Facile discovery of surrogate cytokine agonists.

Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.

A checkpoint function for Nup98 in nuclear pore formation suggested by novel inhibitory nanobodies.

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier.

Single domain antibody: Development and application in biotechnology and biopharma.

Heavy-chain antibodies (HCAbs) are a unique type of antibodies devoid of light chains, and comprised of two heavy chains-only that recognize their cognate antigen by virtue of a single variable domain also referred to as VHH, single domain antibody (sdAb), or nanobody (Nb). These functional HCAbs, serendipitous discovered about three decades ago, are exclusively found in camelids, comprising dromedaries, camels, llamas, and vicugnas. Nanobodies have become an essential tool in biomedical research and medicine, both in diagnostics and therapeutics due to their beneficial properties: small size, high stability, strong antigen-binding affinity, low immunogenicity, low production cost, and straightforward engineering into more potent affinity reagents. The occurrence of HCAbs in camelids remains intriguing. It is believed to be an evolutionary adaptation, equipping camelids with a robust adaptive immune defense suitable to respond to the pressure from a pathogenic invasion necessitating a more profound antigen recognition and neutralization. This evolutionary innovation led to a simplified HCAb structure, possibly supported by genetic mutations and drift, allowing adaptive mutation and diversification in the heavy chain variable gene and constant gene regions. Beyond understanding their origins, the application of nanobodies has significantly advanced over the past 30 years. Alongside expanding laboratory research, there has been a rapid increase in patent application for nanobodies. The introduction of commercial nanobody drugs such as Cablivi, Nanozora, Envafolimab, and Carvykti has boosted confidence among in their potential. This review explores the evolutionary history of HCAbs, their ontogeny, and applications in biotechnology and pharmaceuticals, focusing on approved and ongoing medical research pipelines.

Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.

Nanobodies in cancer.

For treatment and diagnosis of cancer, antibodies have proven their value and now serve as a first line of therapy for certain cancers. A unique class of antibody fragments called nanobodies, derived from camelid heavy chain-only antibodies, are gaining increasing acceptance as diagnostic tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. The small size of nanobodies (∼15 kDa), their stability, ease of manufacture and modification for diverse formats, short circulatory half-life, and high tissue penetration, coupled with excellent specificity and affinity, account for their attractiveness. Here we review applications of nanobodies in the sphere of tumor biology.

Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC50) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.

Nanobody inhibitors of Plexin-B1 identify allostery in plexin-semaphorin interactions and signaling.

Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signaling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. While inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterized a pair of nanobodies that are specific for mouse Plexin-B1 and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signaling and provides a potential innovative route for therapeutic modulation of Plexin-B1.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes.

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Functional insights of Tyr37 in framework region 2 directly contributing to the binding affinities and dissociation kinetics in single-domain VHH antibodies.

Single-domain VHH antibody is regarded as one of the promising antibody classes for therapeutic and diagnostic applications. VHH antibodies have amino acids in framework region 2 that are distinct from those in conventional antibodies, such as the Val37Phe/Tyr (V37F/Y) substitution. Correlations between the residue type at position 37 and the conformation of the CDR3 in VHH antigen recognition have been previously reported. However, few studies focused on the meaning of harboring two residue types in position 37 of VHH antibodies, and the concrete roles of Y37 have been little to be elucidated. Here, we investigated the functional states of position 37 in co-crystal structures and performed analyses of three model antibodies with either F or Y at position 37. Our analysis indicates that Y at position 37 enhances the dissociation rate, which is highly correlated with drug efficacy. Our findings help to explain the molecular mechanisms that distinguish VHH antibodies from conventional antibodies.

Phage display-derived alpaca nanobodies as potential therapeutics for Naja atra snake envenomation.

Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified VHH genes from isolated peripheral blood mononuclear cells and constructed a phage display VHH library of 1.0 × 107 transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

Targeted drug delivery using nanobodies to deliver effective molecules to breast cancer cells: the most attractive application of nanobodies.

Targeted drug delivery is one of the attractive ways in which cancer treatment can significantly reduce side effects. In the last two decades, the use of antibodies as a tool for accurate detection of cancer has been noted. On the other hand, the binding of drugs and carriers containing drugs to the specific antibodies of cancer cells can specifically target only these cells. However, the use of whole antibodies brings challenges, including their large size, the complexity of conjugation, the high cost of production, and the creation of immunogenic reactions in the body. The use of nanobodies, or VHHs, which are a small part of camel heavy chain antibodies, is very popular due to their small size, high craftsmanship, and low production cost. In this article, in addition to a brief overview of the structure and characteristics of nanobodies, the use of this molecule in the targeted drug delivery of breast cancer has been reviewed.

Predicting Long-Term Stability of an Oral Delivered Antibody Drug Product with Accelerated Stability Assessment Program Modeling.

The oral delivery of protein therapeutics offers numerous advantages for patients but also presents significant challenges in terms of development. Currently, there is limited knowledge available regarding the stability and shelf life of orally delivered protein therapeutics. In this study, a comprehensive assessment of the stability of an orally delivered solid dosage variable domain of heavy-chain antibody (VHH antibody) drug product was conducted. Four stability related quality attributes that undergo change as a result of thermal and humidity stress were identified. Subsequently, these attributes were modeled using an accelerated stability approach facilitated by ASAPprime software. To the best of our knowledge, this is the first time that this approach has been reported for an antibody drug product. We observed overall good model quality and accurate predictions regarding the protein stability during storage. Notably, we discovered that protein aggregation, formed through a degradation pathway, requires additional adjustments to the modeling method. In summary, the ASAP approach demonstrated promising results in predicting the stability of this complex solid-state protein formulation. This study sheds light on the stability and shelf life of orally delivered protein therapeutics, addressing an important knowledge gap in the field.

Preparation of divalent camelid single-domain antibody and its application in immunoassays for Salmonella detection in food.

Salmonella-related foodborne infections are commonly caused by the serovars of S. Typhimurium, which can be detected using antibody-based immunoassays. The monovalent variable domain of the camelid heavy chain antibody (VHH) performs excellently in constructing multivalent VHH variants, which generally exhibit higher affinities with antigens and consequently enhance the assay sensitivity. In this study, the divalent variants of VHHs (diVHHs) targeting S. Typhimurium were generated by encoding the monovalent VHH genes assembled in tandem with a flexible linker peptide (G4S)2. Soluble diVHHs were produced in a prokaryotic expression system and purified with a yield of 4.22 mg/L. Benefiting from their stability and antigen-binding abilities towards tested Salmonella serovars, diVHH-based immunoassays were developed. The diVHH-based sandwich immunoassay, using diVHH as capture antibody, exhibited a detection limit of 1.04×102 CFU/mL and enabled as low as 10 CFU/mL S. Typhimurium to be detected after 6 h of enrichment in lettuce. Furthermore, this assay can be applied to spiked lettuce, chicken, and pork samples, showing acceptable recoveries ranging from 83 to 106%. This study presented feasible strategies for VHH multivalence and established a superior sensitivity VHH-based immunoassay for monitoring and analyzing Salmonella contamination in food.

Administration of an AAV vector coding for a P2X7-blocking nanobody-based biologic ameliorates colitis in mice.

BACKGROUND: The pro-inflammatory ATP-gated P2X7 receptor is widely expressed by immune and non-immune cells. Nanobodies targeting P2X7, with potentiating or antagonistic effects, have been developed. Adeno-associated virus (AAV)-mediated gene transfer represents an efficient approach to achieve long-term in vivo expression of selected nanobody-based biologics. This approach (AAVnano) was used to validate the relevance of P2X7 as a target in dextran sodium sulfate (DSS)-induced colitis in mice. RESULTS: Mice received an intramuscular injection of AAV vectors coding for potentiating (14D5-dimHLE) or antagonistic (13A7-Fc) nanobody-based biologics targeting P2X7. Long-term modulation of P2X7 activity was evaluated ex vivo from blood samples. Colitis was induced with DSS in mice injected with AAV vectors coding for nanobody-based biologics. Severity of colitis, colon histopathology and expression of chemokines and cytokines were determined to evaluate the impact of P2X7 modulation. A single injection of an AAV vector coding for 13A7-Fc or 14D5-dimHLE efficiently modulated P2X7 function in vivo from day 15 up to day 120 post-injection in a dose-dependent manner. An AAV vector coding for 13A7-Fc significantly ameliorated DSS-induced colitis and significantly reduced immune cell infiltration and expression of chemokines and proinflammatory cytokines in colonic tissue. CONCLUSIONS: We have demonstrated the validity of AAVnano methodology to modulate P2X7 functions in vivo. Applying this methodological approach to a DSS-induced colitis model, we have shown that P2X7 blockade reduces inflammation and disease severity. Hence, this study confirms the importance of P2X7 as a pharmacological target and suggests the use of nanobody-based biologics as potential therapeutics in inflammatory bowel disease.

Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, against the extracellular domain of the human α7-nAChR were generated through alpaca immunization with cells expressing a human α7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4ß2 and α3ß4. E3 acts as a slowly associating positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not precluding the desensitization of the receptor. An E3-E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with α-bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

A novel cell biological tool to explain mechanics and dynamics in fission yeast.

The Rho guanosine triphosphatase hydrolase enzyme (GTPase) is required for the control of the actin cytoskeleton, but its activation in vivo condition is unknown. The study's goal was to find a new synthetic nanobody VHH (P-36 tagged with mNeonGreen) that interacts strongly with the Rho GTPase. We present the first novel synthetic nanobody, VHH (P-36 tagged with mNeonGreen), tested in fission yeast cells and found to have a particular interaction with Rho1GTPase. Plasmids were constructed by using of certain enzymes to digest the pDUAL-pef1a vector plasmid to produce a protein that was encoded by cloned genes. A varied VHH library was created synthetically, then transformed into yeast cells, and positive clones were chosen using chemical agents. To investigate protein interactions and cellular reactions, several studies were carried out, such as live cell imaging, growth curve analysis, coimmunoprecipitation, structural analysis, and cell therapies. Prism and RStudio were used for the statistical analysis. The presence of VHH (P-36) has no effect on the growth pattern making it an appropriate model for studying cytokinesis in vivo. According to a computational biological study, its affinity to interact with Rho1GTPase with all the complementarity-determining region (CDR) regions found on VHH (P-36) is extremely strong. We were able to track its subcellular target by localization using a fluorescent confocal microscope, ensuring the maintenance of cell polarity and morphology. Spheroplast analysis revealed a circular-shaped cell with an even distribution of Rho1 tagged VHH (P-36), indicating that the interaction occurs near the plasma membrane. The introduction of latrunculin-A (Lat-A) disrupted Rho GTPase localization, demonstrating the control over actin production, and the cell did not show evidence of mitotic phase commencement while Lat-A was present. Finally, this important biological tool can aid in our understanding of the mechanics and dynamics of cytokinesis in relation to Rho1GTPase.

Single-Domain Antibodies-Novel Tools to Study and Treat Allergies.

IgE-mediated allergies represent a major health problem in the modern world. Apart from allergen-specific immunotherapy (AIT), the only disease-modifying treatment, researchers focus on biologics that target different key molecules such as allergens, IgE, or type 2 cytokines to ameliorate allergic symptoms. Single-domain antibodies, or nanobodies, are the newcomers in biotherapeutics, and their huge potential is being investigated in various research fields since their discovery 30 years ago. While they are dominantly applied for theranostics of cancer and treatment of infectious diseases, nanobodies have become increasingly substantial in allergology over the last decade. In this review, we discuss the prerequisites that we consider to be important for generating useful nanobody-based drug candidates for treating allergies. We further summarize the available research data on nanobodies used as allergen monitoring and detection probes and for therapeutic approaches. We reflect on the limitations that have to be addressed during the development process, such as in vivo half-life and immunogenicity. Finally, we speculate about novel application formats for allergy treatment that might be available in the future.

Anti-Idiotypic VHHs and VHH-CAR-T Cells to Tackle Multiple Myeloma: Different Applications Call for Different Antigen-Binding Moieties.

CAR-T cell therapy is at the forefront of next-generation multiple myeloma (MM) management, with two B-cell maturation antigen (BCMA)-targeted products recently approved. However, these products are incapable of breaking the infamous pattern of patient relapse. Two contributing factors are the use of BCMA as a target molecule and the artificial scFv format that is responsible for antigen recognition. Tackling both points of improvement in the present study, we used previously characterized VHHs that specifically target the idiotype of murine 5T33 MM cells. This idiotype represents one of the most promising yet challenging MM target antigens, as it is highly cancer- but also patient-specific. These VHHs were incorporated into VHH-based CAR modules, the format of which has advantages compared to scFv-based CARs. This allowed a side-by-side comparison of the influence of the targeting domain on T cell activation. Surprisingly, VHHs previously selected as lead compounds for targeted MM radiotherapy are not the best (CAR-) T cell activators. Moreover, the majority of the evaluated VHHs are incapable of inducing any T cell activation. As such, we highlight the importance of specific VHH selection, depending on its intended use, and thereby raise an important shortcoming of current common CAR development approaches.

Quantification of human intravenous immunoglobulin from plasma and in process samples by affinity chromatography.

Advances in affinity chromatography now make it possible to analyze immunoglobulin G from plasma and its fractions with a simple chromatographic method. Ligands derived from camelid antibodies have been developed which have affinity to all 4 subclasses of human IgG without a cross reactivity to other immunoglobulins. The commercially available Capture Select FcXL is the basis for a simple method for direct quantification of immunoglobulin G from plasma or from fractions from cold ethanol precipitation. After direct injection of the sample into the column the unbound proteins are washed out with equilibration buffer and eluted with a pH-step. The elution the peak is integrated, and quantity is derived form a standard curve. The limit of detection with 40 µg/mL, and a linearity up to 250 µg/mL allows an analysis of samples ranging from 0.04 to 50 mg/mL using varying injection volume without further dilution and the two-wavelength detection. A full cycle is completed within five minutes. This method can serve as orthogonal method for in-process control but also for process development.