Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics

Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.     

Practical considerations in clinical strategy to support the development of injectable drug-device combination products for biologics

The development of an injectable drug-device combination (DDC) product for biologics is an intricate and evolving process that requires substantial investments of time and money. Consequently, the commercial dosage form(s) or presentation(s) are often not ready when pivotal trials commence, and it is common to have drug product changes (manufacturing process or presentation) during clinical development. A scientifically sound and robust bridging strategy is required in order to introduce these changes into the clinic safely. There is currently no single developmental paradigm, but a risk-based hierarchical approach has been well accepted. The rigor required of a bridging package depends on the level of risk associated with the changes. Clinical pharmacokinetic/pharmacodynamic comparability or outcome studies are only required when important changes occur at a late stage. Moreover, an injectable DDC needs to be user-centric, and usability assessment in real-world clinical settings may be required to support the approval of a DDC. In this review, we discuss the common issues during the manufacturing process and presentation development of an injectable DDC and practical considerations in establishing a clinical strategy to address these issues, including key elements of clinical studies. We also analyze the current practice in the industry and review relevant and status of regulatory guidance in the DDC field.     

Potential application of Leishmania tarentolae as an alternative platform for antibody expression

Through the discovery of monoclonal antibody (mAb) technology, profound successes in medical treatment against a wide range of diseases have been achieved. This has led antibodies to emerge as a new class of biodrugs. As the “rising star” in the pharmaceutical market, extensive research and development in antibody production has been carried out in various expression systems including bacteria, insects, plants, yeasts, and mammalian cell lines. The major benefit of eukaryotic expression systems is the ability to carry out posttranslational modifications of the antibody. Glycosylation of therapeutic antibodies is one of these important modifications, due to its influence on antibody structure, stability, serum half-life, and complement recruitment. In recent years, the protozoan parasite Leishmania tarentolae has been introduced as a new eukaryotic expression system. L. tarentolae is rich in glycoproteins with oligosaccharide structures that are very similar to humans. Therefore, it is touted as a potential alternative to mammalian expression systems for therapeutic antibody production. Here, we present a comparative review on the features of the L. tarentolae expression system with other expression platforms such as bacteria, insect cells, yeasts, transgenic plants, and mammalian cells with a focus on mAb production.     

Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.     

Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy

Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.     

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional ¹H,¹⁵N and ¹H,¹³C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-¹⁵N, 20%-¹³C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.     

重组腺相关病毒载体基因表达效率与辐射的关系

重组腺相关病毒(rAAV)载体被认为是最有希望的临床应用基因治疗载体之一。rAAV载体基因传递的有效性与病毒衣壳蛋白的免疫原性之间的矛盾是制约该类基因药物开发的关键难题,提高其表达效率是rAAV载体研发的主攻方向之一。研究发现,紫外线、γ射线等辐射可以提高rAAV载体的基因表达效率,其作用效果与辐射种类和剂量、细胞种类和状态、载体感染指数和感染时间等因素有关。其机制可能与DNA损伤反应,特别是DNA双链断裂修复有关。对辐射与rAAV载体的表达效率之间关系的深入了解,为两者的联合应用打开方便之门。本文将对UV、γ射线等辐射提高rAAV载体表达效率的机制、影响因素及其在基因治疗领域的应用进展进行综述。     

High-level secretory expression of immunologically active intact antibody from the yeast Pichia pastoris

We have produced a functional murine antibody to dioxin in the culture medium of the methylotrophic yeast Pichia pastoris. Complementary DNA copies encoding the light () and heavy () chains of the dioxin monoclonal antibody, DD1, were each placed under the control of P.pastoris alcohol oxidase (AOX1) promoter and Saccharomyces cerevisiae -mating factor secretion signal sequence. The resulting expression cassettes were assembled into a single plasmid (pPICZDD1) to permit co-expression of both light and heavy chains of the antibody molecule. P.pastoris SMD1168 (pep4, his4) transformed with pPICZDD1 was able to secrete intact antibody into the culture medium. As high as 36 mg l^–1 of the antibody was produced in shake-flask cultures after 96-h induction with methanol. Functional analysis using immunoassay confirmed murine nature of the recombinant antibody and its ability to bind dioxin.     

Enhanced expression of a biosimilar monoclonal antibody with a novel NS0 platform

The precise product quality and lower cost of goods demands of the growing biosimilars industry are driving biomanufacturing innovation. Biosimilar cell lines that produce complex glycoproteins such as monoclonal antibodies must be both highly productive and express a product with critical quality attributes closely matching those of the innovator reference. In this work, a biomanufacturing platform is described that harnesses the commercially‐established NS0 host cell in new ways to create stable, highly productive cell lines with characteristics meeting the current demands. A cholesterol metabolic selection marker and implementation strategy that can be generically applied are shown to yield high expressing cell lines as well as eliminate the need for cholesterol addition, which has been a significant barrier in both stainless steel reactors as well as in single‐use plastic systems. Additionally, for the first time, a multiplex selection strategy was implemented that served to increase NS0 cell line specific productivity >10‐fold and volumetric yields >6‐fold. The best overall performing cell line had a Qp of 28.5 picograms per cell per day and was rapidly adapted to a lean production medium. Yields in l ‐glutamine fed‐batch shaker cultures exceeded 500 mg/L. An initial screening of four feeding strategies resulted in a final 13‐day yield of over 1.4 g/L in small shaker culture. Overall, this work shows both the strategy to develop biosimilar cell lines and the commercial potential of a novel expression system highly suited for the manufacture of biosimilars of reference biologics currently produced in murine cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:455–462, 2018     

The birth pangs of monoclonal antibody therapeutics: The failure and legacy of Centoxin

This paper examines the development and termination of nebacumab (Centoxin®), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor''s development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development.     

人IL17-RD胞外区真核表达及其单克隆抗体的制备

为了进一步研究白介素17受体D (IL-17RD) 在IL-17信号的调节作用,探索是否可以通过单克隆抗体阻断IL-17RD介导的IL-17信号通路而缓解自身免疫疾病,利用昆虫表达载体从Sf9细胞中表达纯化人IL-17RD-ECD蛋白,免疫Balb/C小鼠30 d,取小鼠脾脏细胞并与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株能稳定分泌抗IL-17RD-ECD的杂交瘤细胞株1F8。经过初步鉴定,该细胞株分泌的抗体类型为IgG1+kappa类,经过Western blot     

Utility of immunodeficient mouse models for characterizing the preclinical pharmacokinetics of immunogenic antibody therapeutics

Prior to clinical studies, the pharmacokinetics (PK) of antibody-based therapeutics are characterized in preclinical species; however, those species can elicit immunogenic responses that can lead to an inaccurate estimation of PK parameters. Immunodeficient (SCID) transgenic hFcRn and C57BL/6 mice were used to characterize the PK of three antibodies that were previously shown to be immunogenic in mice and cynomolgus monkeys. Four mouse strains, Tg32 hFcRn SCID, Tg32 hFcRn, SCID and C57BL/6, were administered adalimumab (Humira®), mAbX and mAbX-YTE at 1 mg/kg, and in SCID strains there was no incidence of immunogenicity. In non-SCID strains, drug-clearing ADAs appeared after 4–7 days, which affected the ability to accurately calculate PK parameters. Single species allometric scaling of PK data for Humira® in SCID and hFcRn SCID mice resulted in improved human PK predictions compared to C57BL/6 mice. Thus, the SCID mouse model was demonstrated to be a useful tool for assessing the preclinical PK of immunogenic therapeutics.     

针对猪瘟病毒E2蛋白的嵌合猪源化单克隆抗体的表达及抗病毒活性鉴定

猪瘟(Classical swine fever,CSF)是严重危害养猪业的一种烈性传染病,常造成巨大的经济损失,是世界动物卫生组织要求必须申报的动物疫病之一。猪瘟的病原是猪瘟病毒(Classical swine fever virus,CSFV),CSFV的结构蛋白由衣壳蛋白(C)和囊膜糖蛋白(E~(rns)、E1、E2)构成。E2蛋白是CSFV主要的保护性抗原,可以诱导机体产生中和抗体,从而抵抗CSFV的感染。此前,本团队制备了一株针对CSFV E2蛋白的鼠源单克隆抗体HQ06。文中将HQ06抗体重链和轻链可变区基因与猪源恒定区基因嵌合后克隆至真核表达载体,利用中国仓鼠卵巢(CHO)细胞制备一株针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06。应用ELISA、Western blotting试验证实了c HQ06与CSFV E2蛋白具有良好的反应性;中和试验结果表明c HQ06可以中和CSFV。综上所述,本研究应用CHO细胞稳定表达了具有良好反应性和中和活性的针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06,为研究CSFV E2蛋白结构、功能以及开发新型的CSFV诊断和治疗制剂奠定基础。     

Changes to International Nonproprietary Names for antibody therapeutics 2017 and beyond: of mice,men and more

Active pharmaceutical substances require an International Nonproprietary Name (INN) assigned by the World Health Organization (WHO) to obtain market authorization as a medicinal product. INNs are selected to represent a unique, generic name for a drug enabling unambiguous identification by stakeholders worldwide. INNs may be requested after initiating clinical development of an investigational drug. Pharmaceutical classes are indicated by a common stem or suffix. Currently, INNs for monoclonal antibody-based drugs are recognized by the suffix, -mab, preceded by a source infix such as -xi- (chimeric), -zu- (humanized) or -u- (human) designating the species from which the antibody was derived. However, many technological advances have made it increasingly difficult to accurately capture an antibody's source in its name. In 2014, the WHO and the United States Adopted Names (USAN) Council approached this challenge by implementing changes to antibody source infix definitions. Unfortunately, gaps and ambiguities in the definitions and procedures resulted in inconsistent source category assignments and widespread confusion. The Antibody Society, extensively supported by academic and industry scientists, voiced concerns leading to constructive dialog during scheduled consultations with WHO and USAN Council representatives. In June 2017, the WHO announced that use of the source infix will be discontinued for new antibody INNs effective immediately. We fully support this change as it better aligns antibody INNs with current and foreseeable future innovations in antibody therapeutics. Here we review the changes implemented. Additionally, we analyzed antibody INNs recently assigned under the previous 2014 definitions and provide recommendations for further alignment.     

Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy

Combination therapy is a fast-growing strategy to maximize therapeutic benefits to patients. Co-formulation of two or more therapeutic proteins has advantages over the administration of multiple medications, including reduced medication errors and convenience for patients. Characterization of co-formulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. We present the results of a deamidation study of one monoclonal antibody component (mAb-B) in co-formulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. We developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)–based method to monitor site-specific deamidation. Our approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.     

Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display

The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. On the basis of phage display technology, we recently reported the first humanization of a rabbit monoclonal antibody. The allotypic diversity of rabbit immunoglobulins prompted us to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, we evaluated the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes. Most rabbit light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intrachain disulfide bridges shared with mouse and human kappa light chains. Here we evaluate the impact of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries. We demonstrate that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones with b9 allotype is a rabbit/human Fab that binds with a dissociation constant of 1nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer. Examination of 228 new rabbit antibody sequences allowed for a comprehensive comparison of the LCDR3 and HCDR3 length diversity in rabbits. This study revealed that rabbits exhibit an HCDR3 length distribution more closely related to human antibodies than mouse antibodies.     

An in vitro FcRn- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans

ABSTRACT

A cell-based assay employing Madin–Darby canine kidney cells stably expressing human neonatal Fc receptor (FcRn) heavy chain and β2-microglobulin genes was developed to measure transcytosis of monoclonal antibodies (mAbs) under conditions relevant to the FcRn-mediated immunoglobulin G (IgG) salvage pathway. The FcRn-dependent transcytosis assay is modeled to reflect combined effects of nonspecific interactions between mAbs and cells, cellular uptake via pinocytosis, pH-dependent interactions with FcRn, and dynamics of intracellular trafficking and sorting mechanisms. Evaluation of 53 mAbs, including 30 marketed mAb drugs, revealed a notable correlation between the transcytosis readouts and clearance in humans. FcRn was required to promote efficient transcytosis of mAbs and contributed directly to the observed correlation. Furthermore, the transcytosis assay correctly predicted rank order of clearance of glycosylation and Fv charge variants of Fc-containing proteins. These results strongly support the utility of this assay as a cost-effective and animal-sparing screening tool for evaluation of mAb-based drug candidates during lead selection, optimization, and process development for desired pharmacokinetic properties.     

西藏株小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备

【背景】小反刍兽疫是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,严重威胁我国养羊业的发展。【目的】原核表达PPRVH蛋白,并制备其多克隆抗体。【方法】根据GenBank中PPRV西藏株h基因序列,对其进行密码子大肠杆菌偏爱性优化,采用两步PCR法全化学合成全长h基因。将测序验证正确的h基因克隆至原核表达载体pET-28a、pET-30a、pET-32a,转化E. coli BL21(DE3)并利用IPTG诱导H蛋白表达。以经SDS-PAGE割胶纯化的重组H蛋白免疫新西兰大白兔制备抗PPRV H蛋白多克隆抗体。【结果】重组E. coli [pET-28a(-30a,-32a)-H]表达的重组H蛋白相对分子质量分别约为70、68和86 kD;诱导7 h时PRRV H蛋白表达量最高,而且主要以包涵体形式表达;重组E.coli(pET-30a-H)表达的H蛋白经SDS-PAGE割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组H蛋白发生特异性反应;ELISA法检测抗体效价在1:6400-1:25600之间。【结论】原核表达了PPRVH蛋白,并制备了高效价的抗H蛋白多克隆抗体,为进一步研究PPRV H蛋白的功能及H蛋白的线性B细胞表位作图奠定了基础。     

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019     

一种人源抗狂犬病病毒单克隆抗体的瞬时表达及性质分析

目的瞬时表达人源重组抗狂犬病病毒单克隆抗体,并对抗体性质进行分析。方法 PCR法扩增抗体轻、重链可变区并分别构建真核表达质粒;瞬时转染HEK293 EBNA1细胞;抗体经亲和纯化后,CE-SDS法分析纯化后抗体单体比例,用快速荧光灶抑制试验(RFFIT)分析抗体体外抗狂犬病病毒中和活性,ELISA分析抗体与3aG、CTN、PV及CVS株的结合。结果提取瞬时表达质粒的A260/280 nm比值在2.0～2.1之间纯化后抗体非还原CE-SDS单体纯度为74.4%;还原CE-SDS抗体轻、重链峰合计占比超过95%;抗体RFFIT体外中和活性为293.37 IU/mL,比活达628.21 IU/mg,抗体与3aG、CTN、PV及CVS株交叉反应均为阳性。结论该株抗体具有较高体外抗狂犬病病毒中和活性,与主要疫苗株均有较强结合。