Host cell protein networks as a novel co-elution mechanism during protein A chromatography.

Host cell proteins (HCPs) are process-related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc-based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co-purification of HCPs with the therapeutic protein are either HCP-drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc-based proteins by LC-MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co-eluted with less than three Fc-based proteins indicating a drug-specific co-purification for most HCPs. Only ten HCPs co-purified with over 50% of the 23 Fc-based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co-elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein-protein interaction networks. Here, we propose an additional mechanism for HCP co-elution involving protein-protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.

Fiber chromatographic enabled process intensification increases monoclonal antibody product yield.

The most straightforward method to increase monoclonal antibody (mAb) product yield is to complete the purification process in less steps. Here, three different fiber chromatographic devices were implemented using a holistic approach to intensify the mAb purification process and increase yield. Fiber protein A (proA) chromatography was first investigated, but traditional depth filtration was not sufficient in reducing the contaminant load as the fiber proA device prematurely fouled. Further experimentation revealed that chromatin aggregates were the most likely reason for the fiber fouling. To reduce levels of chromatin aggregates, a chromatographic clarification device (CCD) was incorporated into the process, resulting in single-stage clarification of harvested cell culture fluid and reduction of DNA levels. The CCD clarified pool was then successfully processed through the fiber proA device, fully realizing the productivity gains that the fiber technology offers. After the proA and viral inactivation neutralization (VIN) hold step, the purification process was further intensified using a novel single-use fiber-based polishing anion exchange (AEX) material that is capable of binding both soluble and insoluble contaminants. The three-stage fiber chromatographic purification process was compared to a legacy five-step process of dual-stage depth filtration, bead-based proA chromatography, post-VIN depth filtration, and bead-based AEX chromatography. The overall yield from the five-step process was 60%, while the fiber chromatographic-enabled intensified process had an overall yield of 70%. The impurity clearance of DNA and host cell protein (HCP) for both processes were within the regulatory specification (<100 ppm HCP, <1 ppb DNA). For the harvest of a 2000 L cell culture, the intensified process is expected to increase productivity by 2.5-fold at clarification, 50-fold at the proA step, and 1.6-fold in polishing. Relative to the legacy process, the intensified process would reduce buffer use by 1088 L and decrease overall process product mass intensity by 12.6%.

Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.

Concentration of clarified pool by single-pass tangential flow filtration to improve productivity of protein A capture step: Impact of clarification strategies.

Protein A capture chromatography remains a high-cost and relatively low-productivity step for downstream processing of monoclonal antibodies. As bioprocessing transitions toward intensified processes, maximizing the efficiency of individual steps is key to achieving economic targets. This study was performed to assess the impact of inline concentration of clarified cell culture fluid (CCF), using single-pass tangential flow filtration, on protein A chromatography purification productivity. CCF with varying levels of impurities and turbidity were obtained dependent upon the clarification method. These CCFs were concentrated and processed over a protein A capture step. Productivity increases of 1.8- to 2.6-fold were achieved as compared to a protein A capture step with no CCF concentration. Achieving such targeted improvements requires careful consideration of the multiple components in the clarification strategy before implementation.

Continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1 d-1 (RC-PMCC) and 574 g L-1 d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

Acidic pH or salt treatment can convert soluble antibody aggregates in culture harvest into monomers and improve Protein A chromatography step yield.

While purifying a regular monospecific antibody, we found that the Protein A step yield was much lower than expected. Further studies revealed that the antibody formed large-size aggregates that did not bind to the Protein A resin, hence leading to dropped recovery. In an attempt to solve this low yield issue, we found that mildly acidic pH or ammonium sulfate treatment can partially convert the aggregates into monomers. In addition, when acidic pH treated culture harvest was processed by Protein A chromatography, the yield was restored to the normal range, suggesting that the monomers recovered from aggregates regained Protein A binding capability. Thus, low pH treatment of culture harvest can be potentially used as a general approach for improving Protein A step yield in cases where non-binding antibody aggregates are formed through noncovalent interactions.

SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield.

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.

CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability.

Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high.

Helical Content Correlations and Hydration Structures of the Folding Ensemble of the B Domain of Protein A.

The B domain of protein A (BdpA), a small three-helix bundle, folds on a time scale of a few microseconds with heterogeneous native and unfolded states. It is widely used as a model for understanding protein folding mechanisms. In this work, we use structure-based models (SBMs) and atomistic simulations to comprehensively investigate how BdpA folding is associated with the formation of its secondary structure. The energy landscape visualization method (ELViM) was used to characterize the pathways that connect the folded and unfolded states of BdpA as well as the sets of structures displaying specific ellipticity patterns. We show that the native state conformational diversity is due mainly to the conformational variability of helix I. Helices I, II, and III occur in a weakly correlated manner, with Spearman's rank correlation coefficients of 0.1539 (I and II), 0.1259 (I and III), and 0.2561 (II and III). These results, therefore, suggest the highest cooperativity between helices II and III. Our results allow the clustering of partially folded structures of folding of the B domain of protein A on the basis of its secondary structure, paving the way to an understanding of environmental factors in the relative stability of the basins of the folding ensemble, which are illustrated by the structural dependency of the protein hydration structures, as computed with minimum-distance distribution functions.

Recent advances of affibody molecules in biomedical applications.

Affibody molecules are 58-amino-acid peptides with a molecular weight of about 6.5 kDa, derived from the Z domain of Staphylococcal Protein A. Since they have been used as substitutes for antibodies in biomedicine, several therapeutic affibody molecules have been developed for clinical use. Additionally, affibody molecules have been designed for a range of different applications. This review focuses on the progress made in the last five years in the field of affibody molecules and their potential uses in medical imaging, especially in oncology and cancer treatment. It covers areas such as molecular imaging, targeted delivery of toxic drugs, and their use in combination with nanoparticles. We also highlight some current biomedical applications where affibody molecules are commonly used as a "guide." Due to their many advantages, affibody molecules offer significant potential for applications in both biochemical and medical fields.

pH inactivation of SARS-CoV-2 and SARS-CoV in virus spiked protein A eluates from a mAb purification process.

Biopharmaceutical manufacturing processes may include a low pH treatment step as a means of inactivating enveloped viruses. Small scale virus clearance studies are routinely performed using model enveloped viruses such as murine leukemia virus to assess inactivation at the pH range used in the downstream manufacturing process. Further, as a means of bioburden reduction, chromatography resins may be cleaned and stored using sodium hydroxide and this can also inactivate viruses. The susceptibility of SARS-CoV-2 and SARS-CoV to low pH conditions using protein A eluate derived material from a monoclonal antibody production process as well as high pH cleaning conditions was addressed. SARS-CoV-2 was effectively inactivated at pH 3.0, moderately inactivated at pH 3.4, but not inactivated at pH 3.8. Low pH was less effective at inactivating SARS-CoV. Both viruses were inactivated at a high pH of ca.13.4. These studies provide important information regarding the effectiveness of viral clearance and inactivation steps of novel coronaviruses when compared to other enveloped viruses.

A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells.

Plant viral nanoparticles (VNPs) are attractive to nanomedicine researchers because of their safety, ease of production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits a high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain of staphylococcal Protein A via gene fusion. We selected cetuximab as a model IgG to demonstrate the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells from the Cal33 line of a tongue squamous cell carcinoma. Using confocal microscopy, we observed that fluorescent VNP-cetuximab bound selectively to Cal33 and was internalized, revealing the potential of this nanotool in cancer research.

Creation of a versatile automated two-step purification system with increased throughput capacity for preclinical mAb material generation.

The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification systems require complex flow paths or third-party components that are not found on a standard fast protein liquid chromatography instrument (FPLC) (e.g., Cytiva's ÄKTA) to enable higher throughput. In early mAb discovery there is often a trade-off between throughput and scale where a high-throughput process requires miniaturized workflows necessitating a sacrifice in the amount of material generated. At the interface of discovery and development, flexible automated systems are required that can perform purifications in a high-throughput manner, while also generating sufficient quantities of preclinical material for biophysical, developability, and preclinical animal studies. In this study we highlight the engineering efforts to generate a highly versatile purification system capable of balancing the purification requirements between throughput, chromatographic versatility, and overall product yields. We incorporated a 150 mL Superloop into an ÄKTA FPLC system to expand our existing purification capabilities. This allowed us to perform a range of automated two-step tandem purifications including primary affinity captures (protein A (ProA)/immobilized metal affinity chromatography (IMAC)/antibody fragment (Fab)) followed by secondary polishing with either size exclusion (SEC) or cation exchange (CEX) chromatography. We also integrated a 96 deep-well plate fraction collector into the ÄKTA FPLC system with purified protein fractions being analyzed by a plate based high performance liquid chromatography instrument (HPLC). This streamlined automated purification workflow allowed us to process up to 14 samples within 24 h, enabling purification of ∼1100 proteins, monoclonal antibodies (mAbs), and mAb related protein scaffolds during a 12-month period. We purified a broad range of cell culture supernatant volumes, between 0.1 and 2 L, with final purification yields up to 2 g. The implementation of this new automated, streamlined protein purification process greatly expanded our sample throughput and purification versatility while also enabling the accelerated production of greater quantities of biotherapeutic candidates for preclinical in vivo animal studies and developability assessment.

HED, a Human-Engineered Domain, Confers a Unique Fc-Binding Activity to Produce a New Class of Humanized Antibody-like Molecules.

Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated protein based on the three-dimensional structure of the Z-domain derived from Staphylococcal protein A. This HED retains µM binding activity to the human IgG1CH2-CH3 elbow region. We determined the crystal structure of HED in association with IgG1's Fc. This demonstrated that HED preserves the same three-bundle helix structure and Fc-interacting residues as the Z domain. HED was fused to the single chain variable fragment (scFv) of mAb 4D5 to produce an antibody-like protein capable of interacting with the p185Her2/neu ectodomain and the Fc of IgG. When further fused with murine IFN-γ (mIFN-γ) at the carboxy terminus, the novel species exhibited antitumor efficacy in vivo in a mouse model of human breast cancer. The HED is a novel platform for the therapeutic utilization of engineered proteins to alleviate human disease.

Causes of Industrial Protein A Column Degradation, Explored Using Raman Spectroscopy.

Monoclonal antibodies (mAbs) are used extensively as biotherapeutics for chronic and acute conditions. Production of mAbs is lengthy and expensive, with protein A affinity capture the most costly step, due both to the nature of the resin and its marked reduction in binding capacity with repeated use. Our previous studies using in situ ATR-FTIR spectroscopy indicated that loss in protein A binding capacity is not the result of leaching or degradation of protein A ligand, suggesting fouling is the principal cause. Here we explore binding behavior and resin capacity loss using Raman spectroscopy. Our data reveal a distinct Raman spectral fingerprint for mAb bound to the protein A ligand of MabSelect SuRe. The results show that the drop in static binding capacity (SBC) previously observed for used protein A resin is discernible by Raman spectroscopy in combination with partial least-squares regression. The SBC is lowest (35.76 mg mL-1) for used inlet resin compared to used outlet (40.17 mg mL-1) and unused resin samples (70.35 mg mL-1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (∼18 mg mL-1), binding of mAb is not homogeneous through used resin beads with protein binding preferentially to the outer regions of the bead, in contrast to fully homogeneous distribution through unused control MabSelect SuRe resin beads. Analysis of the Raman spectra indicates that one foulant is irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin beads.

Antibody orientational labeling via staphylococcus A protein to improve the sensitivity of gold immunochromatography assays.

The Cry1Ab toxin is usually expressed in genetically modified crops in order to control chewing pests. Although the gold immunochromatography assay (GICA) based on the double-antibody sandwich method has been developed to detect this toxin, its detection sensitivity needs improvement. In this study, Cry1Ab-51 antibodies were immobilized orientationally in a simple and effective way on colloidal gold nanoparticles (CGNPs) using the affinity of staphylococcal protein A (SPA) towards the fragment crystallizable (FC) fragment of mouse immunoglobulin G (IgG). Lateral flow detection test strips, assembled with probes labeled with orientational methods under optimal operational conditions (new probe), were 10 times more sensitive than test strips assembled with probes labeled by adsorption (conventional probe). Experiments showed that the affinity of the new probe was much higher than the conventional probe. The immunochromatography gold strip (ICG strip) assembled using the new probe was highly specific to Cry1Ab with no cross-reaction with other transgenic proteins, and it was proved that the specificity of the new probe had no change. Furthermore, the ICG strips assembled with the new probe could be stored for 12 months under dry conditions without a significant loss of sensitivity. The orientational labeling of the antibodies with SPA on colloidal gold proved to be suitable for improving the sensitivity of the ICG strips.

Integrated continuous biomanufacturing on pilot scale for acid-sensitive monoclonal antibodies.

In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid-sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 106 cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, ZCa ; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small-scale trials and the pilot-scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial-scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability.

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A.

Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

Affinity-Based Methods for Site-Specific Conjugation of Antibodies.

Conjugation of various reagents to antibodies has long been an elegant way to combine the superior binding features of the antibody with other desired but non-natural functions. Applications range from labels for detection in different analytical assays to the creation of new drugs by conjugation to molecules which improves the pharmaceutical effect. In many of these applications, it has been proven advantageous to control both the site and the stoichiometry of the conjugation to achieve a homogeneous product with predictable, and often also improved, characteristics. For this purpose, many research groups have, during the latest decade, reported novel methods and techniques, based on small molecules, peptides, and proteins with inherent affinity for the antibody, for site-specific conjugation of antibodies. This review provides a comprehensive overview of these methods and their applications and also describes a historical perspective of the field.

Comparison of UV- and Raman-based monitoring of the Protein A load phase and evaluation of data fusion by PLS models and CNNs.

A promising application of Process Analytical Technology to the downstream process of monoclonal antibodies (mAbs) is the monitoring of the Protein A load phase as its control promises economic benefits. Different spectroscopic techniques have been evaluated in literature with regard to the ability to quantify the mAb concentration in the column effluent. Raman and Ultraviolet (UV) spectroscopy are among the most promising techniques. In this study, both were investigated in an in-line setup and directly compared. The data of each sensor were analyzed independently with Partial-Least-Squares (PLS) models and Convolutional Neural Networks (CNNs) for regression. Furthermore, data fusion strategies were investigated by combining both sensors in hierarchical PLS models or in CNNs. Among the tested options, UV spectroscopy alone allowed for the most precise and accurate prediction of the mAb concentration. A Root Mean Square Error of Prediction (RMSEP) of 0.013 g L-1 was reached with the UV-based PLS model. The Raman-based PLS model reached an RMSEP of 0.232 g L-1 . The different data fusion techniques did not improve the prediction accuracy above the prediction accuracy of the UV-based PLS model. Data fusion by PLS models seems meritless when combining a very accurate sensor with a less accurate signal. Furthermore, the application of CNNs for UV and Raman spectra did not yield significant improvements in the prediction quality. For the presented application, linear regression techniques seem to be better suited compared with advanced nonlinear regression techniques, like, CNNs. In summary, the results support the application of UV spectroscopy and PLS modeling for future research and development activities aiming to implement spectroscopic real-time monitoring of the Protein A load phase.