Sources And Manufacture Of Biopharmaceutical Products

The vast majority of biopharmaceutical products currently on the market are produced by recombinant DNA technology in either E. coli or Chinese Hamster ovary (CHO) cell lines (4). Most monoclonal antibody based products are predictably still produced by hybridoma technology, although the technical methodology now exists to facilitate production of antigen-binding antibody fragments by recombinant means (5,6). E. coli represents a popular recombinant expression system for a number of reasons (7,...

Cell Banking And Stability Of The Production Cell Line

A cell bank is maintained which contains hundreds of frozen vials of CHO cells from which the factor IX is expressed. This is the source for initiating each manufacturing campaign, which can produce up to 20 batches of drug substance. Genetics Institute's currently established cell banks are designed to have a sufficient number of vials to meet worldwide commercial demand for factor IX for over 100 years. The banking process for the cells used to manufacture rFIX involved establishing a master...

Adenoviral Vectors

The adenoviruses comprise non-enveloped double-stranded DNA viruses that are responsible for respiratory and eye infections in humans. Though adenoviruses have a natural tropism for the epithelium of the respiratory tract they can also transduce most other cell types giving a broad target cell range. Adenoviral vectors have been the second most popular vehicles of choice for gene delivery after retroviral vectors and, to date, have been employed in 15 of gene therapy clinical trials. In...

Biopharmaceutical Facility Characteristics

The sections above describe validation concepts, approaches and activities that are broadly applicable to most pharmaceutical facilities. The following section considers the specific characteristics of the biopharmaceutical plant and how this impacts on validation. Biopharmaceutical facilities manufacture product typically either by mammalian cell culture, microbial cell culture or extraction from natural sources. One of the major concerns in these facilities is operator and environmental...

Installation Qualification

The Installation Qualification (IQ) is a documentation process which verifies that the physical components of the system have been installed according to design specifications. The IQ will serve as the final major component and system quality audit prior to Operational Qualification (OQ). Traditionally, when the installation and commissioning has been completed, IQ will take place. More recently, the Validation Turnover Package (VTOP) approach has become increasingly used for new systems and...

Construction of the rFIX Expression Vector

For expression in CHO cells, the 2.5-kb Pstl fragment of M13mpll-EX containing the rFEX coding region was initially subcloned into the mammalian expression plasmid p91023(B) (30). The resultant plasmid, designated p91023-IX, was used for studies demonstrating biological activity of rFIX produced in CHO cells and allowed analysis of the requirements for expression, post-translational modification and secretion of active rFEX (17). For development of the production cell line, the 2.5-kb Pstl...

Marketing Plan

As you develop your marketing plan the document should begin with objectives. Make a list of the objectives you wish to fulfil. These should be important corporate, product, financial, and non-financial, targets. These objectives will become the nucleus of your plan. The Marketing Plan should flow from objectives to strategies to programs. The strategies should be constructed to accomplish the objectives the programs should be developed to support the strategies. Think of it this way Objectives...

Dynamic plasma clot lysis model

The activities of reteplase and alteplase were compared in the dynamic plasma clot lysis model (Figure 24 (41)). Figure 24. Schematic description of the dynamic plasma model. In order to avoid the shear stress-induced coagulation of the plasma, the pressure was provided by a buffer compartment filled with 0.01 M Tris HCl, pH 7.4, 0.01 Tween 80. The mixture of buffer and the plasma above the clot was avoided by the installation of a bubble trap. Figure 24. Schematic description of the dynamic...

Genentech v Wellcome Foundation and Genetics Institute 1990

Genentech sued these defendants for infringement of the following three US patents relevant to t-PA (9) - 4,752,603 (the '603 patent) is directed to Human plasminogen activator derived from the Bowes melanoma cell line and it covers the original work carried out by Leuven Research and Development. The claims are limited to material of specific activity of 500,000 IU mg against a specified reference standard. This limitation was necessary because of an earlier publication by one of the inventors...

Other stabilisers and additives for solution formulations

Proteins can also be stabilised by solutes that interact indirectly with the protein molecules. Compounds, which modify bulk solvent properties or act as a scavenger of certain solutes, can also stabilise proteins from certain chemical degradation pathways. The so-called antioxidants, which are scavengers of radicals, can protect cysteine residues from oxidising to disulphide bonds. Commonly used metal chelators such as EDTA chelate transition metal ions to prevent oxidative degradation....

Abciximab The First Platelet Glycoprotein IlbIIIa Receptor Antagonist

Nakada, Harlan F. Weisman Centocor Inc., Malvern, Pennsylvania, USA Key words abciximab, GP Ilb HIa, platelets, ReoPro, monoclonal antibody, antithrombotic therapy. Abstract Platelet aggregation plays a crucial role in the development of the life- threatening thrombosis responsible for such acute coronary syndromes as myocardial infarction and unstable angina pectoris. Although aspirin has traditionally been the mainstay of antiplatelet therapy, it is neither potent...

References

All data described in this Chapter have been published previously in numerous reports. An extensive list of references can be obtained from the The following literature on follitropin beta is especially recommended 1. Hard, K. et al. (1990). Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells. Eur. J. Biochem., 193, 263-271. 2. Mannaerts, B. et al. (1991). Comparative in vitro and...

Transfection and selection of FSHproducing CHO cells

Transfection of CHO-K1 cells with the FSH expression plasmid pKMS.FSHagPg and the selector plasmid pAG60 MT2 (ratio of 10 1) was carried out essentially following a calcium phosphate-DNA co-precipitation protocol. Geneticin resistant cells were subsequently grown in the presence of different concentrations of cadmium chloride (up to 10 pM). A number of pools of Cd2+ resistant cells indeed produced significant amounts of recombinant FSH, as determined by a FSH-specific ELISA. These pools were...

Mode Of Action

There can be no doubt that interferon beta-lb has a strong, relevant and lasting impact on all major aspects of MS. This effect is achieved without jeopardizing the immune status of the patients and without any bone marrow depression or other relevant toxicity. How can this be achieved and what is the mechanism of action of this drug This has not been elucidated and, given the lack of detailed knowledge of MS pathogenesis, the answer will be hard to find. It is generally assumed that the...

The Erythropoietin litigation Further developments

As noted above, one of the parties to this dispute has a patent for a purified form of erythropoietin while the other has a patent covering recombinant techniques for producing the protein. The case throws further light on the question of mutual impact between two patents of this kind (7). The plaintiff Amgen Inc. holds US patent 4,703,008 (the '008 patent) which describes the production of recombinant erythropoietin. The patent states that The present invention provides, for the first time,...

New Centralized System

December 31st, 1997 marked the end of the initial (3 year) introductory phase of the EMEA's existance. By that date, the CPMP had issued scientific opinions for 27 products of biotechnology, 23 of which had also been granted Marketing Authorization by the European Commission (Table 35). The first product of biotechnology to gain approval under the new system was Serono's Gonal F (recombinant follicle stimulating hormone). This product was evaluated by the CPMP in an active time of only 107...

Primary Structure and Posttranslational Modifications

The primary structure of rFIX (see Figure 12) was examined using peptide mapping, mass spectrometry, NH2-terminal sequencing, and COOH-terminal analysis (40). Using these procedures, the entire amino acid sequence of rFIX was confirmed to be identical to the Ala-148 allotype of pdFIX. The observed mass of rFIX as determined by MALDI-TOF MS was 55,290 Da, which is higher than the predicted value calculated for the amino acid sequence alone (47,054 Da) and which reflects the presence of...

Evaluative Tests for Insulin Lispro denotes routine tests

- *Rabbit hypoglycaemia assay for bioidentity - Insulin receptor binding (39) - Amino acid composition and sequence (58) - Peptide mapping by RP-HPLC (28, 59) - *Purity and identity by RP-HPLC and SE-HPLC (59) Crystallisation and X-ray crystal structure (47) Absorption and circular dichroic spectra (33) NMR spectroscopy (61) - Aggregation behaviour by hydrodynamic methods (33, 43, 48) - *E. coli host proteins by immunoassay Residual enzyme activities by immunoassays (54) - *Proinsulin and...

Purity

Using SDS-PAGE, size-exclusion high-performance liquid chromatography (SEC-HPLC), reverse-phase HPLC, and N-terminal sequencing, the purity of rFIX in this study was determined to be higher than that of four different high-purity pdFEX concentrates. SDS-PAGE analysis under nonreducing conditions revealed a single major band for both rFIX and high-purity pdFIX-1, whereas the other pdFIX preparations contained numerous additional bands in significant amounts. NH2-terminal sequence analysis of the...

The rules governing medicinal products in the European Union

In addition to publishing the full text of the actual binding legislation (regulations and directives), the European Commission has facilitated the preparation and publication of several guides, largely designed to assist the pharmaceutical industry and other interested parties interpret meet the requirements of the legislation. The legislation, along with these guides guidelines are published in a series of volumes entitled The rules governing medicinal products in the European Union (Table...

Manufacturing Process for Insulin Lispro

Lilly's experience in manufacturing human insulin in E. coli using rDNA technology is well documented (6, 23, 50-54). Commercial recombinant human insulin was initially made by a chain combination procedure (28), then four years later in 1986 via the proinsulin route (24). In this latter process a 277-residue chimeric fusion protein (Trp LE'- Methionine-Human Proinsulin) is expressed in E. coli followed by chemical cleavage at methionine with cyanogen bromide to release human proinsulin mixed...

Introduction

Haematopoietic growth factors are glycoproteins that act on cells at various stages to produce mature haematopoietic (i.e. blood) cells. There are many hematopoietic growth factors (Figure 14) one factor that has been isolated, purified, cloned, and produced in commercial quantities is granulocyte colony-stimulating factor (G-CSF), a protein that acts on the neutrophil lineage to stimulate the proliferation, differentiation, and function of committed progenitor cells and functionally active...

Recombinant Coagulation Factor IX Bene Fix

Genetics Institute Inc., 87 Cambridge Park Drive, Cambridge, Mass. Keywords Hemophilia, regulatory, manufacturing, clinical, development Abstract Coagulation factor IX is a zymogen that is an essential component of the clotting process deficient factor IX activity results in hemophilia B. To overcome problems associated with plasma-derived blood products, Genetics Institute developed a recombinant coagulation factor IX (rFIX, BeneFix ) produced using a Chinese hamster ovary cell line. The...

Preparation of active reteplase from inclusion bodies

The production of reteplase in E. coli leads to the formation of inclusion bodies (32,35). Lysozyme was added to the E. coli, and the cells were lysed by high-pressure dispersion (LAB 60, APV-Gaulin, L beck, Germany). The inclusion bodies were isolated by centrifugation (20000 g, 1 hour, 4 C) and solubilized with dithioerythritol and guanidium hydrochloride (36). The thiol groups were derivatized with glutathione by incubating the solubilized protein in 0.05 M Tris-HCl, pH 9.3, 6 Mguanidine,...

Managing multiple sources of information

One of the biggest hurdles encountered by the biotechnology and pharmaceutical industry today, is the integration and analysis of information from disparate machines and by researchers located in different research centres around the world. Subsequent integration of this information with external data from collaborators, or with data or from the public domain (such as the Human Genome Project), further complicates the problem. As an example of managing multiple sources of information in a drug...

Follitropin beta Puregon

Recombinant human follicle-stimulating hormone N.V. Orgcmon, P.O. Box 20, 5340BHOss, The Netherlands Key words FSH, infertility, IW, follitropin, Puregon Abstract Follitropin beta (Puregon) is a human follicle stimulating hormone (FSH) produced by means of recombinant DNA technology. Chinese hamster ovary cells were transfected with genes encoding for the a- and p- subunit of FSH. The production of the amino-acid backbone and the very essential post-translational glycosylation process results...

Insulin Lispro Humalog

Bradly Glazer and Kathleen L. Wishner Eli Lilly and Company, Indianapolis, USA Key words Insulin, insulin analogues, insulin lispro, lispro, Humalog, diabetes, diabetes mellitus, rapid acting insulin. Abstract Initially discovered in 1921, insulin was first made commercially available in 1923. Up until the early 1980s, all insulin preparations used medically were obtained by direct extraction from the pancreatic tissue of animals. In 1982, Humulin (recombinant human...

Molecular modifications to produce reteplase

Extensive work on t-PA variants in our laboratories demonstrated that it is possible to produce new molecules with different properties. Reflecting the requirements for an improved thrombolytic, reteplase was designed to retain the plasmin forming activity, the fibrin specificity and to have a longer plasma-half-life which allows bolus injection instead of infusion. The complementary DNA sequences coding for the finger domain, responsible for high affinity fibrin binding, the EGF- and the...

Surrogate Markers

Surrogate markers are often used as the endpoints in clinical trials. A surrogate marker is a measurable entity which is directly related to the actual disease process. For example when we treat patients with hypertension one of the main aims is to prevent stroke. However, it is necessary to treat patients for many years to prevent a stroke occurring. If one used stroke as the endpoint of a 200 patient trial of a new antihypertensive medicine, then one might have to follow these patients for 10...

Erythropoietin litigation

In the Amgen case 3 , Genetics Institute Inc. GI had obtained US patent 4,677,195 for a method of purification of epo from human urine by reverse phase high performance liquid chromatography. But the patent also has broad product claims, including the following - Homogeneous erythropoietin characterised by a molecular weight of about 34,000 Daltons on SDS PAGE, movement as a single peak on reverse phase high performance liquid chromatography and a specific activity of at least 160,000 IU per...

Holding and Distribution Systems

Below is a list of recommendations for design and installation regarding the holding and distribution systems. - Sterile hydrophobic vent filters of 0.2 micron rating should be fitted on storage tanks. In hot services, the filter must be steam jacketed or electrically heated to prevent condensation - WFI should be kept between 65 and 80 C and should be drawn off immediately before use. It should not be allowed to stand for more than 8 hours before use. Ambient water loops must be sanitised...

Morphology

This analysis provides assurance that rFIX WCB and rFIX EOP cells have physical characteristics expected for CHO-derived cells adapted to suspension culture. Cells were examined by light microscopy for cell shape. Examination of rFIX WCB and rFIX EOP cells by light microscopy revealed that the cells are essentially round and highly refractile. These observations are consistent with those made when rFIX MCB cells were similarly examined and support the conclusion that FIX. IF cell morphology is...

Toxicopharmacologic Effects

Prior to clinical trials, insulin lispro was compared to regular human insulin in a variety of receptor-binding and cell-binding functional assays. These studies demonstrated that insulin lispro was equipotent or slightly less potent than regular human insulin in binding to the human insulin receptor. Conversely, studies also found insulin lispro was equipotent or only slightly more potent than regular human insulin in binding to the IGF-I receptor 39, 57, 63, 64 . These binding considerations...

Expression of reteplase in E coli

A complementary DNA library from a Bowes melanoma cell line was screened with a mixture of three oligodeoxynucleotides which were designed on the basis of the published t-PA sequence 26 . A full-length t-PA complementary DNA clone was reconstituted from several overlapping clones. The coding sequence for the finger, EGF and kringle domains of the t-PA nucleotides 199-714 was removed according to the intron-exon organization of the t-PA gene 31 . The coding sequence of reteplase was introduced...

Conclusion De

The processes by which new medicines are developed are continually evolving in parallel with new technologies, medical discoveries and new regulatory requirements. Many discoveries in the pathophysiology of diseases are made by researchers in, or collaboration with, the pharmaceutical industry. The ever spiralling costs of developing new medicines gives great cause for concern, whilst the advances in molecular biology and gene therapy give great hopes for therapeutic advances. As man has a...

Methods For The Determination Of Column Quality Parameters

Equilibrate the column according to standard procedures. 2. Make up a solution of sample in the column quilibrant. The sample should be a substance that can be detected by the downstream detectors but is inert to the column matrix. It is common to use a 2 v v solution of acetone as this generally fufrls the criteria described. It is important that the substance used is approved for use according to standard QC procedures. 3. Add the sample to the column in a manner that minimises the...

Stabilisation to Prevent Acute Damage or Loss

As noted in sections 2, 3 and 7.1, the finished product formulation must be capable of withstanding the stresses encountered during shipping, handling and use of the product to ensure its safety and efficacy. Acute damage to proteins can occur during product handling, transfer and usage, as proteins are unstable to agitation stresses. Mechanical agitation and container movements result in exposure of proteins to shearing forces. These perturbations can cause unfolding of the protein, thereby...

Amidolytic activity

The amidolytic activity of reteplase, indicated by kinetic constants Km and kQit, is similar to that of rt-PA, both for the single- and two-chain forms. Incubation with plasmin i.e. generation of the two-chain forms increases the catalytic efficiency kcJKm of both reteplase and rt-PA by factors of 12 and 17, respectively. These results were obtained in an assay using S-2288 as the substrate 12 . Data is provided in Table 20. Table 20. Kinetic constants for the amidolytic activity of reteplase...

Matrex Cellufine Sulfate Chromatography

The Q-Sepharose FF product peak pool is loaded directly onto the second chromatography column used for purification, Matrex Cellufine Sulfate, a heparin analogue. Matrex Cellufine Sulfate is used for affinity purification of proteins with heparin-binding domains and also behaves as a cation exchange resin due to the negatively charged sulfate groups. Upon completion of loading, the column is washed with buffer 50 mM Tris, 0.2 M NaCl, pH 8.0 to remove loosely bound contaminants, and the bound...