificity, and high sensitivity. Single-parameter optimization cannot achieve simultaneous improvement of yield and quality. Only a trinity systematic development system integrating molecular structure design, host cell line development, and precise culture process regulation can fundamentally address five core challenges including low expression, uncontrolled lactate metabolism, charge heterogeneity, protein aggregation and fragmentation, and unstable glycosylation.
All optimization strategies described in this article are derived from industrial process development and scale-up practices with high implementability and universality. They effectively enable high-yield, low-impurity, and quality-controllable upstream manufacturing of complex biologics, providing solid technical support for process scale-up, technology transfer, and commercial production of complex innovative biotherapeutics.
In the future, upstream processes for complex biologics will develop toward intelligence, digitization, and continuity. The integration of AI algorithms and digital twin technology for process modeling, real-time online monitoring via process analytical technology (PAT), and self-optimizing bioreactors enables real-time closed-loop regulation of process parameters. This innovation transforms upstream culture from traditional experience-driven mode to data-driven precise and intelligent manufacturing, further improving process stability, production efficiency, and product quality consistency.
