Creative Biolabs is specialized in providing custom biotechnology and pharmaceutical services that cover the full scope of biotechnology needs of early drug discovery and drug development. As a trusted provider of the most cost-effective outsourcing solutions, Creative Biolabs has been working for a large number of satisfied clients from biotechnology and pharmaceutical companies as well as government and academic research laboratories all over the world.
Creative Biolabs was founded by scientists who are dedicated to the conquering of cancer. We believe to build up a custom-service-centered business model is important for optimizing the drug development process, leveraging accessible resources, and forming a team of various background to conduct drug discovery in future. Our commitment to this long-term goal motivates us to deliver the highest quality results to our clients with speed.
Creative Biolabs provides a full range of products and services based on our three-dimensional (3D) cell culture platform. With the well-established static and fluidic 3D cell culture systems, our services include, but are not limited to, establishment and optimization of experimental protocols, a series of in vitro analysis and screening (e.g., penetrability analysis, toxicity analysis and targeting analysis), as well as design custom in vitro 3D models (e.g., specific tumor tissue, in vitro organoid, and tissue-/organ-on-a-chip). Creative Biolabs is experienced and dedicated to helping customers in areas of 3D cell culture and analysis.
ADC manufacturing is a multistep process that can be divided into three distinct stages: cGMP production of the antibody, cGMP synthesis of the drug-linker complex, and conjugation to form an ADC. The conjugated antibodies undergo extensive purification and finished as ADC products upon the completion of fill-finishing.
cGMP antibody manufacturing: The Creative Biolabs cGMP antibody manufacturing service platform has the capacity of pilot (5~35 L) to large scale (> 100 L) preparation of therapeutic antibodies or recombinant proteins using various expression systems (hybridoma, mammalian cell lines, insect cell cultures, and microbial expression systems). We also offer process development services under this platform to help establish cGMP stable cell lines, cell banking, upstream process optimization, and downstream scale-up…
cGMP organic synthesis: Creative Biolabs provides customized chemical synthesis service and fine chemicals based on our well-established organic synthesis chemistry pipeline in R&D and cGMP environments. This pipeline enables us in preparing regular or highly tailored linker molecules bearing different release mechanisms, modifying payload drugs for proper conjugation chemistry, preparing new payload derivatives for the purpose of drug development, and formulating the drug-linker complex for ADC manufacturing.
ADC conjugation, purification, and filling: ADC conjugation is achieved in cGMP certified reactors after small-scale conjugation protocol verification. The therapeutic impurities are removed utilizing advanced filtering devices such as ultrafiltration and tangential flow filtration (TFF) systems. The ADC product are purified using single-use chromatography systems and finished by filling into aseptic vials via the cGMP sterile filling pipeline.
Creative Biolabs and our collaborators are also equipped with state-of-the-art analytical platform (MALS/IR, LC/MS/HPLC, UHPLC, CE, icIEF, UV/VIS, Moisture analysis, CCI…) for ADC characterization and ensure the purity and quality of the end products. Process development services are also available for optimizing conjugation strategy, formulation, purification scheme, and scale-up production of ADC to gram scale.
With our Ph.D. level science team and years of experience in the field of ADC development and production, the ADC manufacturing platform at Creative Biolabs will greatly facilitate your ADC projects and push the progress towards clinical trials. Please contact us for more information and a discussion to see how we can be involved in your ADC development projects!
In a broad sense, cancer biomarker is an indicative of the presence of cancer in the body or a molecule secreted by a tumor or a specific response of the body to the presence of cancer. It is usually identifiable or measurable in the blood, urine or other convenient body fluids by PCR, ELISA, and other conventional immune assays. In this sense, biomarkers can be categorized into genetic, epigenetic, proteomic, glycomic, and imaging biomarkers for cancer diagnosis, prognosis, and epidemiology. In a narrow sense of cancer biomarker, it is limited to proteins the most used to challenge in the clinical applications, especially in the CAR-T therapy. Specifically, a cancer biomarker of the CAR-T provides the most prominent signal of cancer cells for distinguishing from normal cells and the most effective target for immune recognition and destruction. As the most basic foundation of therapeutic effect, these cancer biomarkers commonly possess the following characteristics:
Involve in the canceration process, correlate with the disease changes, like deterioration and improvement;
Manifest as a relatively high expression or even unique expression on the surface of cancerous and precancerous tissue than other normal cells to be measured and distinguished easily and reliably for an acceptable safety, especially the mutant proteins, which is the most specific biomarkers for the only origination from an existing tumor;
Relatively stable in response to other unrelated factors.
At Creative Biolabs, we provide almost all the discovered cancer targets and novel ones in the evaluation for CAR-T therapy. Like AFP for liver cancer, BCR-ABL for chronic myeloid leukemia, BRCA1/BRCA2 for breast/ovarian cancer, BRAF V600E for melanoma/colorectal cancer, CA-125 for ovarian cancer, CA19.9 for pancreatic cancer, CEA for colorectal cancer, EGFR for non-small-cell lung carcinoma, HER-2 for breast cancer, KIT for gastrointestinal stromal tumor, PSA for prostate cancer, S100 for melanoma, etc.
As a CAR technology leader, Creative Biolabs has established an unparalleled platform for Chimeric Antigen Receptor (CAR) modified T cell construction, which can satisfy various needs for developing treatments of multiply cancers in preclinical studies and researches.
Creative Biolabs offer TCR repertoire sequencing services. With our powerful denovo TCR (T-cell Receptor) sequencing service, we can sequence different types of TCRs with 100% coverage of V (D) JC regions and >90% accuracy.
With appropriate technical equipment and years of experience in bio-pharmaceuticals, Creative Biolabs provides one-stop customer-oriented services of antibody drug conjugate (ADC) manufacture. We have been dedicating ourselves to helping every custom to facilitate their project in a highly productive and cost-effective way.
With appropriate technical equipment and years of experience in bio-pharmaceuticals, Creative Biolabs provides one-stop customer-oriented services of antibody drug conjugate (ADC) manufacture. We have been dedicating ourselves to helping every custom to facilitate their project in a highly productive and cost-effective way.
Creative Biolabs now provides various strategies for antibody modification and conjugation in antibody-drug conjugate (ADC) development. With state-of-art equipments, advanced techniques and wide antibody platforms, scientists and technicians from Creative Biolabs can perform custom-designed ADCs to meet every client’s requirements.
With substantial experience in T cell receptor (TCR) engineering and TCR transfer gene therapy, Creative Biolabs provides a broad range of assay services to evaluate the antigen specific reactivity and cytotoxicity of TCR-modified T lymphocytes. Taking advantage of state-of-the-art instruments and experienced Ph.D level TCR specialists, we are capable of designing and performing custom analysis service to obtain comprehensive information about the reactivity and cytotoxity of your specific TCRs.
Creative Biolabs is a professional service provider in the field of T-cell receptors (TCRs) developing and analysis, and now offers cytokines release assay service to help customer assess the function of their TCR-modified T cells by determining the cytokines releasing ability, in detail, detecting the quantity and types of cytokines secreted by those engineered T cells.
With many years of experience in Chimeric Antigen Receptor (CAR) field, Creative Biolabs can provide custom CellRapeutics™ canine CAR-T cells or NK cells construction services, which can greatly accelerate your project in canine cancer research.
Creative Biolabs is able to expand our CAR T cells service from human origin to mouse system. Taking the advantage of mouse model diversity, we could greatly facilitate customer’ s research project.
Based on our knowledge and elite research team, Creative Biolabs now expands the service to NHP CAR T cell construction, which could significantly support clients with the fundamental research and new therapeutics development. Additionally, we established a multi-organism, multi-task preclinical test system, which could take on CAR T project from in vitro cytotoxicity test to in vivo safety test in different animal models.
CAR Clinical Trials
To achieve a successful advancement in personalized CAR therapertics for the treatment of cancers, Creative Biolabs provides CellRapeutics™ CAR Clinical Trials service. Since the CAR therapertics holds great promise to improving cancer patients’ health, we strive to move these novel therapeutics into clinical trials based on our years of experience in CAR technology, uncompromised quality standards and unbeatable timelines. For each project, once the preclinical animal models study for safety and efficacy test is complete and ready for clinical trials, Creative Biolabs assembles a strong team with experienced Ph.D level scientists and clinicians, well trained technicians, responsible project managers just in order to deliver the most suitable service according to your budget, timeline, and specific requirements.
CAR Clinical Trials
To speed up the development of CAR therapertics in clinical trials, we provide one-stop, full spectrum service to deliver the most efficient and economic outcomes.
Service & Solutions
• Review of preclinical testing
• Study design and protocol writing
• Ethics & Regulatory
• Project management
• Physician management
• Clinical operations
• Patient enrollment
• Patient treatment
• Patient follow-up
• Data management
• Study summary
• Biostatistics & medical writing
Creative Biolabs offers our expertise in constructing bispecific fusion proteins for your research and development purposes. We work closely with our customers so as to design every project according to their individual requirements.
For many years, Creative Biolabs has devoted itself to developing immunotherapy in hematology to bring effective treatments to people suffering from hematonosis. Now, we can provide bispecific antibody (BsAb) development services for this purpose.
With years of experience in bispecific antibody (BsAb) engineering, Creative Biolabs provides custom BsAbs against various cancers. Our experts can design and perform different BsAbs construction according to the antigens you are interested in, for both scientific and clinical purposes.
BsAbs combine the advantages of both classical monoclonal antibodies and bispecific molecules to achieve an increased antitumor activity. There have been many BsAbs in clinical development such as catumaxomab (Removab®), blinatumomab (Blincyto®), ertumaxomab, solitomab, etc., the first two of which have received market approval. According to your individual requirements, our antibody engineering platform can also provide all of the five classes of BsAbs with different formats. We are experienced in generating BsAbs based on advanced techniques such as BiTE, CrossMab, dock-and-lock, all of which can be used to treat diverse cancers, such as advanced solid tumors, malignant ascites, ALL, neuroblastoma and osteosarcoma. Typically, they can induce anti-tumor responses through various mechanisms: retargeting of T/NK cells to tumor, Fc mediated effector functions, blockage of two antigens (such as pro-angiogenic factors), etc.
Creative Biolabs has been devoted to oncotherapy research for many years. Leveraging advanced immunotherapy technology and antibody engineering platforms, our scientists have developed diverse bispecific antibodies against cancers.
With unchallenged experience in bispecific antibody production, Creative Biolabs offers custom BsAbs for diverse scientific and clinical researches in ophthalmology. We integrate various technology platforms in order to provide high quality BsAbs with first class controllability and operability.
Creative Biolabs provides a full-service for complete evaluating your bispecific antibodies (BsAbs) in vivo and in vitro to help you solve complex preclinical test problems. According to client’s requirements and specific BsAb characteristic, certified experts from Creative Biolabs can design and perform the standard animal and cellular studies to obtain preliminary efficacy, toxicity as well as pharmacokinetic information about your BsAbs. The availability of multiple animal models from rodents to non-human primates (NHP), and various cell models, combined with the state-of-the-art instruments and methodologies, all of the technological superiority enables Creative Biolabs to achieve every customer’s bispecific antibody development goals in a timely manner.
Creative Biolabs has rich experience in antibody engineering and assessment field, which can provide a wide range of analysis services to make you have an overall understanding of the biochemistry features of your bispecific antibodies (BsAbs). Our elaborate characterization platforms based on various analysis techniques are unprecedented in analyzing BsAbs sample purity, molecular weight and molecular structure. With the latest instruments and professional staffs, Creative Biolabs is dedicated to offering first-class biochemistry characterization services to meet your BsAb development goal in a time-saving manner.
The biochemistry characterization of bispecific antibody we can offer includes:
Molecular weight/mass measurement
Molecular structure analysis
Our BsAb characterization platforms include, but not limited to:
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a very common analytical technique for separating and characterizing proteins by electrophoresis, which uses a discontinuous polyacrylamide gel as a support medium, and sodium dodecyl sulfate (SDS) to denature the proteins. This technique can be used to estimate the relative molecular mass, determine the relative abundance of major kinds of proteins in a sample and the protein distribution among different fractions, and assess the purity of protein samples.
Western blot analysis
Western blot, also called immunoblotting, is a useful technique for protein detection. This analysis method combines the separation ability of gel electrophoresis with the specificity of immunoassays, which allows the identification and analysis of a certain kind of protein in the mixture.
SEC -based analysis
Size-exclusion chromatography (SEC) is a chromatographic method, which can be used to separate and characterize certain kinds of proteins in a sample according to their size. SEC works by trapping smaller molecules in the pores of the adsorbent materials, meanwhile the larger molecules simply pass by the pores. As a result, larger molecules flow through the column much faster than smaller ones.
SEC multi-angle light scattering (SEC-MALS) is an analytical technique combining SEC and multi-angle static light scattering (MALS) to provide an absolute measurement of molecular weight. MALS measures the absolute molar mass and size of molecules in solution mainly based on the intensity and the angular dependence of the scattered light. SEC-MALS analysis allows for more accurate mass measurement than either of these two methods alone.
Electrospray ionization mass spectrometry (ESI-MS) is an important technique for structural study or quantitative measurement of bio-molecule in a complex biological sample. This technique can overcome the drawbacks of other conventional techniques and perform an effective study of non-volatile and thermally labile bio-molecules with only a small sample amount.
Creative Biolabs offers customers design and computational strategies for protein engineering to develop proteins of desire properties. We can provide various protein design approaches, including Noncanonical Amino Acids, Design and Synthesis of Artificial Zinc Finger Proteins, computational optimization, selection from combinatorial libraries etc., to meet customers’ specific requirements.
Combinatorial Protein Design
Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids
Design and Synthesis of Artificial Zinc Finger Proteins
Calcium Indicators Based on Calmodulin-Fluorescent Protein Fusions
Engineering Site-Specific Endonucleases
Modulating Protein Characterization
Modulating Protein Structure
Modulation of Intrinsic Properties by Computational Design
Modulating Protein Interactions by Rational and Computational Design
Computer Graphics, Homology Modeling and Bioinformatics
Evolutionary Strategies for Protein Engineering
Protein Library Design and Screening
Protein Mutagenesis Library
Protein Design by Binary Patterning
Versatile DNA Fragmentation and Directed Evolution with Nucleotide Exchange and Excision Technology (NExT)
Degenerate Oligonucleotide Gene Shuffling (DOGS)
Phage Display Systems for Protein Engineering
M13 Bacteriophage Coat Proteins Engineering
Ribosome - Inactivation Display System (RIDS)
Cell Surface Display Systems for Protein Engineering
Cell-Free Display Systems for Protein Engineering
Enhancement of Enzyme Stability
Creative Biolabs offers a full spectrum of bioconjugation services, including protein/antibody biconjugation, oligosaccharide bioconjugation, oligonucleotide bioconjugation, surface immobilization, PEGylation, synthetic polymer modification and liposome conjugation services. Additionally, we also offer custom bioconjugation service to meet your very unique research objectives.
In the development of antibody drug conjugates (ADCs), the design of linkers is of the same essential importance as that of monoclonal antibodies and cytotoxic drugs, because the linker impacts the efficacy and tolerability of ADCs. Now, Creative Biolabs has developed several strategies to generate suitable linkers for the conjugation of different antibodies and drugs.
To generate suitable linkers in ADC development is one of the biggest challenges. The linker needs to be exceedingly stable in systemic circulation before reaching the target but allows for rapid and efficient release from the cytotoxic compound in an active form at the target site. According to the different mechanisms of drug release, linkers in ADC development can be classified into two categories: cleavable linkers and noncleavable linkers. Cleavable linkers rely on the physiological stimuli, which mainly include chemically cleavable linkers and enzymatically cleavable linkers. Chemically cleavable linkers including acid-labile linkers and disulfide linkers are popular in the ADC clinical pipeline. For acid-labile linkers, intracellular release of payloads relies on the different pH between endosomes/lysosomes and blood. The release of disulﬁde-linked drugs is controlled by the factors in intracellular environment. Enzymatically cleavable linkers, peptide linkers and β-glucuronide linkers, are sensitive to enzymes located in cytoplasm. Alternatively, noncleavable linkers require proteolytic degradation. They depend on the internalization more than cleavable linkers do. In ADC development, several noncleavable alkyl and polymeric linkers have been explored. Different linkers have their advantages and limitations. Ultimately, the choice of the linker is unique tailored to the correlate antibody, drug and the disease to be treated. Based on the fully understanding of every client’s requirements, scientists from Creative Biolabs will select the most appropriate linkers for your ADC investigation.
It can be challenging that conjugation of the linker to the antibody at large scale without affecting the structure of the antibody, since proteins are very sensitive to changes in the environment, and changes in the structure of the antibody can affect the performance of the ADC in terms of both efficacy and safety. Because the linker serves as the interface between chemistry and biology, there is always the opportunity for unexpected reactivity during scale up of the conjugation process. Reactivity can be affected by many factors, just as with the scale-up of any process. With most advanced experience, Creative Biolabs has established fairly standard methods used in the production of ADCs, and developed robust, scalable, lab-scale conjugation processes that can be scaled up to provide the desired product consistently from batch-to-batch. After that, we have been able to transform lab-scale into large-scale production of this ADC with consistently high quality. In addition, we can now apply the experience gained in the scale-up of other ADCs to the production in our pipeline according to the most scientific progress and every custom’s interest.
Creative Biolabs offers first-class membrane protein expression service.
Membrane proteins are partially or completely embedded into the cell membrane, and consist of 20~30% of all encoding genes in genome. They participate in different biological processes, including ligand-receptor binding, signal transduction, molecule transportation, intercellular recognition and enzymolysis. Of particular interest, 50-60% available drug candidates target membrane proteins, mainly G-protein coupled receptors (GPCRs) and ion channels.
NanobodiesCreative Biolabs provides the most complete collection of SdAb products the world has ever had. From premade to immunized phage display library, our proprietary SdAb platform allows rapid generation and large-scale production of high affinity novel biological therapeutics that have potential in a wide range of human diseases.
SdAbs are single-domain antibodies derived from the variable regions of heavy chain of Camelidae immunoglobulins, also called VHHs. The SdAb technology was originally developed following the discovery that camelidae, unlike mouse, rat or rabbit, possess fully functional antibodies that lack light chains. Therefore, the size of SdAb is extremely small compared to other forms of antibody fragment, which significantly increase the permeability of SdAb. Thus SdAb is considered of great value for research, diagnostics and therapeutics.
Advantages of SdAbs:
• Subnanomolar High Affinity
• High production yield
• Minimal size (~15 kDa)
• Great stability
• Reversible refolding
• Outstanding solubility in aqueous solutions
To ensure a time efficient and financially economical completion of your project, Creative Biolabs will dedicate a project management team that is powered by a cross-functional group of experts, who will be with you from inception to a final completion with full success. The assigned project manager will be integrated into every aspect of your project.
To build the maximum value into your product, we use our custom project management as a tool to ensure that physical, financial and human resources are efficiently utilized, deadlines and budgets are met and changes are clearly communicated and correctly executed.
Strength of our project management team is represented by, but is not limited to:
All our project managers are Ph.Ds with several years of industry experience.
Team members are available for direct conversation at anytime.
We are proactive in addressing any evolving challenges.
We do everything to meet the unique requirement of your product and business.
We valuate you as the most important expert on the team, and will always keep you being informed and in control of the project.
Creative Biolabs offers a full range of upstream and downstream service package of in vivo ascites production or in vitro cell culture production of monoclonal antibodies following strict cGMP guidelines. Customizable and cost-effective service is available for scale-up cGMP production of monoclonal antibodies from hybridoma cells.
In Vivo Monoclonal Antibody Production through Mouse Ascites
• Mycoplasma testing and decontamination of hybridoma cell lines
• Multiple strains available(including BALB/c, BALB/c hybrid, Nude and SCID mice)
• Ascites fluid develops in around 2 weeks
• 3-5ml of ascites per mouse with an antibody concentration of ~3-10mg/ml
• Downstream purification of ascites fluid
• Typical turnaround time of 5-8 weeks
• Over 5,000 mice capacity (high lot-to-lot consistency)
• Multi-kilogram quantities of mAb
• Liters of ascites
• Batch record with complete information.
Antibody Hybridoma Production
In Vitro Monoclonal Antibody Production through Cell Culture Methods
• Free of endogenous mouse antibodies
• Yield optimization and scale-up production
• Adaption to serum free media
• Multiple production system(spinner flasks, roller bottles, bioreactors)
• 15-50 mg/L for antibody production-in vitro
• Downstream purifications
• Multi-gram quantities of high-quality mAb
• Corresponding CHO stable cell line construction
Creative Biolabs provides custom services for pre-formulation and formulation development studies covering different drugs. At Creative Biolabs, we have formulation experts with extensive expertise and experience to develop a variety of dosage forms. We can perform activity assay through evaluating the physical properties, solubility and stability of your active pharmaceutical ingredient (API), which facilitates identification and selection of the optimal form.
Our services for pre-formulation and formulation development include:
Physico-chemical characterization evaluation
• Particle size distribution and morphology
• Partition coefficient (Log P/LogD)
• Dissociation constant (pKa)
• Solubility in various bio-relevant media, solvents and pH
• Stability under different solvent and pH
• Thermal characterization
• Forced degradation study
Dosage form selection
Comprehensive excipient selection, screening and evaluation
Compatibility of API and excipient
Formulation selection, optimization and finalization
Surface Charge Measurement
With professional science groups on protein assays, Creative Biolabs proudly provides protein surface charge analysis for customers. Analysis details of surface charge include:
• Electrokinetic phenomena
• Interaction between proteins
• Formulation of electric double layer
• Zeta potential
Analytical Methods Development for Validation-surface charge analysis
Methods and Instruments
Capillary electrophoresis (CE) and ion exchanging column (IEX) are the most commonly used methods on surface charge analysis. Combining with these two methods, a complete information including the migrate rate of charged particles, zeta potential and coated strategy will be well performed.
Creative Biolabs not only provides surface charge analysis, we also help customers to establish and validate analytical methods by the guidance of cGMP or GMP. Please click here to obtain more details of our other services.
Creative Biolabs offers unrivaled expertise in the research and development of protein modification. With combination of an extensive background in biopharmaceutical and proprietary development platforms, we can provide various forms of protein modification based on the full understanding of every client`s needs.
Our protein modification services include but are not limited to the following:
Precipitation & Centrifugation
Precipitation & Centrifugation
Creative Biolabs provides customers precipitation and centrifugation method development service. Precipitation and centrifugation are widely used steps in the downstream process of biopharmaceutical products. Our in-home scientists have experience in product purifying, we can perform effective precipitation and centrifugation operation to concentrate and purify products from various media.
Precipitation and centrifugation methods:
Isoelectric point precipitation
Precipitation with miscible solvents
Non-ionic hydrophilic polymers
Polyvalent metallic ions
Creative Biolabs provides customers filtration service to separate solid components from liquid phase. With state-of-art filtration equipment and experience in product purifying, we can compare and select more efficient and suitable filtration strategy depending on the product specifications.
We use the following methods selectively to perform products filtration:
TFF (tangential flow filtration)
CFF (crossflow filtration)
Downstream (Process Development)
Creative Biolabs provides efficient downstream process development services to meet the requirement of product purification. Creative Biolabs has highly qualified groups, advanced technologies and wide platforms to perform product purification and quantification suitable for commercially-available, cost-effective and large-scale manufacturing. Our broad experience in pharmaceutical allows us to develop and optimize downstream process at every stage.
Our downstream process development services include, but not limited to:
Chromatography and elution
Precipitation and centrifugation
Upstream (Process Development)
Creative Biolabs provides all units of upstream process development services to our clients. Creative Biolabs has a professional process development team with wide background of pharmaceutical experience in developing and optimizing robust, reproducible and scalable upstream processes. The goal of our services is to offer the most efficient upstream process development to meet the requirement of pharmaceuticals. Additionally, the use of design of experiments (DOE) helps to enable rapid optimization of process.
Our upstream development services encompass but are not limited to the following aspects:
Raw material and facilities supply
Cell culture optimization
cGMP Stable Cell Line
With substantial experience in protein expression and advanced cell line engineering platform, Creative Biolabs provides comprehensive cGMP stable cell line construction service starting from DNA sequence and ending with the delivery of top quality stable single clones. Owning all the key actuate variables for the service, housing various host cell lines (microbial and mammalian cell lines), vector technology and clone-adopted Media platform, as well as FDA approved cGMP facilities and professional work team, Creative Biolabs is capable of providing custom stable cell line construction service suited to every customer’s specific demands.
Flow Chart Of Creative Biolabs Stable Cell Line Construction Service
Stable cell line construction Procss
Analysis and Verification of DNA
Creative Biolabs always conducts the analysis and verification of DNA from customer to guarantee the quality of DNA used for stable cell line construction.
Construction of High Expression Vector
Creative Biolabs chooses and constructs proper expression vectors based on the property of the gene sequence, Customer’s opinion is always respected.
Creative Biolabs provides cGMP host cell lines covering microbial, insect and mammalian cells to suit customer’s specific demands. Various transfection methods including electroporation, polyethylenimine (PEI) mediated transfection and lipid-based transfection, are available.
Selection of Initial Stable Producing Pools
Creative Biolabs grows transfected cells in the appropriate selective media to generate stable pools. At least top 3 pools move to next cloning step depending on the productivity and quality of the pools.
Semi-solid medium plating or serial dilution is performed on the top 3 pools are for clone-pecking. And at least top 10 clones from each pool (the number can be extended upon request) are selected.
Fed -batch Evalution & Top Clones Selection
The top 10 clones from each pool are further evaluated based on their growth rate to capture 5-6 best clones. Different strategies can be applied to evaluate the productivity of the clones such as quantitative ELISA or fed-batch cultivation. And the final 3 top clones with robust cell growth and high productivity are selected based on the screening data.
Productivity & Stability Study
Creative Biolabs evaluates the yield and the stability of the final top clones for up to 60 generations (the duration can be extended upon request). The genetic stability of the final clones is evaluated by sequencing the full length of the target gene.
Final Deliverables, QC Data& Full Report
Creative Biolabs provides the top stable clone and comprehensive document report covering the stable cell line generation and all QC data.
Cell Banking Service
Creative Biolabs provides professional cGMP compatible custom cell banking service to clients. Under strict cGMP guideline, both master and working cell banks (MCB and WCB) can be manufactured by experienced cell culture specialists in state-of-the-art facilities. We provide cGMP cell banking service covering various species of cell types, including but not limited to mammalian cells, microbial cells, insect and stem cells. With matured cell culture and banking experience, exact quality oversight, client-specific documentation and QA-approved analysis certificate, we are committed to deliver the highest quality of services to our clients.
cGMP Registered and Inspected Facilities
Cell Expansion, Harvest and Cryopreservation under cGMP Conditions
Documentation and Report Facilitating the Regulation Process
Antibody fragments (Fab's) represent important structure for creating new therapeutics. Compared to full antibodies Fab' fragments possess certain advantages, including higher mobility and tissue penetration, ability to bind antigen monovalently and lack of fragment crystallizable (Fc) region-mediated functions such as antibody-dependent cell mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The main drawback for the use of Fab's in clinical applications is associated with their short half-life in vivo, which is a consequence of no longer having the Fc region. To exert meaningful clinical effects, the half-life of Fab's need to be extended, which has been achieved by postproduction chemical attachment of polyethylene glycol (PEG) chain to protein using PEGylation technology.
Advantages of PEGylation
PEGylation is an established tool to increase potency, half-life and solubility of biopharmaceuticals, which can be used for modifying biological compounds such as cytokines, antibody fragments, interleukins, hormones, oligonucleotides and some of proteins and peptides.
PEGylation can be effective in:
• Improving Bioavailability
• Prolonging Blood Circulation
• Maximizing Pharmacokinetics
• Reducing Immunogenicity
• Decreasing Frequency of Dosing
Creative Biolabs provides high-performance activated PEGs for PEGylated drugs from early development stage to commercial use. We have many years of experience in manufacturing high-quality methoxy polyethylene glycol, a key starting material of activated PEG in the pharmaceutical field. Our activated PEGs have a narrow polydisperity and a low diol content with a wide range of molecular weight, ranging from 2kDa to 80kDa.
Our customized PEGylation services include:
• Exploration and establishment of PEGylation process
• Process development and optimization for the production of PEGylated drugs
• Development of analytical methods for PEGylated drugs
• Production of Proof-of-Concept materials
Hi-affinityTM Bacterial Display Technology for Creating Therapeutic Antibodies
Creative BioLabs offers a unique bacterial display technology, Hi-affinityTM, for the development of human antibodies with pM affinity, the highest affinity ever enabled by an in vitro antibody production technology.
Bacterial display (or bacteria display or bacterial surface display) is a protein engineering technique widely used for in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria are screened using flow cytometry or iterative selection procedures (biopanning). Hi-affinityTM is based on a proprietary synthetic bacterial display human antibody library and a unique selection process natural to E. coli. This bacterial surface display technology enables the rapid isolation of target-specific antibodies without the labor intensive screening common to other recombinant and non-recombinant antibody production methods, resulting in unique single-chain variable fragment (scFV) antibodies that have both high specificity and extremely high affinity [Kd up to10-12] for the target antigen.
Hi-affinityTM human scFv antibody library is created by combining a highly diverse collection of synthetically-constructed randomized CDR sequences that are further diversified by random lengths using a unique proprietary technique. The large library has been specifically optimized to eliminate unwanted stop codons and aggregation-prone sequences. scFv molecules are first expressed in E. coli cytoplasm and then translocated and anchored into the bacterial plasma membrane. In the end, binder panels are selected by FACS or panning. Furthermore, multiple rounds of affinity maturation during library screening are incorporated through error prone PCR mutagenesis either directed at CDR or flanking sequences and selection by varying antigen dose.
The Hi-affinityTM platform utilizes a unique antibody selection process that relies on the natural twin-arginine translocation (Tat) system in E. coli. The major limitation challenging conventional phage display technologies is their dependence on the Sec translocation pathway. The Sec pathway transports proteins from the cytoplasm to the periplasm in an unfolded state; consequently, proteins that require a cytoplasmic environment and/or cytoplasmic components for folding, or reach their native state before they interact with the Sec proteins, are not compatible with the Sec pathway. Hi-affinityTM platform overcomes this limitation by exploiting the properties of the Tat pathway which only exports folded proteins that have already attained their native conformation in the cytoplasm.
Hi-affinityTM derived antibodies are intended for research, diagnostic and therapeutic use. The customer owns the exclusive rights to the antibodies including the antibody CDR sequence and any information generated by the customer through using the antibodies.
Creative BioLabs's BacuFlexTM Baculovirus/ Insect Cell Expression Platform was developed by our in-house team of scientists for virus production and expression of recombinant proteins from baculovirus-infected insect cells in flexible scale.
The baculovirus expression vector system (BEVS) is universally recognized as a powerful and versatile system for production of high quality proteins. There are several advantages of insect cells over E. coli such as improved solubility, ability to incorporate post-translational modifications, and higher yields for secreted proteins.
For customers who wish to establish the best possible protocol for their project and then scale up, Creative BioLabs recommends customers to leverage sophisticated BacuFlexTM Platform to explore different conditions for the expression of target proteins.
Mammalian cells have the ability to perform most comprehensive post-translational modifications and to secrete glycoproteins that are correctly folded and contain complex antennary oligosaccharides with terminal sialic acid. Mammalian cells are widely used in the production of recombinant proteins, antibodies, virus, viral-subunit proteins, and gene-therapy vectors.
Protein scientists at Creative BioLabs have experience and expertise in both transient and stable expression using a multitude of cell lines, including CHO and HEK293 expression systems. To meet different needs for protein expression, mammalian cell protein expression can be carried out via the following different approaches:
Transient burst expression
Stable mammalian cell line expression (CHO and HEK293)
CHO expression systems
CHO cells are the most commonly used mammalian cells for protein expression. Our homogeneous stable super-producer clones are obtained without cloning in just one week, whereas classical generation of stable cell lines takes 6 to 9 months. The obtained cell lines can be banked and re-used for further productions. Whereas transient transfection is very expensive at large scale, we are able to offer attractive cost reductions with scale-up.
HEK293 expression systems
Engineered Human Embryonic Kidney cells (HEK293 cells) produce native structure and biologically functional proteins with similar post-translational modification to human body. We provide one-stop-shop protein production services using transient transfection in HEK293 cells from gene sequence to purified proteins. Contracting laboratories have direct access to the scientists working on their projects, and each project is delivered with a specifications sheet and original data on yield, purity, and identity.
Over the years, Creative BioLabs has developed BacTECTM and FoldEZTM technologies to tackle hard-to-express and hard-to-dissolve proteins. With BacTECTM technology, the expression level can be greatly improved for almost all of the proteins. In addition, over 98% of the inclusion bodies can be refolded and solubilized by our proprietary FoldEZTM protein refolding technology.
Our OxiCytoTM competent E.coli strains are excellent options for expressing proteins having complex disulfide bonds.
E. coli Expression
The first choice for the expression of recombinant proteins is typically E. coli. Production of proteins in this bacterial strain is well-established, fast and simple and usually provides high yields. Recent progress in the fundamental understanding of transcription, translation, and protein folding in E. coli, improved genetic tools had made this bacterium more valuable than ever for the expression of complex eukaryotic proteins.
Creative BioLabs’ newly-built facilities supporting a vast array of state-of-the-art equipment are leveraged by our experienced PhD-level scientists to deliver proteins require glycosylation modifications to be expressed via microbial systems quickly and economically.
One of the most popular technologies is BacTECTM system. Creative BioLabs's patented BacTECTM system is capable of achieving soluble expression levels well over 20 g/L. While the technology has shown it can express many types of proteins, it has established a track record for producing high-quality, correctly folded antibody fragments (including Fabs and scFv).
Our FoldEZTM protein refolding technology is designed to determine the optimal refolding conditions by evaluation a series of buffers. We provide refolded protein at purity of 95% or greater.
Exclusively from Creative BioLabs, OxiCytoTM competent E.coli strains are engineered to enable the folding of recombinantly expressed proteins in vivo. The expression of the isomerase, DsbC, within the OxiCyto cytoplasm enhances the correct formation of disulfide bonds in proteins that require them for biological activity.
B. subtilis Expression
Bacillus subtilis is a significant microorganism in the scientific research field. It has been developed as an attractive host for the production of recombinant proteins. Our Bacillus subtilis system is an ideal option for the expression of monomeric protein products at expression levels up to 20g/L. This high yield, combined with our patented fermentation protocols allow for fast process development and scale-up to support early clinical testing.
Our MemProArt™ platform involves three independent methods for membrane protein antigen expression, allowing highly tailored service for customers:
• Cell-based Expression
This is a break-through technology aiming the maximum preservation of the native conformation of membrane protein epitopes, allowing high efficient antibody production.
• Cell-free Expression
This is derived from a unique cell-free expression system, enables membrane proteins directly incorporated into artificial liposomes (both bilayer and planar vehicles) during the in vitro translation step, resulting in “ready-to-use” proteoliposome for immunization.
• Virus-like Particles (VLPs) and Lipoparticles
This resembles the self-assembly of virus, produces protein in correct conformation. Amplifying retroviral (HIV-1 or MLV) gag protein in 293 or CHO-based EBEE™ technique, our patent technique is able to introduce adequate posttranslational modification pattern in the expressed membrane protein, to achieve the highest immunogenicity.
MemProArt™ platform has developed Phage Display Library Screening and hybridoma technique for monoclonal antibody screening and production respectively:
• Phage Display Library Screening
A large library of phage-displayed human antibodies is incubated with mammalian cells presenting target membrane protein, for antigen-antibody interaction. Phages with inert and low-affinity antibody are washed away, while phages with high-affinity antibody are selected and propagated. Creative Biolabs has proprietary technique to greatly increase the affinity during panning procedure, and to select the most effective phage clone for amplification and subsequent application.
• Mouse/Rat Hybridoma Production
Creative Biolabs is able to stably over-express target membrane proteins on the surface of commercially available cell lines (e.g., HEK293, CHOK1, NIH3T3, Sf9). The cells are then used to immunize mice/rats, followed by the isolation of splenic cells. Owing to our unique technique, Creative Biolabs could generate both rat and mouse hybridoma, for rat and mouse monoclonal antibody production.
With our leading MemProArt™ platform and experience for hundreds of successful cases, Creative Biolabs guarantees our customers unparalleled service in anti-membrane protein antibody production.
A hapten is a small molecule that can elicit an immune response only when conjugated with a large carrier such as a protein. Typical haptens include drugs, urushiol, quinone, steroids, etc.
Peptides and non-protein antigens usually need conjugating to a carrier protein (such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin) to become good immunogens). Additionally, haptens should be administered with an adjuvant to ensure a high quality immune response.
Anti-hapten antibodies are increasingly used in:
• Disease diagnostics, prognosis and therapy
• Therapeutic treatment monitorin
• Detection and quantification of drugs and narcotics
• Environmental monitoring
Small molecules, when used as haptens, are not immunogenic. However, on conjugating with carrier molecule they elicit antibody response. The production of anti-hapten antibodies of desired specificity largely depends on the hapten design (preserving greatly the chemical structure and spatial conformation of target compound), selection of the appropriate carrier protein and the conjugation method. We design anti-hapten antibodies based on the HaptenDB information.
Together with Creative Diagnostics, Creative Biolabs provide polyclonal and monoclonal production to haptens & small molecules. We guarantee at least two positive clones for ANTI-HAPTEN monoclonal antibodies development the process of producing anti-hapten antibodies.
Creative Biolabs is professional in raising anti-idiotype antibodies. Anti-idiotype antibodies are antibodies that have specific binding capabilities to an idiotope of another antibody. Theidiotope is the unique set of antigenic determinants (epitopes) of the variable portion of the antibody. Here, it is the complementarity determining regions (CDR) that determine the antigen specificity of the antibody. In some cases it can be the actual antigen-binding site, and in some cases it may comprise variable region sequences outside of the antigen-binding site on the antibody itself. Therefore, anti-idiotype antibodies can be claimed as antibodies that target the antigen-binding site formed by the CDRs of the antibody. In theory, anti-idiotypic antibodies can mimic the original antigen and reproduce/inhibit the immune response raised by the original antigen depending on how to use the anti-idiotypic antibodies. Because of the resemblance of anti-idiotypic antibodies to the original antigen, anti-idiotypic antibodies can be used as the anti-idiotypic vaccine to replace the antigen---if the original antigen is not available.
Antibody characterization is important in research and biopharmaceutical development. All therapeutic proteins produced from current technologies have heterogeneities in the final product because various modifications occur during different stages of production. Due to these heterogeneities, thorough characterization is necessary for the reproducible and safe production of therapeutic proteins.
At Creative Biolabs, antibodies can be completely characterized using a variety of techniques.
Monoclonal antibodies and antibody drug conjugates (ADC's) are one of the most active areas of biologic drug therapies. And thanks to changes in the regulatory framework in the U.S., biosimilar monoclonal antibodies are now entering clinical trials incresing the number of new monoclonal antibody (mAb) drug therapies entering commercial clinical trials. This trend is creating a strong demand for reliable, fast and efficient solutions for mAb characterization.
The basic mAb characterization workflows consists of the following types of analysis:
• molecular weight determination of intact and reduced protein
• peptide mapping
• co- and post-translational modification mapping
• acquired non-biological modifications
• disulfide bond mapping
In addition, Monoclonal and polyclonal antibody binding affinities can be chacterized by Simple Western analysis. Relative specificity of the antibody to the target protein of interest can be monitored. Antibody clonal screening can be performed using nanoliter amounts of material.
Creative BioLabs offers proprietary genetic immunization based polyclonal and monoclonal antibody generation services. This unique antibody development approach involves direct immunization of host animals with plasmid DNA encoding the target protein of interest. The immunized hosts then produce the encoded protein and raise antibodies. Genetic immunization involves introducing the gene in the form of a cDNA directly into an animal which translates this cDNA into protein thus stimulating an immune response against the foreign protein. Protein purification is not necessary for this genetic immunization approach, which can save several months in time over recombinant protein generation followed by antibody production.
In order for genetic immunization to be successful, the cDNA-encoded protein must be secreted by the transfected cells in immunized animals or expressed on the surface of the transfected cells in order to get stimulation of an antibody response.
The foremost advantage of this antibody production approach is its high success rate in generation of high-affinity antibodies recognizing difficult-to-express proteins in their native confirmation, such as GPCRs, ion channels and other multiple membrane spanning proteins. For these proteins, recombinant protein fragments or peptides derived from their extracellular domains may raise antibodies workable in Western blotting but are extremely hard to produce high-affinity antibodies that can recognize their integral proteins in their native form. This point is important in raising antibodies for diagnostic use, in which recognition of the antigens in their native form can be required. For therapeutic antibodies, targeting the antigens in the native conformation with a high-affinity is of course required!
Of note, our technology allows guaranteed antibody development against 7-membrane-spanning GPCR proteins!
High affinity antibodies can result from genetic immunization because of low level of expressed proteins and constant presentation to the immune system; these tend to favor development of high affinity antibodies.
Usually polyclonal antibody development via genetic immunization is tried first since it is an economical way to see whether the protein-encoding plasmid/cDNA will raise the desired antibodies, e.g. antibodies recognizing an integral antigen in its native 3D conformation. If this is successful, monoclonal development is followed.
Next Generation Antibody Sequencing
"Antibody Experts" has developed a novel strategy to determine the complete sequence of an antibody with unparalleled speed and accuracy. The technique is called "Database Assisted Shotgun Sequencing" (DASS) and combines all the advantages of existing methods. Antibody sequencing has now become a routine measurement that typically can be completed within 3-10 working days for IgGs. However, the method is applicable to all antibody formats, namely IgMs, fluorochrome conjugates, immobilized antibodies and mixtures.
In the first step your antibody is fragmented to peptides by a special technique, which generates up to 5000 different peptides per chain. This set of peptides will be analyzed by high end mass spectrometers to generate extensive sequence information. MS/MS spectra are then de Novo sequenced by the latest algorithms and matched against a database with related sequences. Special "in house" data mining tools allow us to extract the sequence information from ten thousends of MS/MS spectra within hours.
Sequencing of the V and J and C segments by "Database Assisted Shotgun Sequencing"
The V and J and C gene segments of antibodies are available in public databases (germline sequences, see figure 1). However, during the maturation of an antibody, the B-cell introduces hypermutations into the sequence to optimize the affinity. Our mapping algorithm is error tolerant and can match the "mutated" peptides to the corresponding germline, reliably.
Due to the high number of peptides, we get sequence information for EVERY peptide bond in the antibody. Typically, 20-70 different MS/MS spectra are generated for each amino acid (AA) position. Hence, even the hardest sequences of proline and arginine rich peptides can be resolved. As the order of all amino acids is clear, there is no need for time consuming techniques like edman sequencing.
We have gained extensive experience in antibody affinity maturation. We usually take scFv as the antibody format in affinity maturation. Also, a monovalent display phagemid system is used to reduce the avidity effects during antigen-binding screening. Two methods, untargeted mutagenesis and oligonucleotide-directed mutagenesis, are employed to construct random or defined sub-libraries to introduce a large number of mutants of the original antibody. Antibody binders of higher affinity are then selected by increasing the screening stringency. By constructing a series of sub-libraries of a scFv/Fab antibody, our proprietary protocol allows increase of the affinity of the scFv antibodies from 10 [-8] to 10 [-9]. We have successfully obtained a scFV antibody that has an extremely high affinity of 10 [-12], whose binding to the antigen is essentially irreversible.
Creative Biolabs a leading service provider that focuses on developing high specific, high affinity monoclonal antibodies for research, diagnostic and therapeutic use. Our service portfolio includes mouse and rat monoclonal antibody production using hybridoma technology, humanized antibody production using phage display technology and a wide range of antibody gene engineering, affinity maturation and humanization services. In addition, we are well-recognized in manufacturing scFv/Fab and full-size IgG antibodies. In addition, OEM services for bulk scale antibody manufacturing, including bacterial production of scFv, diabody, tandem scFv, miniantibody and Fab, and mammalian cell expression of minibody, chimeric IgG and IgG, are also available at the most competitive price in the industry.
Creative Biolabs has extensive experience in antibody humanization. Humanization is important for reducing the immunogenicity of monoclonal antibodies derived from xenogeneic sources (commonly rodent) and for improving their activation of the human immune system. Since the development of the hybridoma technology, a large number of rodent monoclonal antibodies with specificity for antigens of therapeutic interest have been generated and characterized. Rodent antibodies are highly immunogenic in humans, which limits their clinical applications, especially when repeated administration is required. Importantly, they are rapidly removed from circulation and can cause systemic inflammatory effects as well. As a means of circumventing these problems, we have developed three antibody humanization strategies that can preserve the specificity and affinity of the antibody toward the antigen whereas significantly or completely eliminate the immunogenicity of the antibody in humans. The first approach is CDR grafting and the second approach is chain shuffling. These two methods are all based on phage display of humanized scFv variants and selection of high-affinity humanized binders through bio-panning. The third method, humanized IgG library screening, is somehow unique. We will make a library of humanized whole IgG to be displayed on the surface of mammalian cells and then high-affinity binders will be sorted by FACS.
Creative Biolabs has established a solid platform for DNA sequencing of both IgG and IgM types of monoclonal antibodies produced by mouse and rat hybridoma cell lines. The sequences of a monoclonal antibody are important for patent protection and therapeutic approval.
Starting from a hybridoma cell line, we offer the following services:
Validation of the hybridoma cell line in terms of antibody production, antibody isotyping and antigen-binding specificity;
RNA extraction and reverse transcription;
Full length IgG or Fab sequencing by PCR amplification and subcloning of the variable domains.
Of note, hybridoma cells may contain pseudogenes and mRNAs encoding non-functional antibody chains, which might be accidentally amplified by the primer pairs intended to clone the target antibody sequences. Therefore, in a total RNA [as well as cDNA] pool of the hybridoma cells, there are other antibody-like sequences [e.g. other IgG] that can be amplified along with the sequences of the target antibody. In fact, PCR amplifications intended to clone coding sequences [cDNAs] of the mouse/rat monoclonal antibody will inevitably amplify some other antibody-like sequences. This is the key challenge in sequencing the cDNAs of a particular monoclonal antibody. In extreme cases, a small phage display scFv/Fab library is constructed for each monoclonal antibody, and then the right scFv/Fab clones are selected against the original antigen.
Note that, it is even harder to clone the right light chain. This important point is widely forgotten by our peers in the field. In order to make sure the cloned cDNA fragments are derived from the antigen-binding antibody, we usually express the sequenced scFv/Fab and validate its authenticity in an ELISA assay. Frequently, we even express the sequenced VL and VH in full IgG format to make sure the binding specificity and affinity is unchanged in comparison with the parental antibody. We will make our best effort to produce the conjugate you request. Optimal labeling must be determined empirically; and it is technically impossible to guarantee the yield and biological function of the final products. We refund payments for the failed services. Whatever labels you want to add to your protein, just let us know; in most cases we can accommodate your request.
Another feature of our service is that we always keep the original FR1 sequence since we do not design degenerate PCR primers matching this region.
For antibodies intended for therapeutic use, we also sequence the N-terminals of the heavy chain and the light chain using protein sequencing methods to confirm the cDNA sequences cloned from hybridoma cell lines.
Bispecific antibodies (BsAbs) are artificial proteins that contain two different binding specificities within a single molecule, which can specifically bind two different types of antigen.It consists of two heavy and two light chains, one each from two different antibodies. The two Fab regions (the arms) are directed against two antigens.The Fc region (the foot) is made up from the two heavy chains and forms the third binding site. A trifunctional antibody is a monoclonal antibody with binding sites for two different antigens, typically CD3 and a tumor antigen, making it a type of bispecific monoclonal antibody.
The approach offers many advantages over traditional antibody technologies:
• Targeting therapeutics to tissues or specific disease processes
• Mobilizing additional arms of the immune system to fight cancer or infection
• Crosslinking cell-surface receptors to invoke novel biology with powerful therapeutic potential
Creative Biolabs has developed CeativeAb® antibodies in order to increase the efficacy of current therapeutics, based on other antibody formats such as IgGs, Fabs and scFv. Our Creative Discovery PlatformTM provides bi-specific and tri-functional antibodies to satisfy various customers' needs.We also provide further screening and purification of the prepared antibodies.
• Chemically Cross-Linking Antibodies with SPDP.
• Chemically Cross-Linking Antibodies with NonreducibleThioether Bonds.
• Production of Hybrid Hybridoma-Derived Antibodies.
• Selecting Hybrid Hybridomas Using FACS.
• Screening Procedures for Bispecific Antibodies.
• Isolation of Bispecific Antibodies.
The value and potency of this platform is distinguished by the possibility of selecting specific targets and then choosing between different modes of action to create a therapeutic treatment approach for diseases with unmet medical need.
Together with Creative Diagnostics, Creative Biolabs has extensive experience in producing monoclonal antibodies in hamsters. The use of hamster as the host animal offers a unique species alternative in areas where mouse and rat antibodies may not work well. We can employ up to 10 hamsters for each antigen. Immune responses are elicited using immunogens such as transfected cell lines, DNA, recombinant proteins, native proteins, peptides or haptens conjugated to carrier molecules.
We perform splenocyte fusion on one or more animals. We select and subclone the strongly positive clones from the parental cultures by 2 rounds of limiting dilution cloning. Stable clones will be expanded into 24-well plates and then into T-flasks for further characterization. At no additional cost, client could select one clone for in vitro production of approximately 1L of supernantant. Supernatants will be purified using Protein A/G column and pure antibodies will be deliverable. We also offer a full range of contract manufacturing services, including In vitro Antibody and Protein Production in bioreactors and large-scale Antibody Purification.
Creative Biolabs have established a unique platform to develop high-affinity monoclonal antibodies in rats. Compared with commonly used mouse antibodies, rat antibodies do not have the background cross-reaction problems in immune-detection of antigens out of a mouse background, such as a mouse antigen from a mouse animal model. However, Rat monoclonal antibodies are much more difficult to develop compared with mouse monoclonal antibodies, due to the lack of a stable fusion partner cell line for rat hybridoma generation.
Together with Creative Diagnostics, Creative Biolabs has developed a proprietary hybridoma-generation platform for efficient development of high-affinity and high-specificity rat monoclonal antibodies. Our Rat Monoclonal Antibody platform allows us to offer the most comprehensive one-stop custom antibody development services in the industry.
Creative Biolabs provide rat and mouse monoclonal antibodies construction services. In particular, our proprietary mouse immunization approach allows us to provide mouse monoclonal antibodies within 70 days.
To generate monoclonal antibodies that fit your specific purposes, we tailor our protocols in every major step of antibody production, including antigen preparation [peptide synthesis or protein expression in E.coli, yeast, insect or mammalian cells], animal immunization and hybridoma screening.
Creative Biolabs has accumulated extensive experiences in IHC-Positive Hybridoma Screening. For antibodies intended for immunohistochemical [or immunocytochemical] staining, in addition to the native immunogens, e.g. recombinant protein, we may alternatively use denatured antigens in animal immunization and hybridoma screening. Our theory is that antigens to be stained in IHC are denatured [completely or partially], altered [by cross-linking] or precipitated by paraffin embedding and various fixations. We may use fixative-treated immunogens in animal immunization. In the end, we perform hybridoma screening with IHC using paraffin-embedded slides or tissue microarrays at extra charges. Customer may provide the slides or Tissue Arrays; otherwise, we may provide the slides or arrays at customers' expense.
Creative BioLabs offers high-throughput phage display library screening services for selection of receptor-dependent antibodies and peptides that can be internalized into mammalian cells. Internalizing antibodies and peptides specific for cell surface receptors, which upon binding will induce receptor-mediated endocytosis, are highly suitable vehicles for targeted delivery of drugs, toxins, enzymes or DNA into the cytosol of mammalian cells for therapeutic applications. Compared with receptor-independent antibodies and peptides that can penetrate cell membrane without binding to a receptor, receptor-mediated internalizing antibodies and peptides confer cell-specific drug delivery, the fundamental requirement for targeted therapy. Selection of receptor-mediated internalizing antibodies and peptides from phage display library can also be employed to identify cell-specific markers, the endocytosed receptors that are associated with a specific cellular function.
For most soluble protein targets, the antibodies are generated by inoculating animals with purified, full-length protein. Unfortunately, this approach is not effective for most multiple-spanning membrane proteins, whose native structures normally depend on an intact lipid bilayer.
G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, represent a large and important group of proteins involved in many diseases, and are also the target of around half of all modern medicinal drugs.Unfortunately, it is extremely difficult to create antibodies against GPCRs using traditional approaches. Solubilization of the membrane protein immunogens with detergents may lead to major conformational changes; the epitopes are made up of small discontinuous segments; moreover, these proteins in general are expressed at very low levels and it is difficult to generate sufficient material for immunization. Production of GPCRs antibodies are still challenges to overcome.
Creative Biolabs has been pioneered in the Antibody Technology for a long time. After years of extensive optimization, scientist from Creative Biolabs had built up a specific method of producing GPCRs and other membrane protein antibodies. Upon using genomic antibody and engineering technology in each step, including: antigen design, appropriate expression of the antigen, targeting the appropriate immune cells, and use of the right adjuvants. Based on our customers’ need, we would carefully optimized and continue to improve each of these steps to ensure success.
Together with Creative Diagostics, we have developed proprietary MPATTM platform for multi-spanner protein targets. Our antibody platform also enables us to screen in vivo functional antibodies to membrane protein targets.
A domain antibody or sdAb is an antibody fragment consisting of a single monomeric variable antibody domain, and lacking the light chain and CH1 domain of the heavy chain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, nanobody are much smaller than common antibodies (150–160 kDa), and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa). The first single domain antibody were engineered from heavy-chain antibodies found in camelids; these are also called VHH fragments.
Creative Biolabs is the recognized leader in nanobody production and single domain antibody library fields. We are specialize in generating nanobodies against any targets. We also provide a wide range of services, including construction of immunized single domain antibody libraries using llama and camel, construction of synthetic camelised human single domain antibody libraries using DNA synthesis, bio-panning of single domain antibodies libraries and large scale production of recombinant single domain antibodies.
Creative Biolabs offers first class custom PEGylation services that are based on next-generation PEGylation technologies.
PEGylation is the process of covalently attaching one or more chains of PEG [Poly(ethylene glycol)], also known as poly(ethylene oxide) (PEO) or polyoxyethylene (POE)], to a protein molecule, thus creating a PEG-conjugated protein. Therapeutic proteins are either rapidly degraded, have a short circulating life, or are rapidly excreted when introduced into the bloodstream. Consequently, many protein-based biopharmaceuticals require repeated intravenous infusions for therapeutic efficacy. Also, repeated injections of a protein-based biopharmaceutical can result in adverse immunological responses and side effects. As the single proven solution to all these problems, PEGylation has become the biopharmaceutical delivery technology of choice for intravenously administered therapeutic proteins. Covalent attachment of PEG to a therapeutic protein can "mask" the protein from the host's immune system (thus, reduce immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance. These benefits allow significantly less protein (as compared with the un-PEGylated protein) to be administered and yet achieve the same desired therapeutic effect. In addition, a reduction in the amount of protein used in each dose results in lower raw material and production costs. For these reasons, PEGylation becomes mandatory to think about when developing a biopharmaceutical drug. To Date, more than ten PEGylated protein drugs have been approved by FDA and another tens of PEGylated proteins are in clinical trails.
Scientists from Creative Biolabs are professional in construction of various phage display libraries, including cDNA libraries, peptide libraries and scFv/Fab antibody libraries. We normally generate libraries with a diversity of n×108; we are able to generate libraries of n×109-10. In case you need a special cDNA, peptide or antibody library, you can rely on our extensive experience. For peptide libraries with inserts longer than 8-mer [and up to 30-mer], we usually use one of three nucleotide doping strategies [NNK, Split-mix-split synthesis or Tri-mer codon] to maximize the amino acid sequences encoded by the inserted DNA sequences. We can make the both pIII-fusion libraries and pVIII-fusion libraries. For scFv/Fab library construction, we can use both human plasma cells and splenocytes from naïve or immunized mice. We are also professional in producing fully or semi-synthetic antibody libraries.
Our staff scientists have extensive experience in screening of phage display peptide, cDNA and scFv/Fab libraries. In particular, by conducting library screening, panning, for 4 cycles, we normally get scFv/Fab antibodies of an affinity of 10-7. By constructing serial sub-libraries of the isolated scFv/Fab antibodies, our proprietary protocol allows increase of the affinity of the scFV antibodies from 10-8 to 10-9. We have successfully obtained a scFV antibody that has an extremely high affinity of 10-12, whose binding to the antigen is essentially irreversible.
Creative Biolabs develops and commercializes a full range of integrated innovative Bio-fishing services that are based on phage display technology. We have extensive experience in construction of various phage display libraries, including cDNA, peptide and scFv/Fab antibody libraries. We are also professional in screening these libraries against protein, peptide, cells, special surfaces and chemicals. Affinity maturation based on sub-library construction and screening is readily available. In addition, we have built up a series of peptide, human and mouse scFv/Fab phage display libraries that all have a great diversity. We also have a camelised human single domain antibody library constructed. Furthermore, bio-manufacturing of small antibodies in bacterial systems and production of full-size humanized IgG in gene-engineered CHO cells have brought us international recognition. Our Bio-fishing platform enables rapid and effective discovery and efficient production of biological molecules that can provide opportunities for therapeutics, diagnostics and targeting agents.
The process of producing antibodies against a phospho-residue is more complicated than traditional antibody production using peptide immunogens. Phospho-specific antibodies are generated using peptides containing one or more phosphorylated amino acids. There are three residues which can be phosphorylated: Serine (S), Threonine (T) and Tyrosine (Y).
For phospho polyclonal antibody production:
To produce antiserum against Phospho-Peptides includes synthesis of phosphopeptides, conjugation and immunization of rabbits. In many cases, one would need to do affinity purification with a phospho-peptide column. Sometimes, one would also need to cross absorb the antibody with a non-phosphopeptide column in case there are some anti-non-phospho protein antibodies in the antiserum. In this case, synthesis of matching non-phospho peptides is required to make the negative-selection columns. Affinity purified, cross-absorbed polyclonal antibodies that are specific for the phosphor-peptides are usually required for downstream assays.
For phospho monoclonal antibody production:
In comparison with polyclonal antibody production, monoclonal antibody production against Phospho-Peptides is more straightforward; we just use the Phospho-Peptides to immunize the mice, and use Phospho-Peptides to screen for positive hybridoma clones. After that we use non-phospho-peptides to do negative selection. This negative selection is required [although widely forgotten] since peptide phosphorylation [or protein phosphorylation] is never 100% complete.
Creative Biolabs provides a full range of services in custom polyclonal and monoclonal antibody production from gene expression or peptide synthesis to antibody purification and labeling. We have the capacity of producing antibodies in either research quantities or in large scale for diagnostic industry. We do epitope prediction, peptide synthesize, protein expression, polyclonal or monoclonal antibody production as well as antibody purification from cell culture, ascites fluid or whole serum. We also prepare mono-specific polyclonal antibodies by ligand affinity chromatography. Importantly, we can produce monoclonal antibodies within 70 days. Creative Biolabs also has the expertise in handling antigen insufficiency and targeting very impure antigens.
Creative Biolabs has established standard operating procedures in selectively identifying antibodies that bind not only to immunogens but also to the corresponding target antigens in ELISA, Western blotting, Immuno-precipitation, Immuno-fluorescence and Immunohistochemistry. Frequently, polyclonal or monoclonal antibodies raised using peptides and recombinant proteins can not recognize the target antigens, e.g. a protein antigen in tissue, and are essentially useless. To generate antibodies that recognize a native antigen, we employ or predict the 3-D structure of the target protein and design peptide immunogens that display on the protein surface and contain linear continuous epitopes. If a recombinant protein immunogen is used, we usually express it in a soluble form to retrieve the immunogenic properties of its native counterpart. Finally, we use our well-tested ELISA, Western blotting, IP, IF and IHC screening protocols to validate (monoclonal) antibodies that recognize the natural target antigen.
Phage display peptide libraries that display random 5-20 mer peptides have been widely used to determine protease substrate [sequence] specificity for a large number of enzymes and semi-purified cell extracts. This technique has been playing a critical role in designing inhibitors and activators of proteases.
In comparison with phage display peptide libraries used in other fields, phage display peptide libraries used to identify the preferred substrate sequences of a protease usually express a fusion tag at the N-terminus of the peptide sequences, while, like in regular phage display peptide libraries, the minor coat viral protein pIII is located at the C-terminus of the peptide sequences. The N-terminal fusion tag is intended to bind the phage particles to a solid support. We have designed and built up a unique tag, HiAffi-tag, which is derived from Protein G, thus has strong binding affinity to most IgG coated on a solid surface. In contrast to His Tag and Strepavidin Binding Motifs used in such libraries, HiAffi-tag enables the best capture of the phage particles to the solid support and the least steric hindrance for proteolysis of the substrate peptides.
During library screening against a particular protease, phage particles that express sensitive peptide sequences are cleaved off the solid support. After that, the free phages are amplified, immobilized and subjected to protease cleavage again. After several rounds of screening, selected phage clones are DNA sequenced and common features of the peptide sequences are determined. These peptide sequences are good candidates for further identification of the substrates of the protease.
We have also developed proprietary in silicon screening and computer aided rational design methods to use such peptide sequences, which must fit into the active site of the protease, as leads to develop inhibitors and activators of a protease.
Creative BioLabs has established to a unique ribosome display platform that is based on proprietary prokaryotic and eukaryotic ribosome display technologies. The ribosome display platform is a superior in vitro cell-free platform that generates peptide, protein and antibody libraries with an unparalleled diversity up to 1015; as a result, extremely high-affinity binders against a variety of targets can be isolated from the libraries. In particular, our ribosome display antibody libraries can produce antibodies with pM affinity, the highest affinity ever achieved by an antibody production technology.
Ribosome Display Libraries Construction
Creative BioLabs is a leading company in construction of various ribosome display libraries. We are able to generate libraries with a diversity of n*1013-15. Following libraries are frequently made:
• Immune animal libraries • Combinatorial libraries • Random peptide libraries • Constrained flexible libraries Ribosome Display Library Screening
Our staff scientists have extensive experience in screening both prokaryotic and eukaryotic ribosome display libraries. We have developed a robust process for optimization of a full range of ribosome display library screening technologies.
Creative BioLabs has built up a novel intrabody generation platform, SuperTM Intrabody Platform, that is based on an innovative combination of Yeast Two-Hybrid System with antibody library technology.
Intrabodies are antibodiesthat work within cells to bind to intracellular antigens. They are directed against intracellular target molecules and expressed within a specific subcellular compartment as directed by the intracellular localization signals genetically fused to N- or C-terminus. For this reason, intrabodies are defined as antibodies that have been modified for intracellular localization. Because antibodies ordinarily are designed to be secreted from cells, intrabodies require special alterations, including modification of sequences for hyperstability,selection of antibodies resistant to the more reducing intracellular environment, or expression as a fusion protein with maltose binding protein or other stable intracellular proteins. Intrabodies have wide applications in dissecting target protein function, in target validation and functional genomics, as well as acting as potential therapeutic reagents. Intrabodieshave promising applicationsagainst hepatitis B, avian influenza, prion diseases,inflammation, Parkinson's disease, and Huntington's disease.
SuperTMIntrabody Platform developsintrabodiesin the form of single domain antibody, although human antibodies in scFv format have been developed as first generation of intrabodies. Single domain antibodies [also called, sdAb, VHH antibody, domain antibody or nanobody] are antibody fragments consisting of a single monomeric variable antibody domain. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies (150–160 kDa), and even smaller than Fab fragments (~50 kDa) and single-chain variable fragments (~25 kDa).
We have created a large single domain llama antibody library using Matchmaker™ Gold Yeast Two-Hybrid System [Clontech], a GAL4-based two-hybrid assay, in which an antigen protein is expressed as a fusion to the Gal4 DNA-binding domain (DNA-BD), while single domain antibodies engineered from heavy-chain antibodies found in camelids are expressed as fusions to the Gal4 activation domain (AD). As a result, when antigen (bait) and antibody library (prey) fusion proteins interact, the DNA-BD and AD are brought into proximity to activate transcription of reporter genes, allowing the discovery of high-affinity single domain antibodies specific for the antigen.
Since the antibodies discovered on this platform are expressed and selected inside of yeast cells, there are the best intrabodies in the first place that do not require further sequence modification. Also, this platform does not require antigen in a purified protein form to raise antibodies. Instead, the gene of the antigen is expressed into an intracellular protein to capture its antibodies in the yeast cells. As a result, intrabodies discovered from this platform usually recognize the native conformation of the antigen and are of immediate therapeutic potential.
UltraTM Yeast Display Technology
Creative BioLabs offers a unique yeast display technology UltraTM, for the discovery and optimization of antibodies with fM affinity, the highest affinity ever enabled by an in vitro antibody production technology.
Yeast display (or yeast surface display or YDP) is a protein engineering technique, by which libraries of polypeptides are displayed on the surface of yeast cells and high-affinity protein binders are selected by screening the libraries against a target using flow cytometry. UltraTM is based on a proprietary synthetic yeast display human antibody library and a unique selection process natural to yeast. This yeast surface display technology enables the rapid isolation of target-specific antibodies without the labor intensive screening common to other recombinant and non-recombinant antibody production methods, resulting in unique single-chain variable fragment (scFv) antibodies that have both high specificity and extremely high affinity [Kd up to 10-15] for the target antigen.
UltraTM human scFv antibody library is created by combining a highly diverse collection of synthetically-constructed randomized CDR sequences that are further diversified by random lengths using a unique proprietary technique. The large library has been specifically optimized to eliminate unwanted stop codons and aggregation-prone sequences. scFv molecules are first expressed in yeast cytoplasm and then translocated and anchored onto the yeast plasma membrane. In the end, binder panels are selected by FACS or panning. Furthermore, multiple rounds of affinity maturation during library screening are incorporated through error prone PCR mutagenesis either directed at CDR or flanking sequences and selection by varying antigen dose.
The UltraTM platform utilizes a unique antibody selection process that relies on the natural protein translocation system in yeast. The major limitation challenging conventional phage display technologies is their dependence on the Sec translocation pathway. The Sec pathway transports proteins from the cytoplasm to the periplasm in an unfolded state; consequently, proteins that require a cytoplasmic environment and/or cytoplasmic components for folding, or reach their native state before they interact with the Sec proteins, are not compatible with the Sec pathway. In contrast to phage display systems, the UltraTM platform offers the advantage of post-translational modifications and processing of mammalian proteins and, therefore is well suited for expression of human derived antibody fragments. In addition, the platform offers the advantage of protein folding pathways and codon usage that are closely related to mammalian cells, thereby eliminating library biases that might be encountered in phage or bacterial display.
UltraTM yeast display library construction and screening
• Immunized antibody libraries
• Naive antibody libraries
• scFv/Fab antibody libraries
• Semi-synthetic/synthetic antibody libraries
Applications of UltraTM yeast display platform
• De Novo Human Antibody Discovery
• Antibody Humanization
• Peptide Screening
• Protein Interaction
Y2H Library Screening
Creative BioLabs provides a full package of yeast two-hybrid (Y2H) library screening services for quick identification of novel binding partners for membrane or soluble proteins. Y2H screening services are also offered to map binding domains between two proteins.
We provide the services on a strict fee-for-service basis. We are professional in using Gal4 and LexA based Y2H systems. We are able to perform yeast two hybrid screening against integral membrane proteins and membrane-associated/proximal proteins using a modified split-ubiquitin membrane yeast two-hybrid (MYTH) system. We have extensive experience in handling Y2H systems marketed by Invitrogen, Clontech, Stratagene and OriGene. Also, a large number of pre-made Y2H cDNA libraries from these recognized companies are available and free to our customers.
Our Y2H library screening service can be divided into four phases. Phase I is bait vector construction and Phase II is toxicity and self-activation test. The screening will be terminated if the bait gene is toxic to yeast or it is self-activated in yeast. Phase III is large scale library screening. Greater than 5,000,000 clones will be screened for their ability to interact with the bait and potential interactors will be identified. Importantly, we perform phenotypic validation test at Phase IV. To eliminate the possibility that the reporter phenotypes are caused by spontaneous yeast mutations or a mixture of preys during the screen, up to 20 potential interactors are isolated from yeast. The interactor plasmids are then re-transformed individually to yeast strains that express the original bait or unrelated baits to further validate the interactions. This assay is one of the most critical steps for validating specific protein-protein interactions. We also provide an optional service for interaction validation by co-immunoprecipitation in mammalian cells (phase V).
Following the screening experiments, professional Bioinformatics data analysis is available. Integrated methods are used in performing protein interaction data analysis, which including multiple sequence alignment, protein interaction database survey, global analysis of SID, pathway analysis and gene ontology analysis.
Y2H Library Construction
Creative BioLabs also offers quality yeast two-hybrid library construction services. cDNA library or genomic DNA library (for bacteria) construction is an integrated part of our all-in-one yeast two hybrid platform. If there is a regular cDNA library [particular normalized one] or genomic DNA library available, we can take the library, extract the DNA inserts and then subclone them into a Y2H vector. Alternatively, we can start from total RNA or mRNA for cDNA library construction. We reverse-transcribe the mRNAs into cDNAs and clone them into a proper vector. The primary library will have at least 3 x 106primary variant with average insert length 0.5-3kb, which should be far more enough to represent every gene for most organisms.
Y3H Library Screening
Creative BioLabs uses Clontech pBridge Three-Hybrid Vector in combination with its Matchmaker Yeast Two-Hybrid System to offer yeast three-hybrid [Y3H] library screening services.
Y1H Library Screening
Using Clontech's Matchmaker Gold Yeast One-Hybrid (Y1H) Library Screening System, Creative Biolabs also provide quality services for identifying and characterizing novel protein-DNA interactions. The system employs Aureobasidin A (AbA; 1) selection, which provides the most stringent yeast one-hybrid screening strategy available.
We provide both rat and mouse monoclonal antibodies. In particular, our proprietary mouse immunization approach allows us to provide mouse monoclonal antibodies within 70 days.
To generate monoclonal antibodies that fit your specific purposes, we tailor our protocols in every major step of antibody production, including antigen preparation [peptide synthesis or protein expression in E.coli, yeast, insect or mammalian cells], animal immunization and hybridoma screening.
Besides the full range of phage display antibody library construction and screening services, we are also recognized by our downstream services in antibody engineering and antibody biomanufacturing. We have built up our expertise in antibody engineering based around monoclonal antibodies derived from both hybridoma technology and phage display antibody library technology.
Creative BioLabs together with partners who are leading scientists in monoclonal chicken IgY antibody field offers the most recognized custom monoclonal chicken IgY antibody production services. Chicken IgY antibodies are avian equivalents of mammalian IgG antibodies. Compared to mammalian IgG antibodies, chicken IgY antibodies have higher avidity, less cross-reactivity and higher specificity, rendering chicken IgY antibodies more valuable in laboratory experiments and diagnostic assays.
In contrast to rabbit hybridoma technology available in other leading companies, Creative BioLabs has built up a unique rabbit monoclonal antibody production platform that is based on our unparalleled expertise in phage display antibody library technology. We generate rabbit antibody libraries and select high-affinity antigen-specific antibodies by phage display following Dr. Christoph Rader's methodology. Usually, two or more rabbits of the b9 allotype are immunized with one antigen, total RNA from spleen lymphocytes and bone marrow cells are purified, and then the first-strand cDNAs are synthesized. cDNA from spleen and bone marrow samples derived from multiple groups of animals immunized by different antigens can be pooled to make a unified library for all the antigens. Chimeric rabbit/human Fab libraries are made in our pC Display phagemid vectors by fusing rabbit Vκ/Vλ to human Cκ and rabbit VH to human CH1 of human IgG1.
To create stable cell lines is a key method in reducing the production costs of biopharmaceuticals. We use a modified FRT/Flp-In recombinase system to produce human proteins in CHO cells and 293 cells for stable expression. We have pre-selected CHO cell lines [and 293 cell lines] that contain a single stably integrated FRT site at a transcriptionally active genomic locus and we have proven that they can produce high level expressers of recombinant proteins.
We have also developed a unique series of high-yield vector system, in which two GC-rich regions flanking the gene expression cassette makes the promoter and enhancer continuously exposed to transcriptional machinery by preventing chromatin condensing. This revolutionary ground-breaking technology allows the available highest yields in all common mammalian cell protein production host cell lines, such as 293, CHO and NSO. Antibody yields of up to 2.0- 5.5 grams/liter [80-120pg/cell/day] have been achieved using this system in chemically defined, animal component-free medium. The selection strategy has been designed to identify cell lines that are highly productive and have the ability to grow well in suspension culture in a state-of-the-art, fed-batch fermentation process, in which cell density can reach 1.5×107/mL.
We are a recognized service provider in converting small gene-engineered scFv or Fab human/mouse antibodies derived from phage display antibody library screening or mouse/rat hybridoma cell lines into full-size recombinant human or mouse IgGs with Fc fragments of various functions, e.g. ADCC, or non-ADCC. We have established a solid platform for industrial scale production of recombinant IgG in mammalian cells.
Of note, to mimic the behavior of a recombinant human IgG antibody [targeting a human protein] in humans the best, specificity-matching recombinant mouse IgG antibodies derived from phage display mouse scFv/Fab libraries [targeting the mouse protein] are frequently produced and used in animal models.
We use a modified FRT/Flp-In recombinase system to produce human IgG in CHO cells for stable expression of humanized/recombinant human IgG. The same approach is used in stable expression of recombinant mouse IgG. We have pre-selected CHO cell lines that contain a single stably integrated FRT site at a transcriptionally active genomic locus and we have proven that they can produce high level expressers of recombinant human IgGs [targeting various antigens]. Unfortunately, even starting from these pre-selected cell lines, significant amount of time and effort are required to create a stable cell line that can express recombinant human [mouse] IgG at a cost-effective level, e.g. 800mg/L.
In the end of production, the recombinant antibody will be three-step purified (protein A/G followed by two polishing steps, antibody monomer >99%, endotoxin level ‹0.5 EU/mg antibody, protein A residual ‹ 10 PPM, which is equivalent to antibody for human use). Here, we do not disclose our process technology. We also do not provide the stable cell lines. However, the cell lines will be archived for a period of 2 years free of charge to client in case future production is needed.
Isothermal Titration Calorimetry (ITC) Services
Creative Biolabs offers Isothermal Titration Calorimetry (ITC) Services, which provide incomparable sensitivity with high quality binding data for biomolecular interactions of interest. Our ITC services have been used extensively in studying macromolecule interactions, with studies looking at antibody-antigen, protein-protein, protein-ligand, DNA-ligand and RNA-macromolecule studies.
In biology, particularly in studies relating the structure of macromolecules to their functions, two of the most important questions are (i) how tightly does a small molecule bind to a specific interaction site and (ii) if the molecule is a substrate and is converted to a product, how fast does the reaction take place? ITC is a quantitative technique that can determine the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution. ITC is now routinely used to directly characterize the thermodynamics of macromolecule binding interactions and the kinetics of enzyme-catalyzed reactions.
ITC has advantages over other techniques such as fluorescence assays, NMR and SPR for studying complex formation in terms of ease of use and cost. It does not require any fluorescent probes or radioactive tags for data analysis. Immobilization and chemical modification of protein is not required. Also, it does not have limitations associated with clarity of the solution, molecular weight, temperature or pH. It is one of the best methods for determining the thermodynamic parameters of ligand binding.
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For label-free interaction measurements, Creative BioLabs provide Surface Plasmon Resonance (SPR) service based on our unique ProteOn™ XPR36 protein interaction array system. Creative Biolabs have extensive experience in providing SPR services. You can count on us to assist you all through your project.
SPR is an optical phenomenon which allows monitoring binding of any two unlabeled molecules in real time. The SPR signal is based on changes in the refractive index at the surface of a gold sensor chip as an analyte flows in a microfluidic channel and binds to a ligand immobilized on the sensor chip. Monitoring the changes in the SPR signal over time results in a plot of the binding response versus time called sensogram. Fitting of the sensogram data allows calculation of binding parameters such as KD, Kon and Koff. On top of the kinetic constants, SPR measurements can be applied for
Determination of specificity
Studying thermodynamics of interaction
Investigating mechanism of action
Epitope characterization or affinity screening
Our ProteOn™ XPR36 system is a unique application of SPR that uses enhanced microfluidics and a 6 × 6 interaction array on the sensor chip to characterize the binding of multiple analytes to multiple ligands, in a single step, on a single chip. It means that Proteon allows to measure 36 interactions at the same time, which saves not only time, but also your sample.
Experienced staff at Creative Biolabs will help you all through your project, including method development, validation, analysis of delivered samples and transfer to the client, upon request. We can provide ready-to-publish or ready-to file data in any format required. Please see below for a list of services we provide.
Label-free interaction studies:
Protein - protein interactions for biological complexes
• Kinetic parameters
• Mechanism of action
• Competition assays
Protein -DNA interactions
Low molecular weight compounds with receptors and many others according to the client’s needs
Service dedicated for comparability studies of biosimilars:
Binding to the recombinant target
Binding to Fcγ receptors (FcγRI, FcγRII, FcγRIII) and neonatal Fc receptor
Differential Scanning Calorimetry (DSC) Services
Creative Biolabs is a recognized service provider in Differential Scanning Calorimetry (DSC). DSC is a powerful analytical tool for characterizing the stability of proteins and other biomolecules directly in its native form with the aid of suitable thermodynamic models.
DSC technique is very convenient requiring only minimal assay development, NO labeling or immobilization, and permits screening of large numbers of samples at low concentrations minimizing the consumption of valuable samples. It does this by measuring the heat change associated with the molecule’s thermal denaturation when heated at a constant rate. The resulting data provide critical guidance for biopharmaceutical development, from protein engineering, through pre-formulation and process development, to formulation of the final product, driving productivity in biopharmaceutical research. We have reference samples meeting your requirement which are well-defined heat capacity over the range of temperatures to be scanned.
Key services we can supply include:
• Monitoring protein affinity, specificity and activity.
• Observing protein fusion and crystallization.
• Characterization and selection of the most stable proteins or potential candidates in biotherapeutic development and formulation.
• Studying protein-ligand interactions.
• Rapid optimization of purification and manufacturing conditions.
• Quick stability assays for target proteins to be used for screening.
Mutant Library Construction
In vitro molecular optimization is a very efficient means of generating mutant proteins with improved or novel properties, identifying regulatory sequences, and probing for structurally and functionally critical residues. Mutant libraries constructed using the in vitro molecular optimization method provide one useful approach to the systematic study of protein properties, regulation, and function.
Creative BioLabs's strong expertise in de novo gene synthesis allows us to synthesize complex protein libraries without any dramatic increase in cost. Our mutant library services include site-directed mutagenesis libraries, sequential permutation scanning libraries, and randomized and degenerated libraries.
Site-Directed Mutagenesis Libraries
Creative BioLabs combines its expertise in de novo gene synthesis and site-directed mutagenesis into an excellent site-directed mutagenesis library construction service. The site-directed mutagenesis library offers a great platform for protein function and active center studies. In these libraries, any given residue can be substituted with any of other 19 common amino acids, creating systematic combinations of amino acid mutations that reveal any significant pattern.
Scanning Point Mutation Libraries
Scanning point mutation is a systematic means of improving protein performance. It outperforms standard alanine/cysteine scanning by replacing each amino acid with all 20 amino acids simultaneously. This technique provides a detailed profile of each amino acid at the position. For each codon of interest, a small, site-saturated library is constructed. This library can be delivered as a pool or in a separated format for any substitution variant (19 in total). The application of Creative BioLabs's expertise in de novo gene synthesis to the field of sequential permutation scanning allows us to provide superb sequential permutation scanning library construction services.
The key point in protein array technology is that arrayed proteins should be correctly folded, functional and of the greatest flexibility for experimental design. For this purpose, the proteins spotted on our arrays were all expressed by yeast cells in a secreted form and purified under native conditions. These protein arrays support a broad range of applications and enable rapid profiling of thousands of biochemical interactions. Highly sensitive and reproducible results are routinely obtained by our staff scientists during custom services. In addition, comprehensive Bioinformatics support including GO analysis and Pathway analysis is included in our custom protein array services.
Randomized and Degenerated Libraries
With our advanced degenerated oligonucleotide techniques, Creative BioLabs can generate any form of randomization or degeneration of the full-length gene in a synthetic DNA fragment. This permits controlled, highly precise randomization within oligonucleotides.
Creative BioLabs's in vitro library synthesis technology can introduce random substitutions on a controlled level with maximum flexibility. The mutation frequency can be set to any value between 1 and 20 mutations per kb. A peer group of 48, 96, or 192 individual transformants is sequence-verified.
Truncation technology incrementally trims genes around a defined core region. Genes can be truncated from both the 5' and the 3' ends simultaneously to produce an incremental truncation library. Unlike other methods, our process preserves the open reading frame, avoiding out-of- frame mutations.
• Optimize solubility
• Evaluate minimal functional-protein size
• Identify functional domains
• Screen for inhibitors
• Map epitopes
• More functional variants
• No system-based out-of-frame mutations
• Low ancillary mutation rates—>90% sequence integrity (depending on length of the gene;
• unintended mutants will have amino acid substitutions in most cases)
• High success rates—receive all possible truncation variants; library diversity of up to ~40,000
Anti-drug Antibodies (ADA) Assays
Creative Biolabs provides one-stop, customized and optimized detection and characterization assays for Anti-drug Antibodies (ADA) of new drug candidates. Depending on the molecular and structural characteristics of the active ingredients, the according adverse immunological reactions may vary widely. Therefore, our experienced scientists ensure that, the detection and characterization assays for ADA are 100% absolutely developed, customized and optimized for each drug.
Our customized Anti-Drug Antibody assay development & validation service includes:
Positive control formation - polyclonal or monoclonal antibody production against the active ingredients
Assay development & validation - “bridging”, direct or competitive ELISA development & cytokine profile & sensitivity, specificity and precision evaluation in compliance with FDA requirement
Confirmation assay (characterization and titration)
Characterization assay (class/isotypes of antibodies, neutralizing yes/no)
Possible changes in the PK that might be correlated with the effects induced by ADA (the pharmacokinetics (PK) of the active ingredients is available).
Figure: Bridging ELISA for ADA testing (Daniel Kramer, 2013, Open Scientific EIP Symposium München)
Immunogenicity Safety Studies are mandatory in pre-clinical and clinical development of biopharmaceuticals. It is well known that the Anti-Drug Antibodies (ADA) may induce undesirable side effects, especially in biopharmaceuticals, such as therapeutic antibodies and growth factors. With more than ten years experience in providing excellent services for biopharmaceutical industrials, Creative Biolabs is experienced, professional and reliable in ADA assay development and validation to accelerate the advancement of your new drug, from preclinical stage to clinical, and eventually to commercial market.
Epitope Mapping by X-ray Crystallography
The gold standard approach, also the most unambiguous method for epitope mapping, is X-ray co-crystallography. X-ray co-crystallography allows mapping of both linear and conformational epitopes. By co-crystallizing the monoclonal antibody with its respective antigen, the epitope can be defined immediately with high accuracy.
Staffed by dedicated scientists with combined expertise in antibody engineering and protein X-ray crystallography, Creative Biolabs have extensive experience in crystallizing antibody/antigen complexes and resolving their 3D structures. In particular, we are able to map the epitopes of a large variety of antibody forms, including intact IgG, Fab, scFv, F(ab')2, diabody, minibody, miniantibody, tandem scFv and single domain antibody as well as protein A scaffold.
Please refer to our website at Creative Biolabs for more information regarding resolving the structure of an antigen/antibody complex.
Structure of APS Kinase co - crystallized with ligands ADP and APS.
Creative Biolabs provides monoclonal antibodies sequencing services in a variety of species, offering full sequence coverage of the antibodies with 100% accuracy. Functional biosimilar monoclonal antibodies can be sequenced by subunit with 100% coverage as well as excellent precision based on our world’s number one platform of de novo antibody sequencing using our proprietary “Database Assisted Shotgun Sequencing" (DASS) technology, all of which have been proven with abundant successful cases.
Creative Biolabs can supply you the services on bio-manufacturing with traditional or novel bioreactors, both of them are certificated and licensed. A novel but certified bioreactor improves the traditional ones through oxygen transfer method. The new designed bioreactor can eliminate the blades which are caused by rotating blades and air sparging, and it can attract oxygen from ambient air efficiently. After process development, large scale fermentation or called cGMP manufacturing will be involved.
Creative Biolabs provides Custom Cell Lysate Service to meet your scientific research needs. Our cell lysates with excellent quality can be used as assay standards in Western blot, ELISA and a series of bio-analytical assays.
Creative Biolabs has established a proprietary platform for cell lysate preparation with very stable quality and a fast turnaround time. We can provide cell lysates at very high concentration of up to 20 mg/mL with affordable prices, which are far more cost-effective than cell lysates from other resources. Scale-up production is also available.
Creative Biolabs offers Dual-polarization interferometry (DPI) Services, which provide exquisite sensitivity to give previously unavailable insights into the structural changes taking place in molecular systems as they function and interact. DPI resolves protein structure to subatomic dimensions in real time and is typically used to characterize biochemical interactions by quantifying any conformational change at the same time as measuring reaction rates, affinities and thermodynamics.
Creative Biolabs is the recognized leader in single domain antibody library fields. A wide range of unique services are available, including construction of immunized single domain antibody libraries using llama and camel, construction of synthetic camelised human single domain antibody libraries using DNA synthesis, bio-panning of single domain antibodies libraries and large scale production of recombinant single domain antibodies.
As an industry leading expert in antibody engineering and bio-pharmaceuticals, Creative Biolabs is committed to developing new generation of pharmaceuticals against various diseases for both scientific and clinical purposes. Antibody-drug conjugates (ADCs) are a class of novel APIs with high potency. Our experienced scientists and technicians can provide comprehensive custom services to develop numerous ADC products for every stage of development, from antibody expression to cGMP manufacturing.
TCR Transgene Expression Assay
Lymphocyte Culture and Proliferation Assay
Cytokines Release Assay
TCR/CD3 Stability Assay
Lymphocyte Antigen Reactivity and Cytotoxicity
Creative Biolabs offers a series of assay services for TCR validation. In order to obtain high quality TCR-modified T cells prior to scientific studies, preclinical researches and clinical trials, a serial assays related to TCR construction are needed. Transgene expression, cytokines release, TCR/CD3ζ stability etc. are performed so as to evaluate the TCR gene transfection efficiency and the reactivity of engineered lymphocytes to antigen. With advanced technologies and experienced researchers, scientists from Creative Biolabs can design and operate a full range of validation assay services according to your T cells engineered objective.
Our TCR validation services include but are not limited to:
➤ TCR transgene expression assay
Ideal gene transfer means that exogenous DNA can be incorporated into host genome and stable long-term expression. To ensure the best outcome, the expression level of TCR gene should be tested at different stages during the whole process of TCR engineered T cell therapy, including post-transfection with TCR gene, cell preparation and clinical treatment period. Generally, the methods include western blot (WB), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), immune-histochemical (IHC), etc.
➤ Lymphocyte culture and proliferation assay
The failure of T cell proliferation and persistence in response to tumors is one of the reasons that immune systems are unable to eliminate cancers. In the strategy of TCR gene therapy, sufficient amount of activated T cells is crucial to perform effective immune responses. Trypan Blue staining and CFSE-based flow cytometry are the generally used methods to access cell activation and proliferation.
➤ Cytokines release assay
The level of cytokines released by modified T cells is one of the indicators reflecting the functional activity of T cells in vivo. The cytokines mainly are IFN-ɣ, IL-12 and GM-CSF. And the widely used methods include enzyme-linked immuno spot assay (ELISpot), intracellular cytokine staining assay (ICS), and flow cytometry.
➤ TCR/CD3ζ stability assay
Increased stability of the TCR/CD3complex contributes to the enhanced antitumor response activity of T cells. The TCR/CD3 complex stability assay can be achieved via immunoprecipitation (IP) and immunoblot.
➤ TCR pairing
In the process to assess TCR pairing, flow cytometry-based FRET, immunoprecipitation, western blotting and probes-based approach can be used.
➤ Lymphocyte antigen reactivity and cytotoxicity
The cytotoxicity of modified T cells can be measured by the chromium release assay and the calcein-AM release assay.
➤ Animal experiments
Creative Biolabs provides multiple animal models for the in vivo function and safety evaluation of T-cell receptor (TCR) gene transfer therapy.
Creative Biolabs possesses unchallenged experience in bispecific T-cell engager (BiTE) synthesis. We elaborately integrate multiple platforms for providing customers an expected BiTE with high affinity and low immunogenicity for both academic and clinical purposes.
Creative Biolabs also possesses experience in synthesis of bispecific intrabodies. Our elaborately integrated multiple platforms can provide customers an expected bispecific intrabody with high affinity and low immunogenicity for both academic and clinical applications.
Creative Biolabs offers our expertise in constructing bispecific fusion proteins for your research and development purposes. We work closely with our customers so as to design every project according to their individual requirements.
As a leading custom service provider in antibody field, Creative Biolabs now offers a range of specifical analysis services for your bispecific antibodies (BsAbs). BsAbs have great potential for treating human diseases, especially for cancers, thus it is essential to perform comprehensive analysis to ensure their quality and function during development. Creative Biolabs has established various bispecific antibody analytical platforms, and we can perform quality assessment, characteristic analysis as well as preclinical research based on our strict standards just in order to meet clients’ requirements about therapeutic BsAb discovery or manufacturing. Enriched experience, profound expertise and state-of-the-art analytical instruments, all of these can ensure Creative Biolabs offer top-quality analysis services for bispecific antibody.
"Stress-free experience from to start to finish of the project. Once the sequence was provided, Creative BioLabs started the project right away and delivered the antibody earlier than anticipated."
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